WO2022140613A1 - Methods and compositions for the treatment of perianal fistula in crohn's disease - Google Patents

Methods and compositions for the treatment of perianal fistula in crohn's disease Download PDF

Info

Publication number
WO2022140613A1
WO2022140613A1 PCT/US2021/064970 US2021064970W WO2022140613A1 WO 2022140613 A1 WO2022140613 A1 WO 2022140613A1 US 2021064970 W US2021064970 W US 2021064970W WO 2022140613 A1 WO2022140613 A1 WO 2022140613A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
liter
mscs
million
composition
Prior art date
Application number
PCT/US2021/064970
Other languages
French (fr)
Inventor
Amy LIGHTNER
Erik John Woods
Hannah Marie MILLER
Brian H. Johnstone
Original Assignee
Ossium Health, Inc.
Cleveland Clinic
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ossium Health, Inc., Cleveland Clinic filed Critical Ossium Health, Inc.
Publication of WO2022140613A1 publication Critical patent/WO2022140613A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
    • C12N2500/62—DMSO
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/99—Serum-free medium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/73—Hydrolases (EC 3.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2523/00—Culture process characterised by temperature

Definitions

  • CD Crohn’s disease
  • An aspect of the present disclosure is a method for treating a perianal fistula in a subject. Th method comprising injecting into the perianal fistula a first dose of a composition comprising more than about 10 million human mesenchymal stem cells (MSCs) obtained from a non-living source.
  • MSCs human mesenchymal stem cells
  • the subject is afflicted with an autoimmune disease, e.g., associated with Crohn’s Disease.
  • an autoimmune disease e.g., associated with Crohn’s Disease.
  • the method further comprises injecting into the fistula an at least second dose of a composition comprising more than about 10 million MSCs.
  • the first dose and/or the at least second dose independently comprise more than about 50 million MSCs.
  • the first dose and/or the at least second dose independently comprise more than about 100 million MSCs.
  • the at least second dose is administered about one month, two months, three months, four months, five months, or six months after the first dose.
  • the subject has an at least second perianal fistula.
  • the at least second perianal fistula may be injected with a first dose of a composition comprising more than about 10 million MSCs.
  • the at least second perianal fistula may be injected with an at least second dose of a composition comprising more than about 10 million MSCs.
  • the at least second fistula may be injected with a first dose of a composition comprising more than about 50 million MSCs and/or an at least second dose of a composition comprising more than about 50 million MSCs.
  • the MSCs are allogenic to the subject.
  • the MSCs are HLA matched or mis-matched to the subject.
  • the MSCs and subject may have fewer than six HLA matches out of eight HLA markers, fewer than four HLA matches out of six HLA markers, or fewer than five HLA matches out of ten HLA markers.
  • the MSCs are obtained from one or more vertebral bodies. [0012] In various embodiments, the MSCs are obtained from a single donor.
  • the composition comprises from about 0.5 % CD45+ cells to about 10 % CD45+ cells, e.g., the composition comprises less than about 5% CD45+ cells.
  • the composition comprises from about 70 % CD105+ cells to about 100 % CD105+ cells, e.g., the composition comprises at least 90% CD105+ cells.
  • the composition comprises from about 70 % CD 166+ cells to about 100 % CD166+ cells, e.g., the composition comprises at least about 90% CD166+ cells.
  • the MSCs are vertebral body bone marrow MSCs (vBM-MSCs).
  • the MSCs are vertebral body bone adherent MSCs (vBA- MSCs). In some cases, the vBA-MSCs are separated from a vertebral body or a fragment thereof by incubation of the vertebral body or the fragment thereof in a collagenase solution. [0018] In embodiments, the MSCs are vertebral body bone adherent MSCs (vBA-MSCs). [0019] In embodiments, the MSCs are both vBA-MSCs and vBM-MSCs.
  • the MSCs have been cultured for at least one passage, at least two passages, at least three passages, at least four passages, or at least five passages.
  • the MSCs have been cryopreserved before injecting into the perianal fistula.
  • the MSCs have been warmed to a temperature of about 0 °C to about 4 °C at least 12 hours before injecting into the perianal fistula.
  • the MSCs may have been warmed to a temperature of about 0 °C to about 4 °C at least 24 hours before inj ecting into the perianal fistula.
  • the MSCs after being cryopreserved, the MSCs are contacted with a growth medium at a suitable temperature and suitable time sufficient to provide an at least 25% to at least 250% increase in cell number.
  • the MSCs have been warmed to a temperature of about 0 °C to about 4 °C at least two days, three days, four days, five days, six days, or a week before injecting into the perianal fistula.
  • the MSCs are warmed to about room temperature before injecting into the perianal fistula.
  • the MSCs are warmed to about body temperature before injecting into the perianal fistula.
  • the composition comprising the MSCs includes two or more of Plasma-Lyte® A, Human Serum Albumin (HSA), and DMSO, e.g., the composition comprising the MSCs includes Plasma-Lyte® A, HSA, and DMSO.
  • HSA Human Serum Albumin
  • the composition comprising the MSCs includes at least about 2 million cells per ml, e.g., the composition comprising the MSCs includes at least about 3 million cells per ml, 4 million cells per ml, 5 million cells per ml, 6 million cells per ml, 7 million cells per ml, 8 million cells per ml, 9 million cells per ml, 10 million cells per ml, 15 million cells per ml, 20 million cells per ml, 30 million cells per ml, 40 million cells per ml, or 50 million cells per ml.
  • the composition comprising the MSCs includes at least about 2 million MSCs per ml, e.g., the composition comprising the MSCs includes at least about 3 million MSCs per ml, 4 million MSCs per ml, 5 million MSCs per ml, 6 million MSCs per ml, 7 million MSCs per ml, 8 million MSCs per ml, 9 million MSCs per ml, 10 million MSCs per ml, 15 million MSCs per ml, 20 million MSCs per ml, 30 million MSCs per ml, 40 million MSCs per ml, or 50 million MSCs per ml.
  • the composition has an endotoxin level of less than about 1, 2, 3, 4, or 5 Endotoxin Units (EU)/ml, e.g., the composition has an endotoxin level of less than about 2 EU/ml.
  • EU Endotoxin Units
  • the injecting is via a 22G needle.
  • each dose comprises at least about 1 ml of the composition, e.g., each dose comprises at least about 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml of the composition.
  • the injecting is into the submucosal layer of the bowel wall.
  • the injecting is along the length of the fistula tract and/or in parallel to the wall of the tract and/or no deeper than 2 mm from the fistula tract wall.
  • the subject has received a previous medical and/or surgical therapy for treating perianal fistula.
  • the previous medical therapy may comprise one or more of systemic immune suppression (e.g. azathioprine, methotrexate, 6- mercaptopurine) or biologic (e.g. anti-TNF, anti-integrin, anti-interleukin) therapies.
  • the previous surgical therapy may comprise a surgical repair including seton placement, glue or plug insertion, local tissue flaps, and/or ligation of intersphincteric fistula tract repair.
  • the non-living source from which the MSCs were ultimately obtained from a human cadaver.
  • the subject experiences a complete healing as assessed by radiographic healing (MRI with an absence of a fluid collection >2 cm in 3 of 3 dimensions, lack of edema, a lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain) and/or clinical healing (100% cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening).
  • radiographic healing MRI with an absence of a fluid collection >2 cm in 3 of 3 dimensions, lack of edema, a lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain
  • clinical healing (100% cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening).
  • the subject experiences a partial healing as assessed by radiographic healing (MRI with an absence of a fluid collection >2 cm in 2 of 3 dimensions, lack of edema, lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain) and/or clinical healing (Greater than or equal to 50 % cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening).
  • radiographic healing MRI with an absence of a fluid collection >2 cm in 2 of 3 dimensions, lack of edema, lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain
  • clinical healing Greater than or equal to 50 % cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening).
  • compositions for use in any herein- disclosed method are a composition for use in any herein- disclosed method.
  • compositions comprising about at least 10 million human mesenchymal stem cells (MSCs) obtained from a non-living source.
  • MSCs human mesenchymal stem cells
  • the composition comprises less than 5% CD45+ cells.
  • the composition comprises at least 90% CD 105+ cells.
  • the composition comprises at least 90% CD 166+ cells.
  • the human MSCs comprise vertebral body bone marrow MSCs (vBM-MSCs), adherent vertebral body MSCs (vBA-MSCs), or both.
  • vBM-MSCs vertebral body bone marrow MSCs
  • vBA-MSCs adherent vertebral body MSCs
  • the MSCs have been cultured for at least one passage, at least two passages, at least three passages, at least four passages, or at least five passages.
  • the MSCs are obtained from one or more vertebral bodies. [0044] In various embodiments, the MSCs are obtained from a single donor.
  • the MSCs have been cryopreserved. In some cases, the MSCs have been warmed to a temperature of about 0 °C to about 4 °C at least 12 hour, 24 hours, two days, three days, four days, five days, six days, or a week before use in treating a perianal fistula in a subject.
  • the MSCs may be warmed to about room temperature before use in treating a perianal fistula in a subject.
  • the MSCs may be warmed to about body temperature before use in treating a perianal fistula in a subject.
  • the composition comprises at least about 2 million cells per ml, 3 million cells per ml, 4 million cells per ml, 5 million cells per ml, 6 million cells per ml, 7 million cells per ml, 8 million cells per ml, 9 million cells per ml, 10 million cells per ml, 15 million cells per ml, 20 million cells per ml, 30 million cells per ml, 40 million cells per ml, or 50 million cells per ml.
  • the at least about 2 million cells, 3 million cells, 4 million cells, 5 million cells, 6 million cells, 7 million cells, 8 million cells, 9 million cells, 10 million cells, 15 million cells, 20 million cells, 30 million cells, 40 million cells, or 50 million cells are MSCs.
  • the MSCs were ultimately obtained from a human cadaver.
  • FIG. 1 is a schematic showing an illustrative manufacturing process for mesenchymal stromal cells (MSCs).
  • FIG. 2 depicts surface marker expression of cells derived from previously expanded live donor BM-MSC (Ex LD BM-MSC), freshly isolated live donor BM-MSC (LD BM-MSC) and deceased donor BM-MSC (DD BM-MSC).
  • FIG. 3 depicts the population double times of cells derived from previously expanded live donor BM-MSC (Ex LD BM-MSC), freshly isolated live donor BM-MSC (LD BM-MSC) and deceased donor BM-MSC (DD BM-MSC).
  • FIG. 4 depicts the CFU-F potential of cells derived from previously expanded live donor BM-MSC (Ex LD BM-MSC), freshly isolated live donor BM-MSC (LD BM-MSC) and deceased donor BM-MS. (DD BM-MSC).
  • FIG. 5 depicts the ability of cells derived from previously expanded live donor BM- MSC (Ex LD BM-MSC), freshly isolated live donor BM-MSC (LD BM-MSC) and deceased donor BM-MSC (DD BM-MSC) to form adipocytes, chrondrocyes and osetocytes.
  • compositions for treating a perianal fistula in a subject comprise injecting into the perianal fistula a first dose of a composition comprising more than about 10 million human mesenchymal stem cells (MSCs) obtained from a non-living source.
  • MSCs human mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • Human bone marrow contains a population of nonhematopoietic progenitor cells, referred to as mesenchymal stem/stromal cells (MSCs).
  • MSCs mesenchymal stem/stromal cells
  • BM-MSCs are multipotent cells that reside in the perivascular space in bone marrow, and play key roles in supporting hematopoiesis and mediating tissue homeostasis and repair.
  • BM-MSC have been obtained via percutaneous aspiration of living donor BM from the iliac crest. Deceased human donor vertebral bodies are a much more plentiful source of MSC. MSC within the medullary cavity reside both within the BM and trabecular bone compartments.
  • MSCs are clonogenic and multipotent, with the capacity to differentiate into mesodermal cell types, including bone, cartilage, and fat under defined conditions.
  • MSCs exhibit low immunogenicity: MSCs express low levels of major histocompatibility complex (MHC) class I antigens, and are negative for MHC class II molecule, HLA-DR, and for the co-stimulatory molecules CD40, CD80, and CD86 essential for immune recognition. Functionally, MSCs do not elicit a proliferative response to T cells and do not stimulate clinically significant immune responses following allogeneic transplantation in vivo. These immune characteristics allow MSCs to be used in the allogeneic setting between unrelated, human leukocyte antigen- unmatched donor and recipient.
  • MHC major histocompatibility complex
  • MSCs have been shown to attenuate inflammatory and immunological processes relevant to GVHD.
  • MSCs demonstrate immunosuppressive activity in T cell-driven immune responses in animal models of allogenic skin graft rejection and GVHD.
  • MSCs suppress T-cell proliferation in response to allo-antigenic and mitogenic stimulation, and stimulate an increase in the regulatory T cell (Treg) population.
  • Tregs play an important role in inhibiting allogeneic T cell response.
  • MSCs alter the cytokine secretion profile of immune cells (dendritic cells, naive and effector T cells, natural killer cells), decreasing expression of pro-inflammatory cytokines (e.g.
  • MSCs may mediate tissue protection and repair at sites of injury by secretion of soluble factors that are known to mediate processes such as inhibition of apoptotic cell death, recruitment of endogenous stem cell populations and angiogenesis.
  • an Organ Procurement Organization performs evaluation and donor recoveries according to 21 CFR ⁇ 1271.
  • a donor screening and monitoring process may include completion and assessment of a Uniform Donor Risk Assessment Interview (UDRAI).
  • UDRAI Uniform Donor Risk Assessment Interview
  • the UDRAI comprises of flowcharts, guidance documents, and questionnaires which are used to screen potential donors related to medical history, behavioral history, travel history and social history.
  • Serology testing may be completed in a CLIA/CMS-approved laboratory using FDA-cleared test kits to rule out viral pathogens.
  • Donors should be negative or non-reactive for HIV-1, HIV-2, hepatitis B virus (HBV, surface and core antigen), hepatitis C virus (HCV), Syphilis (Treponema pallidum), human T- lymphotropic virus types 1 and 2 (HTLV-1, HTLV-2), West Nile Virus, Chagas (Trypanosoma cruzi). Toxoplasmosis, and Epstein-Barr Virus (EBV). Donors may be screened for Cytomegalovirus (CMV). In some embodiments, a donor is under 30 years old, non-smoker, and confirmed CMV negative.
  • CMV Cytomegalovirus
  • the methods and composition described herein comprises mesenchymal stem cells (MSCs) obtained from a non-living source.
  • MSCs mesenchymal stem cells
  • the MSCs are obtained from a donor bone.
  • the donor bone is vertebral bodies.
  • the methods described herein can be used on the ilium, a combination of the vertebral bodies and ilium, or other bones suitable for extraction of bone marrow and cells from the marrow, even donor bones with lower expected yields.
  • the donor bones can be procured according to fixed protocols for clinical recovery. Bones can be recovered by surgeons or by personnel at a trained OPO (organ procurement organization) using an osteotome and mallet from consented organ and tissue donors. Unprocessed bones are preferably wrapped in sponges and towels soaked in saline to ensure moisture retention during hypothermic shipment on wet ice at a temperature of 0 to 10 °F to a processing facility.
  • OPO organ procurement organization
  • the process for preparing the donor bone can occur soon after the bone is obtained from the deceased donor or can occur after the donor bone has been shipped in a hypothermic environment to a processing facility. Since the donor bone can experience prolonged periods of ischemia during recovery and shipment to the processing facility, care must be taken to track the length and type of ischemia — e.g., warm ischemia and cold ischemia. As described in more detail herein, bone subject to predetermined periods of warm ischemia and/or cold ischemia are suitable for obtaining meaningful quantities of viable bone marrow cells.
  • the bone is debrided in an ISO-5 (class 100) environment (biosafety cabinet) with an ISO-7 (class 10,000) background (clean room), with special care taken to sterilize the bag containing the donor bone, such as by spraying with 70% isopropanol.
  • the debridement is conducted manually using scalpels, osteotomes and gouges.
  • a spinal segment including multiple vertebral levels will be provided.
  • the spine segment runs from T8 to L5, for ten vertebral bodies.
  • the pedicles are removed using either a tissue processing band saw or a bone saw, such as the Stryker System 6 Saw (Stryker, Kalamazoo, MI), or with a hand tool. Special care is taken to avoid breaching the cortical bone which would expose the cancellous bone, to ensure that the hypoxic cancellous bone marrow remains protected throughout the entire debriding process. The anterior element of the vertebral bodies remain, while the pedicles and posterior elements are discarded.
  • the vertebral bodies (VB) are separated at the intervertebral discs.
  • the intervertebral disc and soft tissue remaining on each vertebral body is removed with a scalpel, scissors and/or osteotomes, leaving clean, separated VBs.
  • the soft tissue can be removed with gouges and a scalpel, with special care again taken to ensure that the cortical bone is not breached. Any anatomical pathologies or injuries of the bone are noted and recorded as part of the batch record for the marrow ultimately obtained from the bones. Bones damaged during the recovery process are discarded.
  • cadaver bones undergo a “pre-processing” to reduce contaminants carried by the cadaver bone and which risk transferring the contamination to the facility that the bone is processed.
  • two technicians perform different aspects of the pre-processing.
  • a first technician opens a package containing harvested cadaver bones, preferably contained in a sealed, inner bag.
  • the second technician wearing sterile gloves, removes the cadaver bone from the package and places the tissue in a first (rinse) basin.
  • the second technician scrubs all surfaces of cadaver bone vigorously with an about 4% chi orhexi dine gluconate solution for about 3 minutes while in or above the rinse basin.
  • the first technician wearing sterile gloves, pours sterile saline onto the scrubbed cadaver bone, with the runoff being captured in the rinse basin.
  • a sufficient amount of saline is poured onto the cadaver bone to rinse all of its surfaces.
  • the rinsed cadaver bone is then placed on a sterile cloth adjacent to the rinse basin.
  • the saline rinse may be repeated as necessary.
  • Alcohol e.g., 70% isopropyl alcohol
  • a sufficient amount of alcohol is poured onto the cadaver bone to contact all of its surfaces.
  • the alcohol runoff is captured in the rinse basin.
  • the cadaver bone is placed in an open container which is sprayed with alcohol and, then the open container and bone is transferred to a hood, where further processing of the bone can take place.
  • An aspect of the present disclosure comprises a method for processing bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone from a deceased donor; contacting the bone with a bleach solution for at least about 10 minutes to at least about 25 minutes, wherein the bone is submerged in the bleach solution; extracting the bone marrow or the derivative thereof from the bone, wherein at least 90% of CD34+ cells comprised in the bone marrow or the derivative thereof are viable.
  • the bone marrow or derivative thereof is contacted with the bleach solution for at least about 25 minutes.
  • the bleach solution comprises 10% bleach.
  • the bone is a vertebral body.
  • the hydrogen peroxide is a 3% hydrogen peroxide solution.
  • the method further comprises transferring the bleached bone product from a container comprising the bleach solution to a container containing the hydrogen peroxide solution.
  • the method further comprises a step of agitating the bleached bone product within the hydrogen peroxide solution.
  • the submerging the bleached bone product in a solution comprising hydrogen peroxide comprises: submerging the bleached bone product in a container containing the hydrogen peroxide solution; detecting foam or froth associated with the bleached bone product; and repeating the submerging until no foam or froth is detected.
  • the method further comprises manually removing soft tissue from a bleached bone product that is associated with foam or froth.
  • an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance visibility of any foam or froth associated with the bleached bone product.
  • the bone in the bleach solution are agitated (e.g., shaken).
  • the VBs are placed into a sterile bag and submerged in a 10% bleach solution, yielding a concentration of 5,000 ppm free chlorine, for a predetermined period, typically 5 or more minutes.
  • Bleach has a broad spectrum of anti-microbial activity, does not leave a toxic residue, is unaffected by water hardness and is fast acting.
  • Bone marrow from each group of VBs processed at different duration of bleach treatment can be tested by flow cytometry to assess the viability of the cells isolated from the bone marrow. Soaking the VBs for more than 10 minutes yields no significant difference in cell viability compared to when the VBs are soaked for up to 25 minutes.
  • an increase in bleaching time improves the ultimate product. For example, increasing the soaking of the VBs in bleach for longer period of time allows the bleach to fill the cavity or crevice of the VBs to further decontaminate or sterilize the VBs.
  • Bone marrow from each group of VBs processed at different duration of bleach treatment can be tested by flow cytometry to assess the viability of the cells isolated from the bone marrow (Table 1) As seen from Table, soaking the VBs for more than 10 minutes yields no significant difference in cell viability compared to when the VBs are soaked for up to 25 minutes.
  • the bleach treatment comprises using 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or higher percentage of bleach.
  • the bleach treatment comprises contacting the VBs with bleach for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11, minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, or longer duration.
  • the viability of the bone marrow cells isolated from the VBs treated with the bleach treatment is not decreased at any duration of bleach treatment described herein compared to bone marrow cells isolated from the VBs without the bleach treatment. In some embodiments, the viability of the bone marrow cells isolated from the VBs treated with 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, or longer duration of the bleach treatment is not decreased or is decreased by less than 3% compared to the viability of the bone marrow cells isolated from the VBs treated with the 10 minutes bleach treatment.
  • the viability of the bone marrow cells isolated from the VBs treated with more than 10 minutes decreased by less than 2% compared to the viability of the bone marrow cells isolated from the VBs treated with the 10 minutes bleach treatment. In some embodiments, the viability of the bone marrow cells isolated from the VBs treated with more than 10 minutes decreased by less than 1% compared to the viability of the bone marrow cells isolated from the VBs treated with the 10 minutes bleach treatment.
  • bleach treatment provides surface sterilization of the bone, but does not penetrate the BM-containing compartment. Therefore, the bleach treatments disclosed herein do not substantially reduce the yield of viable cells obtained from BM.
  • the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least about 80% to about 95%. In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95%. In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least about 80%, about 85%, about 90%, or about 95%.
  • the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least at least about 80%, about 85%, or about 90%. In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least at most about 85%, about 90%, or about 95%.
  • the bones are transferred to another sterile bag and submerged in a 3% hydrogen peroxide (H2O2) solution.
  • the H2O2 solution comprises PLASMA-LYTETM (a multiple electrolyte injection that is a sterile, nonpyrogenic isotonic solution that is a base source of water and electrolyte-balanced crystalloids for the cells, obtained from Baxter Healthcare, Ltd.).
  • the H2O2 solution comprises PLASMA- LYTETM and Human Serum Albumin (HSA) which is a stabilizing reagent and storage agent (it may be diluted in the H2O2 solution to achieve 2.5% HSA).
  • HSA Human Serum Albumin
  • the bag is closed and shaken briefly to ensure that the entire surface of the bone is in contact with the solution.
  • Most living cells include catalase, which is an enzyme that catalyzes the breakdown of H2O2 into H2O and O2. This breakdown manifests as foam or froth when the H2O2 solution contacts soft tissue but not bone.
  • the foam level can be observed as an indication of the amount of soft tissue remaining on the bone. This observation can be performed manually by a human processor or, in another embodiment, by an automated processor.
  • the automated processor incorporates a visualization device, such as a camera, and object recognition software that can determine foam levels within the bag.
  • the addition of an inert contrast dye can help the human or automated processor detect the foam level.
  • the bone is returned for further processing to remove all of the remaining soft tissue from the bone.
  • the bones are transferred to a new sterile bag.
  • the bag is filled with IL of PLASMA-LYTETM (multiple electrolytes injection obtained from Baxter Healthcare, Ltd.), or other suitable sterile, nonpyrogenic isotonic solution.
  • the bag is closed and shaken briefly to ensure that the entire bone is contacted with the PLASMA-LYTETM.
  • the method further comprises a step of agitating the bleached bone product within the hydrogen peroxide solution.
  • the submerging the bleached bone product in a solution comprising hydrogen peroxide comprises: submerging the bleached bone product in a container containing the hydrogen peroxide solution; detecting foam or froth associated with the bleached bone product; and repeating the submerging until no foam or froth is detected.
  • the method further comprises manually removing soft tissue from a bleached bone product that is associated with foam or froth.
  • an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance visibility of any foam or froth associated with the bleached bone product.
  • the bleaching step and the hydrogen peroxide steps may be repeated multiple times.
  • the H2O2 solution not only helps surface sterilize the bone, it helps break down any residual bleach into salt, oxygen, and water.
  • the cadaver bone may be rinsed with water, a saline, or with a cryoprotectant solution. Then the surface sterilized cadaver bone may be placed in a closed container comprising a cryoprotectant solution and the pressure is reduced.
  • Ischemia time of the donor bone impacts the viability of the cells extracted using the processes described herein.
  • total ischemia is defined as the interval starting at time of death (the point at which the donor's arterial system was cross-clamped and circulation ceased) and ending with the start of the recovery of cells from the bone.
  • this total interval can be separated into three successive and mutually exclusive time components: (a) Warm Ischemia Time (WIT) - beginning at time of death and ending either when bones are recovered and packed on ice or when the body is placed in a cooler; (b) Body Cooling Time (BCT) - beginning when the body is placed in the cooler and ending when bones are packed on ice; and (c) Cold Ischemia Time (CIT) - beginning when bones are packed on ice and ending when processing begins for extraction of cells, such as HSPCs.
  • WIT Warm Ischemia Time
  • BCT Body Cooling Time
  • CIT Cold Ischemia Time
  • Other variables can include bone type (such as vertebral bodies and ilia), donor sex and donor age.
  • the outcome variables are: the proportion of a particular cell population, such as CD34+ cells, that are viable, the total number of colony forming units (CFUs) per 10 5 nucleated cells detected following cell processing, and the number of CFU granulocyte macrophages (CFU-GM) detected per 10 5 nucleated cells.
  • an ordinary least squares (OLS) beta regression model can be used to predict the outcome variables, with linear regression models used for CFU and CFU-GM and a beta regression model used for the proportion of viable CD34+ cells, or %CD34+, where 0 ⁇ (%CD34+) ⁇ 1.
  • the beta regression equation for predicting %CD34+ is:
  • the regression models are based on un-adjusted models that only account for the ischemia-based variables and not the experience, bone type, donor sex and donor age variables.
  • a fully adjusted model for %CD34+ that accounts for all of the variables. The results of these models are depicted in Tables 2-4.
  • coefficient Pi attempts to quantify the effect of the number of donors processed (i.e., experience) on cell quantity and viability.
  • coefficient P2 corresponds to the experience at a particular facility based on the assumption that facilities can have different learning trajectories. Either or both of these coefficients may be modified or even eliminated.
  • the models disclosed herein predict viability in which an 80% CD34+ (or at least 70%) cell viability threshold is determined to be acceptable.
  • cryoprotectant Surprisingly, by immersing a cadaver bone in a closed container of cryoprotectant and applying an intermittent vacuum to the closed container, the cryoprotectant infiltrates the cadaver bone significantly more rapidly that would occur by passive diffusion. Such effective infiltration of cryoprotectant contributes to reduced ice crystal formation during freezing of the cadaver bone and, ultimately, extraction of viable bone marrow cells that have replicative potential.
  • Cadaver bone can be contacted with a cryoprotectant solution for a length of time and under conditions sufficient to allow infiltration of a cryoprotectant solution into the cadaver bone.
  • Methods for cry opreserving bone is described below and elsewhere.
  • the conditions sufficient to allow infiltration of a cryoprotectant solution involve use of vacuum-assisted infiltration of a cryoprotectant into a cadaver bone as disclosed in PCT/US2021/042064, the contents of which are incorporated by reference in its entirety.
  • cadaver bone is submerged in a cryoprotectant solution and without use of a vacuum.
  • An aspect of the present disclosure is a method for cry opreserving a cadaver bone using vacuum to assist infiltration of a cryoprotectant into the cadaver bone.
  • the method comprises steps of: (a) placing a cadaver bone in a closed container comprising a cryoprotectant solution; (b) reducing the pressure in the closed container, and optionally, holding the closed container at reduced pressure, to remove at least a portion of the water present in the cadaver bone; (c) raising the pressure in the closed container and holding the closed container at a raised pressure to allow infiltration of the cryoprotectant solution into the cadaver bone; (d) removing the cadaver bone from the closed container; and (e) chilling the cadaver bone to a temperature at least below 0 °C, thereby cryopreserving the cadaver bone.
  • cryoprotectant Surprisingly, by immersing a cadaver bone in a closed container of cryoprotectant and applying an intermittent vacuum to the closed container, the cryoprotectant infiltrates the cadaver bone significantly more rapidly that would occur by passive diffusion.
  • the cryoprotectant With respect to PCT/US2021/042064, compare FIG. 2 with FIG. 4A and FIG. 4B and FIG. 3 with FIG. 5.
  • Such effective infiltration of cryoprotectant contributes to reduced ice crystal formation during freezing of the cadaver bone and, ultimately, extraction of viable bone marrow cells that have replicative potential.
  • Steps (b) and (c) may occur only once or steps (b) and (c) may be repeated at least once, at least twice, at least four times, at least five times, or at least six times.
  • repeating the reduced pressure and the raised pressure may increase infiltration of a cryoprotectant into a cadaver bone. See, e.g., FIG. 5 of PCT/US2021/042064.
  • a cadaver bone e.g., vertebral body
  • a cadaver bone is bisected, cut into quarters, or more extensively divided prior to vacuum-assisted infiltration of the cryoprotectant.
  • the reduced pressure in the closed container may any pressure value from about -400 mmHg to about -800 mmHg.
  • the pressure requirement should be sufficient to remove at least a portion of the water present in the cadaver bone.
  • the reduced pressure in the closed container may have a value of about -400 mmHg, -425 mmHg, -450 mmHg, -475 mmHg, -500 mmHg, - 525 mmHg, -550 mmHg, -575 mmHg, -600 mmHg, -625 mmHg, -650 mmHg, -675 mmHg, - 700 mmHg, -725 mmHg, -750 mmHg, -775 mmHg, or - 800 mmHg.
  • the reduced pressure in the closed container is from about -400 mmHg to -500 mmHg.
  • the closed container it takes from about one minute to about 10 minutes for the closed container to reach a desired reduced pressure once the pressure in the closed container begins reducing.
  • the closed container is may take less than 1 minute, about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or about 10 minutes and any length of time in between (e.g., a fraction of a minute, e.g., about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds, and any number of seconds therebetween) to reach the desired reduced pressure.
  • the cadaver bone reaches the desired reduced pressure rapidly, e.g., from about one second to about one minute.
  • the cadaver bone is held at the reduced pressure once the reduced pressure has been reached.
  • the cadaver bone may be held for from less than one minute to about 50 minutes.
  • the closed container is held at reduced pressure for less than one minute, about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 minutes, 49 minutes, or about 50 minutes and any length of time in between (e.g., a fraction of a minute, e.g., about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds, and any number of seconds therebetween).
  • the cadaver bone is not held at any length of time in between (e.g
  • step (c) the pressure of the closed container is raised until the pressure is from about 0 mmHg to about 760 mmHg.
  • the pressure is raised to up to standard atmospheric temperature.
  • the exact raised pressure may be any amount within the specified range, e.g., 0 mmHg, 50 mmHg, 100 mmHg, 150 mmHg, 200 mmHg, 250 mmHg, 300 mmHg, 350 mmHg, 400 mmHg, 450 mmHg, 500 mmHg, 550 mmHg, 600 mmHg, 650 mmHg, 700 mmHg, or 750 mmHg.
  • the raised pressure must be high enough to allow infiltration of the cryoprotectant solution into the cadaver bone.
  • the closed container may be held at the raised pressure for less than about two hours. As examples, for less than one hour, less than one-half hour, about one-half hour, or less time. In some embodiments, the closed container is held at the raised pressure for ten minutes. The duration that the closed container is held at the raised pressure must be long enough to allow infiltration of the cryoprotectant solution into the cadaver bone.
  • the closed container is held at the raised pressure for about, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, or about 30 minutes, and any length of time in between (e.g., a fraction of a minute, e.g., about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds, and any number of seconds therebetween).
  • the closed container and the cryoprotectant contained therein may be at room temperature. Alternately, the closed container and the cryoprotectant contained therein may be below room temperature, e.g., as low as 4 °C. The closed container and the cryoprotectant contained therein may be above room temperature, e.g., as high as 37 °C.
  • cryoprotectant any suitable cryoprotectant may be used in a cryoprotectant solution.
  • cryoprotectant include dimethyl sulfoxide (also known as DMSO, C2H6OS, and ME2SO); 1, 2 propane diol; propylene glycol; ethylene glycol; glycerol; foramamide; ethanediol or butane 2, 3 diol; hydroxyethyl starch (HES); dextran; sucrose; trehalose; lactose; raffinose; ribotol; mannitol; and polyvinylpyrrolidone (PVP).
  • the cryoprotectant is DMSO.
  • the cryoprotectant solution may comprise from about 10% DMSO to about 30% DMSO, e.g., about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or about 30% DMSO.
  • the cryoprotectant solution may comprise about 20% DMSO.
  • a higher percentage of cryoprotectant is preferred, e.g., percentages that are two times higher than equivalent cell suspension values to help drive osmotic penetration.
  • the cryoprotectant solution may have water or a saline as base.
  • the saline is isotonic to human tissues.
  • the saline is a 0.9% saline solution. Any commercially-available saline solution may be used: sodium chloride solution, PBS, HEPES, Ringers or Lactate.
  • the saline may be 0.9% sodium chloride.
  • the cryoprotectant solution may further comprise a protein.
  • the protein may be a human albumin (e.g., HSA) or a constituent of a human platelet lysate.
  • HSA human albumin
  • An example of a commercially-available human platelet lysate product is Stemulate (from Cook Regentec).
  • the cryoprotectant solutions comprises about 10% protein, e.g., 10% human platelet lysate or 10% albumin.
  • the cryoprotectant solution comprises about 20% DMSO and about 10% human platelet lysate in 0.9% NaCl.
  • the cryoprotectant solution comprises about 40% DMSO and about 10% human platelet lysate in 0.9% NaCl.
  • the cryoprotectant solution comprises about 60% DMSO and about 10% human platelet lysate in 0.9% NaCl.
  • the cryoprotectant solution comprises about 80% DMSO and about 10% human platelet lysate in 0.9% NaCl.
  • the cryoprotectant solution comprises about 100% DMSO in 0.9% NaCl.
  • the method may comprise a step of increasing the pressure in the closed container comprising a cryoprotectant to above 760 mmHg by introducing a compressed gas (e.g., nitrogen, xenon, CO2, argon, H2S, or helium), a gas released by sublimination (e.g., CO2 via dry ice), or a gas provided by evaporation (e.g., nitrogen via liquid nitrogen), thereby permeating gas into the cadaver bone.
  • a compressed gas e.g., nitrogen, xenon, CO2, argon, H2S, or helium
  • a gas released by sublimination e.g., CO2 via dry ice
  • a gas provided by evaporation e.g., nitrogen via liquid nitrogen
  • the gas is CO2 , e.g., compressed CO2.
  • the gas is nitrogen, e.g., compressed nitrogen.
  • the time required for gas infiltration into a vertebral body is less when the gas
  • the cadaver bone is placed in a closed container that lacks a cryoprotectant solution.
  • the method comprises a step of increasing the pressure in the closed container (which lacks a cryoprotectant solution) to above 760 mmHg by introducing a compressed gas (e.g., nitrogen, xenon, CO2, argon, H2S, or helium), a gas released by sublimination (e.g., CO2 via dry ice), or a gas provided by evaporation (e.g., nitrogen via liquid nitrogen), thereby permeating gas into the cadaver bone.
  • a compressed gas e.g., nitrogen, xenon, CO2, argon, H2S, or helium
  • a gas released by sublimination e.g., CO2 via dry ice
  • a gas provided by evaporation e.g., nitrogen via liquid nitrogen
  • any method disclosed herein may be adapted by comprising initial steps of placing a cadaver bone in closed container that lacks a cryoprotectant solution and increasing the pressure in the closed container to above 760 mmHg by introducing a compressed gas, a gas released by sublimination, or a gas provided by evaporation; in a later step, a cryoprotectant solution is added to the closed container.
  • the gas is CO2 , e.g., compressed CO2.
  • the gas is nitrogen, e.g., compressed nitrogen.
  • a compressed gas e.g. , nitrogen, xenon, CO2, argon, H2S, or helium
  • a gas released by sublimination e.g., CO2 via dry ice
  • a gas provided by evaporation e.g., nitrogen via liquid nitrogen
  • the closed container comprises solid materials, e.g., metal, plastic, or other polymers.
  • the closed container comprises a foam material, e.g., Styrofoam.
  • a cadaver bone is infiltrated with a cryoprotectant without use of a vacuum.
  • an intact vertebral body a vertebral body that has been bisected, cut into quarters, or more extensively divided is submerged into a cryoprotectant solution for a length of time and under conditions sufficient to allow infiltration of the cryoprotectant solution into the cadaver bone.
  • the bone or bone fragment is placed, e.g., submerged, in a cryoprotectant solution and incubated for 1 hour at about 4°C.
  • the incubation period is about 1 hour to about 3 hours.
  • the incubation period is about 1 hour to about 1.5 hours, about 1 hour to about 2 hours, about 1 hour to about 2.5 hours, about 1 hour to about 3 hours, about 1.5 hours to about 2 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 3 hours, about 2 hours to about 2.5 hours, about 2 hours to about 3 hours, or about 2.5 hours to about 3 hours.
  • the incubation period is about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours.
  • the incubation period is at least about 1 hour, about 1.5 hours, about 2 hours, or about 2.5 hours.
  • the incubation period is at most about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours.
  • cryoprotectant any suitable cryoprotectant may be used in a cryoprotectant solution.
  • cryoprotectant include dimethyl sulfoxide (also known as DMSO, C2H6OS, and ME2SO); 1, 2 propane diol (also known as propylene glycol); ethylene glycol; glycerol; foramamide; ethanediol, butane 2, 3 diol; hydroxyethyl starch (HES); dextran; sucrose; trehalose; lactose; raffinose; ribotol; mannitol; and polyvinylpyrrolidone (PVP).
  • the cryoprotectant is DMSO.
  • the cryoprotectant solution may comprise from about 5% DMSO to about 100% DMSO, e.g, about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,
  • the cryoprotectant solution may comprise about 20% DMSO. In some embodiments, the cryoprotectant solution may comprise about 40% DMSO or 60% DMSO. In some embodiments, a higher percentage of cryoprotectant is preferred, e.g., percentages that are two times higher than equivalent cell suspension values to help drive osmotic penetration.
  • the cryoprotectant solution may have water or a saline as base.
  • the saline is isotonic to human tissues.
  • the saline is a 0.9% saline solution. Any commercially available saline solution may be used: sodium chloride solution, PBS, HEPES, Ringers or Lactate.
  • the saline may be 0.9% sodium chloride.
  • the cryoprotectant solution may further comprise a protein.
  • the protein may be a human albumin (e.g., HSA) or a constituent of a human platelet lysate.
  • HSA human albumin
  • An example of a commercially available human platelet lysate product is StemulateTM (from Cook® Regentec).
  • the cryoprotectant solutions comprises about 10% protein, e.g., 10% human platelet lysate or 10% albumin.
  • the cryoprotectant solution comprises about 20% DMSO and about 10% human platelet lysate in 0.9% NaCl.
  • the cryoprotectant solution comprises about 40% DMSO and about 10% human platelet lysate in 0.9% NaCl.
  • the cryoprotectant solution comprises about 60% DMSO and about 10% human platelet lysate in 0.9% NaCl.
  • the cryoprotectant solution comprises about 80% DMSO and about 10% human platelet lysate in 0.9% NaCl.
  • the cryoprotectant solution comprises about 100% DMSO in 0.9% NaCl.
  • the cadaver bone After the pressure in the closed container comprising the cadaver bone and cryoprotectant solution has been raised and held for a suitable amount of time to allow infiltration of the cryoprotectant solution into the cadaver bone, the cadaver bone, now infiltrated with cryoprotectant, is removed from the closed container.
  • the cadaver bone then undergoes an initial chilling period.
  • the cadaver bone is placed in a static minus 80 freezer set at a temperature of colder than about - 60 °C or colder than about -100 °C, e.g., from about - 70 °C to about -80 °C.
  • the cadaver bone undergoes an initial chilling period.
  • the cadaver bone is initially chilled in a static minus 80 freezer set at a temperature of about - 86 °C.
  • Data showing the dynamics of the initial chilling period is shown in PCT/US2021/042064 at FIG. 6A.
  • the static freezer is set at a range of temperature from about -60 °C, about -65 °C, about -70 °C, about -75 °C, about -80 °C, about -82 °C, about -84 °C, about -86 °C, about -88 °C, about -90 °C, about -95 °C, or about -100 °C.
  • the freezer can be set at a range of temperature from at least about -60 °C, about -65 °C, about -70 °C, about - 75 °C, about -80 °C, about -82 °C, about -84 °C, about -86 °C, about -88 °C, about -90 °C, or about -95 °C.
  • the freezer can be set at a range of temperature from at most about -65 °C, about -70 °C, about -75 °C, about -80 °C, about -82 °C, about -84 °C, about -86 °C, about -88 °C, about -90 °C, about -95 °C, or about -100 °C.
  • the cadaver bone may be initially chilled at a rate of from about -0.3 °C/min to about -5 °C/min. In some embodiments, the cadaver bone is initially chilled at a rate of from about -0.4 °C/min to about -0.9 °C/min. As examples, the initial chilling rate may be about - 0.3 °C/min, -0.4 °C/min, -0.5 °C/min, -0.6 °C/min, -0.7 °C/min, -0.8 °C/min, -0.9 °C/min, to about -1 °C/min.
  • the initial chilling rate may be about -1 °C/min, -2 °C/min, -3 °C/min, -4 °C/min, or about -5 °C/min.
  • the minus sign means that the temperature is dropping by the stated amount.
  • the duration of the initial chilling period may vary from a few hours to overnight.
  • the time should be sufficient for the cadaver bone to reach a temperature of colder than about -50 °C, e.g., at -60 °C to -80 °C.
  • the bone reaches the desired temperature in about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or about 12 hours.
  • the cadaver bone is initially chilled in the minus 80 freezer for at least 12 hours or at least overnight.
  • the cadaver bone is not held in a static freezer having its temperature set to from about - 5 °C to about - 15 °C and for a period of time between about 1 and about 30 minutes.
  • the cadaver bone may temporarily acquire a temperature of between about - 5 °C and about - 15 ° C, but this occurs as the temperature of the cadaver bone is continuously dropping towards the desired temperature, e.g., colder than about -50 °C.
  • the cadaver bone undergoes a subsequent chilling period.
  • the cadaver bone is placed in liquid nitrogen or in liquid nitrogen vapor, e.g., at a temperature of about -200 °C.
  • Data showing the dynamics of the subsequent chilling period is shown in PCT/US2021/042064 at FIG. 6B.
  • the subsequent chilling period may occur in a suitable static freezer that is capable of maintaining temperatures equivalent to liquid nitrogen yet without use of liquid nitrogen, e.g., a cryogenic freezer.
  • the cadaver bone is cooled at a rate of from about -2 °C/min to about -6 °C/min.
  • the cadaver bone is initially chilled at a rate of about -2 °C/min, -2,2 °C/min, -2,4 °C/min, -2,6 °C/min, -2,8 °C/min, -3 °C/min, -3,2 °C/min, -3,4 °C/min, -3,6 °C/min, -3,8 °C/min, -4 °C/min, -4,2 °C/min, -4,4 °C/min, -4,6 °C/min, -4,8 °C/min, -5 °C/min, -5,2 °C/min, -5,4 °C/min, -5,6 °C/min, -5,8 °C/min, -5 °C/min, -5,2 °
  • the cryopreserved cadaver bone may be held in liquid nitrogen, in liquid nitrogen vapor, or in a suitable static freezer indefinitely.
  • the cryopreserved cadaver bone may be held for at least a day, at least a week, at least a month, at least a year, at least five years, or at least 20 years.
  • the cryopreserved cadaver bone may be held in liquid nitrogen, in liquid nitrogen vapor, or suitable static freezer for hundreds or thousands of years.
  • the two-step chilling of cadaver bone method improves the viability of the extracted bone marrow cells (hematopoietic stem cells (HSCs; CD34+ cells) and/or mesenchymal stromal/stem cells (MSCs)) relative to methods that do not use the two-step chilling method. Therefore, using the methods of the present disclosure, a greater number of viable cells (HSCs and/or MSCs) are obtained relative to standard methods.
  • HSCs hematopoietic stem cells
  • MSCs mesenchymal stromal/stem cells
  • the methods of the present disclosure provide from about 1% more viable cells to about 100% more viable cells, e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,
  • the methods of the present disclosure provide from about 101% more viable cells to about 200% more viable cells, e.g., about 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 10%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%,
  • the methods of the present disclosure provide from about 2-fold more viable cells to about 10-fold more viable cells, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, about 10-fold, or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein.
  • the methods of the present disclosure provide 2-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold, or 9-fold to 10-fold more viable cells than from methods that do not use two-step chilling, as disclosed herein.
  • the methods of the present disclosure provide from about 10-fold more viable cells to about 100-fold more viable cells, e.g., about 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, about 100-fold or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein.
  • the methods of the present disclosure provide 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40-fold, 40-fold to 50-fold, 50-fold to 60-fold, 60-fold to 70-fold, 70-fold to 80-fold, 80-fold to 90-fold, or 90-fold to 100-fold more viable cells than from methods that do not use two-step chilling, as disclosed herein.
  • the methods of the present disclosure provide from about 100-fold more viable cells to about 1000-fold more viable cells, e.g., about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, about 1000-fold or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein.
  • the methods of the present disclosure provide 100-fold to 200 fold, 200-fold to 300-fold, 300-fold to 400-fold, 400-fold to 500-fold, 500-fold to 600-fold, 600-fold to 700-fold, 700-fold to 800-fold, 800-fold to 900-fold, or 900-fold to 1000-fold more viable cells than from methods that do not use two-step chilling, as disclosed herein.
  • the methods of the present disclosure provide from about 1000-fold more viable cells to about 10000-fold more viable cells, e.g., about 1000-fold, 2000-fold, 3000- fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, about 10000-fold or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein.
  • the methods of the present disclosure provide 1000-fold to 200 fold, 2000-fold to 3000-fold, 3000-fold to 4000-fold, 4000-fold to 5000-fold, 5000-fold to 6000-fold, 6000-fold to 7000-fold, 7000-fold to 8000-fold, 8000-fold to 9000-fold, or 9000- fold to 10000-fold more viable cells than from methods that do not use two-step chilling, as disclosed herein.
  • Another aspect of the present disclosure is a method for rapidly warming cadaver bone for providing bone marrow or a derivative thereof.
  • PCT/US2021/042064 discloses methods for rapidly warming cryopreserved bone; the contents of which are incorporated by reference in its entirety. These disclosed methods may be useful in the methods of the present disclosure.
  • the method for rapidly warming cadaver bone comprise steps of: obtaining a cryopreserved cadaver bone; dividing the cryopreserved cadaver bone to obtain fragments of the cryopreserved bone; transferring the fragments of the cryopreserved bone into a grinding medium having a temperature of from about 35 °C to 45 °C for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20 °C.
  • the cryopreserved cadaver bone is cryopreserved by an above-described method, e.g., comprising vacuum-infiltration of a cryoprotectant and/or using a two-step chilling method.
  • the cryopreserved cadaver bone is not cryopreserved by an above-described method, e.g., without vacuum-infiltration of a cryoprotectant and/or not using a two-step chilling method.
  • a cryopreserved cadaver bone transferred into a grinding medium without having been divided into fragments.
  • the cryopreserved cadaver bone has a temperature of at least below 0 °C when transferred into a grinding medium.
  • the method comprises dividing the cryopreserved cadaver bone to obtain fragments of the cryopreserved bone.
  • the cryopreserved cadaver bone has a temperature of below 0 °C when dividing into fragments.
  • a custom bone cutting tool as described in US 2019/0343112, which is hereby incorporated by reference in its entirety, is used to divide the cryopreserved cadaver bone into smaller pieces.
  • Another bone cutting tool may be used in combination, or in lieu of the custom bone cutting tool as described in US 2019/0343112.
  • the elements of the bone cutting tool are formed of medical grade stainless steel.
  • the steel is preferably hardened steel capable of withstanding the forces required to cut through frozen bone.
  • the tool is subjected to steam sterilization, which can be deleterious to the steel.
  • the surfaces of the stainless-steel elements are passivated to prevent oxidation of the steel elements during sterilization.
  • the manual bone-cutting device for dividing the cryopreserved cadaver bone is capable of generating up to 1000 Ibf when less than 50 Ibf is applied.
  • a manual bonecutting device comprises: a force transmission mechanism, wherein the force transmission mechanism comprises an elongated force transducing member pivotally coupled to a gear mechanism; and a manually operable handle coupled to an end of the elongated force transducing member, wherein the end is opposite of the gear mechanism.
  • the manual bonecutting device comprises an upper cutting element and/or a lower cutting element. Its upper cutting element and/or lower cutting element each comprises one or more cutting blades that radiate outwards from a central portion of the upper cutting element and/or the lower cutting element. When the one or more cutting blades divide the cryopreserved cadaver bone into fragments that are generally sector-shaped.
  • the manual bone-cutting device divides the cryopreserved cadaver bone into fragments of the cryopreserved bone.
  • the fragments of the cryopreserved bone are transferred into a grinding medium having a temperature of from about 35 °C to about 45 °C for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20 °C.
  • whole cryopreserved bone which has not been divided, is transferred into a grinding medium having a temperature of from about 35 °C to about 45 °C for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20 °C.
  • the surface temperature of the cadaver bone fragments is higher than 20 °C, e.g., 25 °C or higher.
  • a volume of grinding medium is warmed and held at a temperature of from about 35 °C to about 45 °C, for example, by placing a container holding the grinding medium on a hot plate or in a water bath.
  • 300ml, 500ml or one liter of grinding medium is used to warm the cadaver bone.
  • the grinding medium has a temperature of about 37 °C to about 40 °C when the fragments of the cryopreserved bone are transferred to the grinding medium.
  • the cadaver bone fragments are warmed to a surface temperature of about 20 °C at a rate of from about 100 °C/min to about 500 °C/min.
  • the warming rate is greater than about 300 °C/min, e.g., about 300 °C/min, 310 °C/min, 320 °C/min, 330 °C/min, 340 °C/min, 350 °C/min, 360 °C/min, 370 °C/min, 380 °C/min, 390 °C/min, 400 °C/min, 410 °C/min, 420 °C/min, 430 °C/min, 440 °C/min, 450 °C/min, 460 °C/min, 470 °C/min, 480 °C/min, 490 °C/min, and about 500 °C/min.
  • the warming rate is from about 400 °C/min to about 500 °C/min.
  • the cadaver bone fragments are warmed to a surface temperature of about 20 °C in less than one minute. In some cases, the cadaver bone fragments are warmed to a surface temperature of about 20 °C in about one minute or more, e.g., about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or about 10 minutes.
  • Data showing the dynamics of the fast warming is shown in PCT/US21/42064 at FIG. 15.
  • the warming rate will be slower than when bone fragments are warmed.
  • the cadaver bone is warmed to a surface temperature of about 20 °C at a rate of from about 100 °C/min to about 250 °C/min.
  • the fast warming rate of the present disclosed methods prevents ice recrystalization during thawing of the bone fragments (or whole cadaver bone) and improves the viability of the extracted bone marrow cells (hematopoietic stem cells (HSCs; CD34+ cells) and/or mesenchymal stromal/stem cells (MSCs)) relative to methods that do not use the rapid warming method. Therefore, using the methods of the present disclosure, a greater number of viable cells (HSCs and/or MSCs) are obtained relative to standard methods.
  • HSCs hematopoietic stem cells
  • MSCs mesenchymal stromal/stem cells
  • a fresh cadaver vertebral body (VB) or a warmed, previously cryopreserved VB is prepared for grinding.
  • a saw and/or anvil shears are used to cut the VBs are cut into smaller pieces, such as 1.5 cm 2 pieces, that are small enough for fragmenting with a bone grinder.
  • a custom bone cutting tool as described in US 2019/0343112, which is hereby incorporated by reference in its entirety, is provided is used to cut the VBs into the smaller pieces.
  • Another custom bone cutting tool can be used in combination, or in lieu of the custom bone cutting tool as described in US 2019/0343112.
  • the additional bone cutting tool is described in US2020/0325451, which is hereby incorporated by reference in its entirety.
  • the elements of the bone cutting tool are formed of medical grade stainless steel.
  • the steel is preferably hardened steel capable of withstanding the forces required to cut through bone.
  • the tool is subjected to steam sterilization, which can be deleterious to the steel.
  • the surfaces of the stainless-steel elements are passivated to prevent oxidation of the steel elements during sterilization.
  • the grind media uses PLASMA-LYTETM-A as a base with heparin, human serum albumin (HSA), and a nuclease (Merck KGAA Corporation). Heparin is used as an anticoagulant. Other anticoagulants at various quantities can also be used. HSA provides a protein source to prevent cell adherence and adsorption to surfaces, as well as reactive oxygen scavenging.
  • the amount of heparin in the grind media is about 5 U/ml to about 15 U/ml. In some embodiments, the amount of heparin in the grind media is about 5 U/ml to about 6 U/ml, about 5 U/ml to about 7 U/ml, about 5 U/ml to about 8 U/ml, about 5 U/ml to about 9 U/ml, about 5 U/ml to about 10 U/ml, about 5 U/ml to about 11 U/ml, about 5 U/ml to about 12 U/ml, about 5 U/ml to about 13 U/ml, about 5 U/ml to about 14 U/ml, about 5 U/ml to about 15 U/ml, about 6 U/ml to about 7 U/ml, about 6 U/ml to about 8 U/ml, about 6 U/ml to about 9 U/ml, about 6 U/ml to about 10 U/ml, about 6 U/ml to
  • the amount of heparin in the grind media is about 5 U/ml, about 6 U/ml, about 7 U/ml, about 8 U/ml, about 9 U/ml, about 10 U/ml, about 11 U/ml, about 12 U/ml, about 13 U/ml, about 14 U/ml, or about 15 U/ml. In some embodiments, the amount of heparin in the grind media is at least about 5 U/ml, about 6 U/ml, about 7 U/ml, about 8 U/ml, about 9 U/ml, about 10 U/ml, about 11 U/ml, about 12 U/ml, about 13 U/ml, or about 14 U/ml.
  • the amount of heparin in the grind media is at most about 6 U/ml, about 7 U/ml, about 8 U/ml, about 9 U/ml, about 10 U/ml, about 11 U/ml, about 12 U/ml, about 13 U/ml, about 14 U/ml, or about 15 U/ml.
  • heparin is omitted from a grind medium.
  • the amount of Benzonase® in the grind media is about 1 U/ml to about 10 U/ml. In some embodiments, the amount of Benzonase in the grind media is about 1 U/ml to about 2 U/ml, about 1 U/ml to about 3 U/ml, about 1 U/ml to about 4 U/ml, about 1 U/ml to about 5 U/ml, about 1 U/ml to about 6 U/ml, about 1 U/ml to about 7 U/ml, about 1 U/ml to about 8 U/ml, about 1 U/ml to about 9 U/ml, about 1 U/ml to about 10 U/ml, about 2 U/ml to about 3 U/ml, about 2 U/ml to about 4 U/ml, about 2 U/ml to about 5 U/ml, about 2 U/ml to about 6 U/ml, about 2 U/ml to about 7 U/ml, about 2
  • the amount of Benzonase in the grind media is about 1 U/ml, about 2 U/ml, about 3 U/ml, about 4 U/ml, about 5 U/ml, about 6 U/ml, about 7 U/ml, about 8 U/ml, about 9 U/ml, or about 10 U/ml. In some embodiments, the amount of Benzonase in the grind media is at least about 1 U/ml, about 2 U/ml, about 3 U/ml, about 4 U/ml, about 5 U/ml, about 6 U/ml, about 7 U/ml, about 8 U/ml, or about 9 U/ml.
  • the amount of Benzonase in the grind media is at most about 2 U/ml, about 3 U/ml, about 4 U/ml, about 5 U/ml, about 6 U/ml, about 7 U/ml, about 8 U/ml, about 9 U/ml, or about 10 U/ml.
  • the amount of Benzonase® or Denarase® in the grind media is about 11 U/ml to about 55 U/ml. In some embodiments, the amount of Benzonase in the grind media is about 11 U/ml to about 15 U/ml, about 11 U/ml to about 20 U/ml, about 11 U/ml to about 25 U/ml, about 11 U/ml to about 30 U/ml, about 11 U/ml to about 35 U/ml, about 11 U/ml to about 40 U/ml, about 11 U/ml to about 45 U/ml, about 11 U/ml to about 50 U/ml, about 11 U/ml to about 55 U/rnl, about 15 U/rnl to about 20 U/rnl, about 15 U/rnl to about 25 U/ml, about 15 U/ml to about 30 U/ml, about 15 U/ml to about 35 U/ml, about 15 U/ml to about 15 U/ml to
  • the amount of Benzonase in the grind media is about 11 U/ml, about 15 U/ml, about 20 U/ml, about 25 U/ml, about 30 U/ml, about 35 U/ml, about 40 U/ml, about 45 U/ml, about 50 U/ml, or about 55 U/ml. In some embodiments, the amount of Benzonase in the grind media is at least about 11 U/ml, about 15 U/ml, about 20 U/ml, about 25 U/ml, about 30 U/ml, about 35 U/ml, about 40 U/ml, about 45 U/ml, or about 50 U/ml.
  • the amount of Benzonase in the grind media is at most about 15 U/ml, about 20 U/ml, about 25 U/ml, about 30 U/ml, about 35 U/ml, about 40 U/ml, about 45 U/ml, about 50 U/ml, or about 55 U/ml.
  • the amount of Benzonase® in a grind medium is about 3 U/ml and the amount of heparin in the grind medium is about 10 U/ml.
  • Denarase® reagent (C-Lecta GmbH) is equivalent to Benzonase® reagent in the same quantity in the present process.
  • HSA is present in the grind media at about 0.5 % to about 5 %. In some embodiments, HSA is present in the grind media at about 0.5 % to about 1 %, about 0.5 % to about 1.5 %, about 0.5 % to about 2 %, about 0.5 % to about 2.5 %, about 0.5 % to about 3 %, about 0.5 % to about 3.5 %, about 0.5 % to about 4 %, about 0.5 % to about 4.5 %, about 0.5 % to about 5 %, about 1 % to about 1.5 %, about 1 % to about 2 %, about 1 % to about 2.5 %, about 1 % to about 3 %, about 1 % to about 3.5 %, about 1 % to about 4 %, about 1 % to about 4.5 %, about 1 % to about 5 %, about 1.5 % to about 2 %, about 1.5 % to about 2.5 %, about 1.5 % to about 3 %, about 1 % to about
  • HSA is present in the grind media at about 0.5 %, about 1 %, about 1.5 %, about 2 %, about 2.5 %, about 3 %, about 3.5 %, about 4 %, about 4.5 %, or about 5 %. In some embodiments, HSA is present in the grind media at least about 0.5 %, about
  • HSA is present in the grind media at most about 1 %, about 1.5 %, about 2 %, about 2.5 %, about 3 %, about 3.5 %, about 4 %, or about 4.5 %.
  • HSA is present in the grind media at most about 1 %, about 1.5 %, about
  • Another pitcher of 300-500 ml of grind media is retained for collecting the bone fragments after grinding, and another supply of about 100 ml of the grind media is retained for rinsing through the grinder during the grinding process to prevent bone fragments from sticking to the surface of the pitcher of the grinding components.
  • the additional grind media may have different quantities of heparin, HSA, and Benzonase as compared to the initial grind media.
  • An electric bone grinder or a purpose-built bone grinder such as the grinder of Biorep Technologies Inc, (Miami, FL) can be used in an ISO-5 environment within an ISO- 7 clean room. Bone types are kept separate if both VB and ilium from the same donor are being processed. The bone is kept submerged in grind media at all times during and after the grinding process. Once all of the donor bone pieces are ground, the chamber of the bone grinder is thoroughly rinsed with fresh processing media. The bone fragments are discharged from the grinder into the pitcher containing grind media.
  • bone marrow and bone grindings are shaken for 10 minutes at 150 RPM.
  • the contents of the pitcher are transferred to sterile bags.
  • the contents of the sterile bags are filtered to extract the solid components.
  • the contents of each bag are passed through a series of stainless-steel sieves.
  • a No. 40 (425 pm) sieve is stacked on top of a No. 80 (177 pm) sieve, which is seated over a catch-pan to receive the liquid filter contents.
  • the sterile bags containing the output from the grinder is swirled and then poured evenly over the sieve stack or filtration sets. The filtering process is observed to ensure that excessive clumping is not occurring, which can signal the presence of soft tissue or other contaminants.
  • Bone fragments retained on the surface of the sieves are distributed evenly on the sieves and rinsed with 250 ml of fresh processing medium.
  • the processing medium used for rinsing is the grind media described above or PLASMA-LYTETM with 2.5% HSA.
  • the sieved bone marrow product which can be approximately 1000 ml in a well-performed process, is transferred to sterile packs for subsequent processing and analysis. The contents of each bag are visually inspected to confirm that the contents do not include any visible bone fragments or soft tissue.
  • the rinse media can contain the various amounts of HSA as described for the grind media.
  • the rinse media can contain, additionally, heparin and/or Benzonase.
  • the amount of Benzonase® in a rinse medium is about 3 U/ml and the amount of heparin in the rinse medium is about 10 U/ml.
  • the contents of each bag are passed through bone marrow filtration units.
  • the system includes a stand configured to support a sterile collection bag which contains the bone fragments and media from the grinding operation described above.
  • the stand includes a container hanger configured to engage the cap of the sterile bag to suspend the container.
  • the bottom of the bag includes a discharge assembly that includes a pre-filter projecting into the body of the collection bag.
  • the pre-filter is an 850pm filter.
  • the bone marrow passes first through an 800 pm pre-filter.
  • the filter is connected to an output tube that is connected by a container claim to the input line of a first in-line filter.
  • the first in-line filter is a 200pm or a 500pm filter.
  • the output line of the first in-line filter is connected to the input line of a second in-line filter.
  • the second in-line filter is a 200pm or a 500pm filter.
  • the two in-line filters are initially both 500pm for a first pass through the filter system. A second rinse is then performed on the grindings with the two in-line filters being 200pm. This double-pass filtration results in a cleaner suspension and enhances removal of fat from the suspension.
  • the second in-line filter has an output line that can be engaged to a sterile bag, such as bag for the second filtration pass.
  • the output line of the second in-line filter can be engaged to a container clamp of a transfer pack container.
  • the transfer pack container can be a 600-2000 ml bag to accommodate the filtered bone marrow product, which can be approximately 1000 ml in a well -performed process.
  • the method comprises mechanically agitating the bone grindings and/or bone grinding filtrate during the grinding and filtration portion of the processing of the bone marrow.
  • the bone marrow can be obtained from a deceased donor.
  • the bone marrow can be obtained from a sample (e.g. bone or VB) that was previously chilled.
  • the bone marrow can be obtained from a sample (e.g. bone or VB) that was previously chilled but not frozen.
  • the bone marrow can be obtained from a sample (e.g. bone or VB) that is thawed.
  • the bone marrow can be processed for obtaining bone marrow cells.
  • the bone marrow cells can be hematopoietic stem cells (HSCs).
  • the bone marrow cells can be mesenchymal stem cells (MSCs).
  • Aspect disclosed in the present disclosure comprises a method for processing bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone or bone fragment from a deceased donor, optionally, processing the bone into bone fragments; mechanically grinding the bone or bone fragment in the presence of a grinding solution to generate a plurality of bone grindings; placing the plurality of bone grindings on a shaker at about 100 to about 200 rounds per minute (“RPM”) for about 1 to about 20 minutes; and removing the solution from the shaker, wherein the solution comprises the bone marrow or the derivative thereof and wherein the bone marrow or the derivative thereof comprises at least about 1,500,000 CD34+ cells/ml of the bone marrow or the derivative thereof.
  • RPM rounds per minute
  • the method further comprises contacting the solution with a rinse media and repeating the placing of the bone grindings on the shaker and then removing the solution from the shaker. In some embodiments, the method further comprises repeating step placing the bone grinding on the shaker and then removing the solution from the shaker one or more times.
  • the at least about 1,500,000 CD34+ cells/ml of the bone marrow or the derivative thereof comprises at least 85% viable CD34+ cells. In some embodiments, the method further comprises the at least about 1,500,000 CD34+ cells/ml of the bone marrow or the derivative thereof comprises at least 90% viable CD34+ cells.
  • the mechanical agitation can comprise agitating the bone grindings in a linear fashion. In some embodiments, the mechanical agitation can comprise agitating the bone grindings in a three-dimensional fashion. In some cases, the mechanical agitation of the bone grindings can comprise orbital shaking (via an orbital shaker) such as placing the bone grinding on a shaker.
  • the bone grindings can be mechanically agitated by the shaker at a rate at least about 10 rounds per minute (RPM), 20 RPM, 30 RPM, 40 RPM, 50 RPM, 60 RPM, 70 RPM, 80 RPM, 90 RPM, 100 RPM, 110 RPM, 120 RPM, 130 RPM, 140 RPM, 150 RPM, 160 RPM, 170 RPM, 180 RPM, 190 RPM, 200 RPM, 210 RPM, 220 RPM, 230 RPM, 240 RPM, 250 RPM, or more.
  • the bone grindings can be mechanically agitated by centrifugation (e.g. spinning).
  • the bone grindings can be spun at least 10 RPM, 20 RPM, 30 RPM, 40 RPM, 50 RPM, 60 RPM, 70 RPM, 80 RPM, 90 RPM, 100 RPM, 110 RPM, 120 RPM, 130 RPM, 140 RPM, 150 RPM, 160 RPM, 170 RPM, 180 RPM, 190 RPM, 200 RPM, 210 RPM, 220 RPM, 230 RPM, 240 RPM, 250 RPM, or more.
  • the bone grindings can be spun at least 300 RPM, 400 RPM, 500 RPM, 600 RPM, or more.
  • the bone grindings can be mechanically agitated by both shaking and spinning.
  • the mechanical agitation of the bone grindings can be for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or longer.
  • the mechanical agitation of the bone grindings increases the yield of the bone marrow cells obtained.
  • the yield of the bone marrow cells obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to yield of bone marrow cells obtained without the mechanical agitation.
  • the mechanical agitation of the bone grindings increases the viability of the bone marrow cells obtained.
  • the viability of the bone marrow cells obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the viability of bone marrow cells obtained without the mechanical agitation.
  • the mechanical agitation of the bone grindings increases the number of CD34 expressing bone marrow cells obtained.
  • the number of CD34 expressing bone marrow cells obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the number of CD34 expressing bone marrow cells obtained without the mechanical agitation.
  • the mechanical agitation of the bone grindings increases the number of CD45 expressing bone marrow cells obtained by the methods described herein.
  • the number of CD45 expressing bone marrow cells obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the number of CD45 expressing bone marrow cells obtained without the mechanical agitation.
  • the agitation can occur before the filtration steps described previously.
  • the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least about 100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,050,000, 1,100,000, 1,150,000, 1,200,000, 1,250,000, 1,300,000, 1,350,000, 1,400,000, 1,450,000, 1,500,000, 1,550,000, 1,600,000, 1,650,000, 1,700,000, 1,750,000, 1,800,000, 1,850,000, 1,900,000, 1950,000, 2,000,000, or more than 2,000,000 CD34+ cells/ml.
  • the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least about 1,500,000 CD34+ cells/ml to about 2,000,000 CD34+ cells/ml. In some embodiments, the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least about 1,500,000 CD34+ cells/ml to about 1,750,000 CD34+ cells/ml, about 1,500,000 CD34+ cells/ml to about 2,000,000 CD34+ cells/ml, or about 1,750,000 CD34+ cells/ml to about 2,000,000 CD34+ cells/ml.
  • the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least about 1,500,000 CD34+ cells/ml, about 1,750,000 CD34+ cells/ml, or about 2,000,000 CD34+ cells/ml. In some embodiments, the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least at least about 1,500,000 CD34+ cells/ml, or about 1,750,000 CD34+ cells/ml. In some embodiments, the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least at most about 1,750,000 CD34+ cells/ml, or about 2,000,000 CD34+ cells/ml.
  • the viability of the CD34+ cells is at least about 70% to about 95%. In some embodiments, the viability of the CD34+ cells is at least about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95%.
  • the viability of the CD34+ cells is at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the viability of the CD34+ cells is at least at least about 70%, about 75%, about 80%, about 85%, or about 90%. In some embodiments, the viability of the CD34+ cells is at least at most about 75%, about 80%, about 85%, about 90%, or about 95%.
  • a small quantity of bone marrow such as 0.3 mL is extracted from the sterile pack using a syringe at an injection site and conducting inversion mixing before pulling the sample.
  • the sample can be tested by a hematology analyzer, such as a Sysmex Hematology Analyzer, to determine the total nucleated cell (TNC) content of the sample, as an indicator of the TNC content of the bone marrow being subsequently processed.
  • TNC total nucleated cell
  • the bone marrow product collected from the filtering is essentially a fatty emulsion.
  • the fat content of the suspension obtained from the sieve filtering approach disclosed above is greater than the fat content of the suspension obtained from the doublepass filtration system.
  • the suspension obtained from the filtering is recovered into 250 ml bags which are hermetically sealed with tube welders. Pairs of sterile bags and taring sticks are mounted within a centrifuge with bag ports facing down, and balanced. Volume compensating plates are used to prevent creasing of the bags during centrifugation.
  • the bags are centrifuged at 500xg for 15 minutes at room temperature to concentrate the cells, preferably to 2-3xl0 8 /ml. After centrifugation is complete, each bag is individually hung on a ring stand. The distinct layers within the bag are visible, with the fat layer clearly delineated on top of the supernatant with the bone marrow pellet at the bottom.
  • a new sterile bag is welded to the bag removed from the centrifuge.
  • a bag clamp or clip is placed on the bag just below the fat layer, to clamp off or squeeze the bag closed beneath the fat layer. The pellet is then drained from the centrifuge bag into the new sterile bag, with the bag clip preventing passage of the fat layer.
  • the pellet is agitated as it is drained to resuspend all of the pellet. After about half of the pellet has drained into the new bag, the tubing is closed with a hemostat or tube sealer. The second centrifuge bag is then welded to the new bag containing the pellet, and the contents of this second centrifuge bag are drained into the new bag.
  • the result is new sterile bags containing the bone marrow centrifuged to remove the fat. These bags of de-fatted bone marrow are then centrifuged at 500xg for 15 minutes at room temperature, with volume compensating plates to prevent creasing of the bags. Each bag is removed and suspended on a ring stand and a waste bag is welded to the bag, and a plasma extractor is used to remove the supernatant into the waste bag. The tubing is clamped with a hemostat when the pellet rises or breaks. The tubing is then sealed and severed to remove the pellet — containing bag from the waste bag, which is discarded. A Luer connection is welded to the pellet-containing bag.
  • the pellets from each bag are combined into a bulk bag using a large syringe.
  • the pellet-containing bags are rinsed into the bulk bag using a rinse media.
  • the bulk bag is inverted several times to ensure that all of the pellet is resuspended.
  • a small quantity of the processed BM such as 0.5 mL, can be removed for quality control testing for density and cell count.
  • the test sample can also be evaluated for human leukocyte antigens, CCR5delta 32 mutation and apolipoprotein (APOE), among other things.
  • APOE apolipoprotein
  • the centrifuge settings at one or more steps can be increased.
  • the centrifuge is spun at about 400 g to about 650 g.
  • the centrifuge is spun at about 400 g to about 450 g, about 400 g to about 500 g, about 400 g to about 550 g, about 400 g to about 600 g, about 400 g to about 650 g, about 450 g to about 500 g, about 450 g to about 550 g, about 450 g to about 600 g, about 450 g to about 650 g, about 500 g to about 550 g, about 500 g to about 600 g, about 500 g to about 650 g, about 550 g to about 600 g, about 550 g to about 650 g, or about 600 g to about 650 g.
  • the centrifuge is spun at about 400 g, about 450 g, about 500 g, about 550 g, about 600 g, or about 650 g. In some embodiments, the centrifuge is spun at least about 400 g, about 450 g, about 500 g, about 550 g, or about 600 g. In some embodiments, the centrifuge is spun at most about 450 g, about 500 g, about 550 g, about 600 g, or about 650 g. In some embodiments, the centrifuge is spun for about 10 minutes to about 40 minutes.
  • the centrifuge is spun for about 10 minutes to about 15 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 35 minutes, about 10 minutes to about 40 minutes, about 15 minutes to about 20 minutes, about 15 minutes to about 25 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 35 minutes, about 15 minutes to about 40 minutes, about 20 minutes to about 25 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 35 minutes, about 20 minutes to about 40 minutes, about 25 minutes to about 30 minutes, about 25 minutes to about 35 minutes, about 25 minutes to about 40 minutes, about 30 minutes to about 35 minutes, about 30 minutes to about 40 minutes, or about 35 minutes to about 40 minutes.
  • the centrifuge is spun for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes. In some embodiments, the centrifuge is spun for at least about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some embodiments, the centrifuge is spun for at most about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes. In some embodiments, the centrifuge is stopped without the use of a brake. In some embodiments, the centrifuge is stopped with a brake. In some embodiments, the centrifuge brake is set at about 25% to about 100%.
  • the centrifuge brake is set at about 25% to about 50%, about 25% to about 75%, about 25% to about 100%, about 50% to about 75%, about 50% to about 100%, or about 75% to about 100%. In some embodiments, the centrifuge brake is set at about 25%, about 50%, about 75%, or about 100%. In some embodiments, the centrifuge brake is set at least about 25%, about 50%, or about 75%. In some embodiments, the centrifuge brake is set at most about 50%, about 75%, or about 100%.
  • fat removal can be occur using a commercial cell processing device (e.g., COBE® 2991 cell processor, TerumoBCT). See the World Wide Web (al) terumobct.com/2991. Such commercial cell processing devices may also concentrate cell products.
  • a commercial cell processing device e.g., COBE® 2991 cell processor, TerumoBCT. See the World Wide Web (al) terumobct.com/2991.
  • COBE® 2991 cell processor TerumoBCT
  • Such commercial cell processing devices may also concentrate cell products.
  • the freeze media is a solution of a rinse media and a cry opreservation composition.
  • the cryoprotectant can be a cell-permeable media, such as dimethyl sulfoxide (DMSO); 1, 2 propane diol (also known as propylene glycol); ethylene glycol; glycerol; foramamide; ethanediol or butane 2, 3 diol; and/or a non-permeable media, such as hydroxyethyl starch (HES), dextran, sucrose, trehalose, lactose, raffinose, ribotol, Mannitol or polyvinylpyrrolidone (PVP).
  • DMSO dimethyl sulfoxide
  • 1, 2 propane diol also known as propylene glycol
  • ethylene glycol ethylene glycol
  • glycerol foramamide
  • ethanediol or butane 2, 3 diol and/or a non-permeable media
  • Each bone donor can also provide at least three surrogate vials, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more surrogate vial.
  • the greater number of cry opreservation bags obtained from a donor the greater number of surrogate vials that can be prepared, so that each cryopreservation bag has at least one vial, preferably, two, three, four, or more vials per cryopreservation bag.
  • HSA also provides cryoprotection through oncotic pressure, cell surface protein stabilization and reactive oxygen scavenging.
  • the cryoprotectant is DMSO.
  • the rinse media can be an electrolyte medium, such as PlasmaLyte, Isolyte, IMDM or other electrolyte solutions suitable for infusion.
  • the freeze media can also include concentrations of oxyrase to reduce oxygen content to less than atmospheric, such as to less than 3% of atmospheric concentrations. The addition of oxyrase produces a hypobaric composition that can facilitate cry opreservation.
  • a bone marrow product is cryopreserved in a freeze media, wherein said freeze media comprises an electrolyte formulation, human serum albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof.
  • HSA human serum albumin
  • DMSO dimethyl sulfoxide
  • said freeze media and/or rinse media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 3% to about 4%, about
  • said freeze media and/or rinse media comprises about 1% to about 5% HSA. In some embodiments, said freeze media and/or rinse media comprises about 2.5% HSA. [0196] In some embodiments, said freeze media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about
  • said freeze media comprises about 1% to about 10% DMSO. In some embodiments, said freeze media comprises about 2.5% DMSO,
  • said electrolyte formulation is Plasmalyte A.
  • a rinse medium and/or freeze medium lacks heparin.
  • the rinse media is fresh.
  • the freeze media is ⁇ 25°C before adding it to the bone marrow bulk bag.
  • the freeze media may be added to the bone marrow bulk bag at a predetermined rate (10% of the Freeze Media volume per minute) based on the following formula:
  • the elapsed time for adding the cryoprotectant to the bone marrow bulk bag does not exceed 9-11 minutes.
  • Described herein, in some aspects, is a method for processing (e.g. isolating) CD34+ cells obtained from bone marrow or bone marrow derivative.
  • the bone marrow or bone marrow derivative can be fresh (e.g. never frozen) or thawed from being previously frozen.
  • the bone marrow or bone marrow derivative can be ground by the methods and systems described herein.
  • ground bone marrow or bone marrow cells can be contacted with the stabilization buffer described herein.
  • the stabilization prevents formation of aggregates of the bone marrow cells.
  • the bone marrow cells contacted and suspended in the stabilization buffer can be isolated by attaching to antibody such as a conjugated antibody.
  • antibody such as a conjugated antibody.
  • bone marrow cells expressing CD34+ can be isolated and enriched by contacting the bone marrow cells with the CD34 antibody conjugated with iron, where the bone marrow cells expressing CD34 are then trapped a magnetic separation column (e.g. “CliniMACS®”).
  • the bone marrow cells not expressing CD34 are can be washed away.
  • the trapped CD34+ bone marrow cells can be harvested by removing the magnetic field and eluting the targeted CD34+ bone marrow cells.
  • Such approach does not require isolating the bone marrow cells with a Ficoll gradient.
  • Aspect described in the present disclosure comprises a method for processing a population of CD34+ cells obtained from bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone or bone fragment from a deceased donor, optionally, processing the bone into bone fragments; extracting the bone marrow or derivative thereof from the bone or bone fragment; and contacting the bone marrow or derivative thereof with a stabilization buffer, wherein the stabilization buffer comprises more than about 3 U/ml of a nuclease; performing a CD34+ cell isolation assay to generate a cellular composition comprising the population of CD34+ cells, wherein the composition comprising the population of CD34+ cells comprises at least about 80,000 CD34+ cells/750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer.
  • the at least about 80,000 CD34+ cells/750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 70% viable CD34+ cells. In some embodiments, the at least about 80,000 CD34+ cells/750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 80% viable CD34+ cells. In some embodiments, the at least about 80,000 CD34+ cells/750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 90% viable CD34+ cells.
  • stabilization buffer comprising: at least 5 U/ml of an anticoagulant; and more than 3 U/ml of a nuclease.
  • stabilization buffer comprises more than about 5 U/ml of a nuclease.
  • the stabilization buffer comprises more than about 10 U/ml of a nuclease.
  • the stabilization buffer comprises more than about 15 U/ml of a nuclease.
  • the stabilization buffer comprises more than about 20 U/ml of a nuclease.
  • the stabilization buffer comprises about 20 U/ml of a nuclease.
  • the nuclease is Benzonase® or Denarase®.
  • the stabilization buffer further comprises more than about 10 U/ml of an anticoagulant. In some embodiments, the stabilization buffer further comprises about 10 U/ml of an anticoagulant. In some embodiments, the anticoagulant is heparin. In some embodiments, the stabilization buffer further comprises human serum albumin (HSA). In some embodiments, the stabilization buffer comprises 0.5% HSA.
  • the stabilization buffer comprises nuclease.
  • the nuclease is Benzonase® or Denarase®.
  • the stabilization buffer comprises nuclease at about 3 U/ml, 4 U/ml, 5 U/ml, 6 U/ml, 7 U/ml, 8 U/ml, 9 U/ml, 10 U/ml, 11 U/ml, 12 U/ml, 13 U/ml, 14 U/ml, 15 U/ml, 16 U/ml, 17 U/ml, 18 U/ml, 19 U/ml, 20 U/ml, 21 U/ml, 22 U/ml, 23 U/ml, 24 U/ml, 25 U/ml, 26 U/ml, 27 U/ml, 28 U/ml, 29 U/ml, 30 U/ml, 50 U/ml, 100 U/ml, 200 U/ml, or more U/ml.
  • the stabilization buffer comprises an anticoagulant.
  • the anticoagulant is Heparin.
  • the stabilization buffer comprises anticoagulant at about 0.1 U/ml, 0.2 U/ml, 0.3 U/ml, 0.4 U/ml, 0.5 U/ml, 0.6 U/ml, 0.7 U/ml, 0.8 U/ml, 0.9 U/ml, 1.0 U/ml, 2.0 U/ml, 3.0 U/ml, 4.0 U/ml, 5.0 U/ml, 6.0 U/ml, 7.0 U/ml, 8.0 U/ml, 9.0 U/ml, 10 U/ml, 11 U/ml, 12 U/ml, 13 U/ml, 14 U/ml, 15 U/ml, 16 U/ml, 17 U/ml, 18 U/ml, 19 U/ml, 20 U/ml, 21 U/ml, 22 U/ml, 23 U/ml, 24 U/ml
  • a stabilization buffer lacks heparin.
  • the stabilization buffer comprises about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05% HSA, 0.1% HSA, 0.2% HSA, 0.3% HSA, 0.4% HSA, 0.5% HSA, 0.6% HSA, 0.7% HSA, 0.8% HSA, 0.9% HSA, 1.0% HSA, 1.5 % HSA, 2% HSA, 2.5% HSA, 5% HSA, 10% HSA, 20% HSA, or more HSA.
  • the method comprises contacting the bone marrow or the bone marrow cells with the stabilization buffer described herein.
  • An aspect of the present disclosure comprises a method for processing a population of CD34+ cells comprised in bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone or bone fragment from a deceased donor, optionally, processing the bone into bone fragments; extracting the bone marrow or derivative thereof from the bone or bone fragment; and contacting the bone marrow or derivative thereof with a stabilization buffer, wherein the stabilization buffer comprises more than about 3 U/ml of a nuclease; performing a CD34+ cell isolation assay to generate a cellular composition comprising the population of CD34+ cells, wherein the composition comprising the population of CD34+ cells comprises at least about 80,000 CD34+ cells/750 ul of the bone marrow or the derivative thereof contacted with the stabilization buffer.
  • processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the yield of the bone marrow cells obtained from the methods described herein compared to the yield of the bone marrow cells processed in the absence of the stabilization buffer.
  • processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the yield of the bone marrow cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to yield of bone marrow cells processed in the absence of the stabilization buffer.
  • processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the viability of the bone marrow cells obtained from the methods described herein compared to the viability of the bone marrow cells processed in the absence of the stabilization buffer. In some instances, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the viability of the bone marrow cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to viability of bone marrow cells processed in the absence of the stabilization buffer.
  • processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the number of CD34+ bone marrow cells compared to the number of CD34+ bone marrow cells processed in the absence of the stabilization buffer.
  • the number of CD34+ bone marrow obtained from processing with the stabilization buffer is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the number of CD34+ bone marrow obtained from processing in the absence of stabilization buffer.
  • processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the number of CD45+ bone marrow cells compare to the number of CD45+ bone marrow cells processed in the absence of the stabilization buffer.
  • the number of CD45+ bone marrow obtained from processing with the stabilization buffer is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the number of CD45+ bone marrow obtained from processing in the absence of stabilization buffer.
  • cellular compositions comprising CD34+ cells derived from bone marrow samples processed with the stabilization buffers described herein have an increased amount of CD34+ cells, as compared to cellular compositions generated from known CD34+ isolation methods.
  • the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 CD34+ cells/750 ul of bone marrow or a derivative thereof contacted with the stabilization buffers described herein.
  • the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 cells/750 ul to about 100,000 cells/750 ul.
  • the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 cells/750 ul to about 75,000 cells/750 ul, about 70,000 cells/750 ul to about 80,000 cells/750 ul, about 70,000 cells/750 ul to about 85,000 cells/750 ul, about 70,000 cells/750 ul to about 90,000 cells/750 ul, about 70,000 cells/750 ul to about 95,000 cells/750 ul, about 70,000 cells/750 ul to about 100,000 cells/750 ul, about 75,000 cells/750 ul to about 80,000 cells/750 ul, about 75,000 cells/750 ul to about 85,000 cells/750 ul, about 75,000 cells/750 ul to about 90,000 cells/750 ul, about 75,000 cells/750 ul to about 95,000 cells/750 ul, about 75,000 cells/750 ul to about 100,000 cells/750 ul, about 80,000 cells/750 ul to about 85,000 cells/750 ul, about 75,000 cells/750 ul to
  • the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 cells/750 ul, about 75,000 cells/750 ul, about 80,000 cells/750 ul, about 85,000 cells/750 ul, about 90,000 cells/750 ul, about 95,000 cells/750 ul, or about 100,000 cells/750 ul. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least at least about 70,000 cells/750 ul, about 75,000 cells/750 ul, about 80,000 cells/750 ul, about 85,000 cells/750 ul, about 90,000 cells/750 ul, or about 95,000 cells/750 ul.
  • the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least at most about 75,000 cells/750 ul, about 80,000 cells/750 ul, about 85,000 cells/750 ul, about 90,000 cells/750 ul, about 95,000 cells/750 ul, or about 100,000 cells/750 ul.
  • the CD34+ cells derived from bone marrow samples processed with the stabilization buffers described herein also exhibit higher viability as compared to cellular compositions generated from known CD34+ isolation methods.
  • the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70% to about 95%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70% to about 95%.
  • the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95%.
  • the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least at least about 70%, about 75%, about 80%, about 85%, or about 90%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least at most about 75%, about 80%, about 85%, about 90%, or about 95%. Viability may relate to either or both of functional viability which measures the cells’ ability to proliferate and routine viability which relates to the numbers or percentages of live cells, e.g., as measured by Trypan Blue.
  • a method for selecting CD34 expressing (CD34+) cells from deceased donor bone marrow using density reduced Ficoll and an immunomagnetic CD34+ cell isolation kit is provided.
  • cell isolation using density reduced Ficoll prior to CD34 selection is beneficial to obtain high purity and viability CD45/CD34+ cells from freshly prepared deceased donor bone marrow.
  • Ficoll at conventional density has been found to be optimal for CD45/CD34+ cell selection from thawed cryopreserved deceased donor bone marrow.
  • Vertebral sections obtained from a recently deceased donor were processed as described above.
  • the bone is cleaned of all soft tissue and then cut into small pieces that were immediately submerged into 500 ml of grinding media.
  • the grinding media can be PLASMA-LYTETM A injection pH 7.4, multiple electrolytes, injection type 1 USP (PLASMA-LYTETM) containing 2.5% human serum albumin (HSA), 3 U/ml denarase, and 10 U/ml heparin.
  • the sectioned VB are ground using a bone grinder, filtered and rinsed with rinse media (such as PLASMA-LYTETM with 2.5% HSA).
  • the entire cell suspension is centrifuged to concentrate cells to 2-3xl0 8 /ml and the cell concentration is extracted.
  • a portion or all of the resulting BM preparation can be used immediately for CD34 selection, while the remainder can be prepared for cry opreservation.
  • the cryopreserved portion involves adding a final concentration of 10% DMSO and 5% HSA to the BM cells and bringing the preparation to - 86°C, either by passive cooling or by controlled cooling at a rate of approximately -l°C/min, after which the cryopreserved portion is plunged into liquid nitrogen.
  • Ficoll-Paque PLUS is added to the BM preparation to separate the desired CD34+ cell component of the bone marrow.
  • the Ficoll density is reduced from the conventional density.
  • the density is reduced by mixing Ficoll-Paque PLUS (density 1.077 g/mL, GE Company) with Plasma Lyte-A Injection pH 7.4 (Baxter Healthcare 2B2544X) in specific proportions to obtain an overall density of less than 1.077 g/ml, particularly 1.063 — 1.052 g/ml.
  • the density of 1.063 g/ml was found to be optimal for isolation of CD34+ cells, taking into account quantity, viability and purity of the CD34+ cells.
  • 5 ml of the 1.063 g/ml density Ficoll solutions is pipetted into 15-ml centrifuge tubes, and the BM solution generated from VBs of deceased donors is carefully layered over the Ficoll gradient.
  • the tubes are centrifuged for 30 min at 400 g without break at room temperature. After centrifugation, buffy coat cells are harvested carefully, and the cells are washed in phosphate-buffered saline (PBS) containing 0.5% HSA and 2mM Ethylenediaminetetraacetic acid (EDTA) (MACS buffer, Miltenyi).
  • PBS phosphate-buffered saline
  • EDTA Ethylenediaminetetraacetic acid
  • centrifugation is performed for 5 min at 400 g, and the resulting cell pellets are resuspended in 10 ml PBS, followed by a second centrifugation for 5 min at 400 g.
  • Nucleated cells in the isolated buffy coat can be counted using a Sysmex XP-300.
  • a Cellometer Vision (Nexcellom) or flow cytometer can be used to determine cell counts of purified CD34 cells.
  • 20 microliters of AOPI can be added to 20 microliters of cells and after mixing total viable cells can be determined.
  • the CD34+ cells can be selected by a positive immune separation method using a CliniMAX system (Miltenyi, Bergisch Gladbach, Germany) or an EasySep CD34 kit (Stemcell Technologies, Vancouver, BC, Canada) in accordance with the protocol of the manufacturer.
  • the lower Ficoll density contemplated in the present disclosure leads to more optimum cell recovery. Optimization is based on purity, viability and yield of selected CD34 cells. A target of >90% purity and >90% viable CD34+ cells is preferred. While lower Ficoll densities resulted in greater purity and fewer dead cells, it was surprisingly found that a greater portion of the CD34+ cells present in the deceased donor whole bone marrow before selection are lost using the lower Ficoll densities to prepare buffy coat. Thus, the high viability and purity of CD45/CD34+ cells achieved at the conventional Ficoll density gradient also leads to a large loss in yield (approximately 60% loss of input CD34+ cells).
  • the optimal density of Ficoll for selection of CD45/CD34+ cells at >90% purity and viability is less than 1.077 and particularly 1.063- 1.052.
  • This Ficoll density provides a higher yield of CD45/CD34+ cells with similar purity and cell viability to the conventional Ficoll density approach.
  • the CD34+ cells can be initially acquired from a freshly prepared deceased donor bone marrow using the reduced density Ficoll-Paque described above.
  • the BM can be cryogenically frozen and then the CD34+ cells can be acquired later using conventional density Ficoll-Paque. This approach essentially allows selective recovery of cells from deceased donor bone marrow — either before freezing using the modified Ficoll density or after freezing and thawing using conventional Ficoll density.
  • Bone marrow is a well-known source for mesenchymal stromal/stem cells (MSCs) which can be harvested from bone marrow obtained using the methods described above.
  • MSCs are self-renewing, multipotent progenitor cells with multilineage potential to differentiate into cell types of mesodermal origin, such as adipocytes, osteocytes, and chondrocytes.
  • MSCs can migrate to sites of inflammation and exert potent immunosuppressive and antiinflammatory effects through interactions between lymphocytes associated with both the innate and adaptive immune system.
  • MSCs can be used in treating osteogenesis imperfect, cartilage defects, myocardial infarction, Crohn's disease, multiple sclerosis, autoimmune disease such as Lupus, liver cirrhosis, osteo arthritis, and rheumatoid arthritis.
  • Matched HSC/MSC units which can be used in co-transplant for treatment of graft vs. host disease (GVHD), and for hematopoietic stem cell transplant support.
  • a method for preparing a composition of human MSCs from bone may include providing a bone derived from a deceased donor, grinding the bone into one or more ground bone segments, filtering the one or more ground bone segments and extracting the human MSCs from the one or more ground bone segments.
  • the MSCs may be recovered from thawed or cryopreserved VB bone fragments.
  • the MSC cells are readily released from the VB; these MSCs are referred to herein as bone marrow vertebral body mesenchymal stem cells (vBM-MSCs).
  • the extracted human MSCs may be vertebral bone marrow MSCs (vBM-MSCs), adherent vertebral body mesenchymal stem cells (vBA-MSCs), or both.
  • the extracted human MSCs are derived from a bone or fragments thereof that has already been processed to remove bone marrow or derivates thereof associated with the bone or fragment thereof (e.g. bone marrow derived cells, hematopoietic stem cells).
  • the extracted human MSCs are derived from a bone or fragments thereof that has been processed for bone marrow and/or bone marrow-derived cells (e.g. hematopoietic stem cells) as described herein.
  • the extracted human MSCs are derived from the bone grindings and/or segments described herein following filtration and/or extraction and/or isolation of bone marrow and/or bone marrow-derived cells as described herein.
  • the processing and extraction of viable vBA-MSCs from the bone and/or derivates thereof results in significant improvements in cell yield, especially with respect to total cell yield (vBA-MSCs and hematopoietic stem cells) per weight of bone derived from a donor, and viability of cells with respect to the state of the art.
  • the vBA-MSCs described herein can be combined with bone marrow-derived MSCs (vBM-MSCs) isolated from bone marrow isolated and processed as described herein.
  • Some methods recover MSCs from enzymatically digested vertebral body (VB) bone fragments that are the byproduct of the VB grinding and elution of the methods described herein.
  • VB vertebral body
  • a mixture of both collagenase and neutral protease is used to obtain the highest possible yields of vertebral bone adherent MSC (vBA-MSC).
  • the MSCs can be recovered from cryopreserved VB bone fragments that are later processed according to the present disclosure.
  • recombinant Clostridium histolyticum collagenase comprised of the two active isoforms, is used in effective amounts in the MSC extraction process.
  • the mixture of cells liberated by digesting VB bone fragment is cultured on tissue-coated plastic in the presence of Mesencult medium to select proliferative vBA-MSC.
  • Freshly digested preparations as well as different passages of vBA-MSC can be characterized by flow cytometry, colony forming unitfibroblast (CFU-F) potential, population doubling time (PDT) and trilineage (adipogenic, chondrogenic and osteogenic) differentiation in vitro.
  • the mesenchymal stem cells can be recovered or cultured in Alpha-MEM supplemented with human platelet lysate and epidermal growth factor and/or fibroblast growth factor.
  • the extraction of human MSCs may include contacting the bone or derivatives thereof with a digestion solution.
  • the digestion solution may include one or more distinct enzymes.
  • the one or more distinct enzymes may include one or more collagenases and neutral proteases.
  • the combination of one or more collagenases and neutral proteases is used to obtain the highest possible yields of vBA-MSC.
  • the present disclosure thus contemplates a method for optimizing digestion and MSC recovery from vertebral bone fragments using a combination of purified collagenase and neutral protease.
  • the collagenase is DE collagenase (Vitacyte), which is comprised of purified Clostridium histolyticum collagenase and Paneibacillus polymyxa neutral protease.
  • optimal neutral protease concentration and collagenase concentrations (Cl and C2 collagenase) and optimal ratio of solution volume (mis) to bone fragment weight (mgs) are determined.
  • a collagenase may include Clostridium histolyticum further comprising two active isoforms, Cl and C2.
  • one or more collagenases comprising isoforms Cl and C2 may be present in the digestion solution at a ratio comprising more collagenase isoform Cl than collagenase isoform C2.
  • the ratio of collagenase isoform Cl to collagenase isoform C2 may be about 30 to about 70: about 10 to about 29.
  • the ratio of collagenase isoform Cl to collagenase C2 may be 35: 15.
  • the mass ratio of Cl and C2 for each concentration may be 70:30, 54:46, 37:63, 82: 18, 54:46, and 90: 10.
  • the neutral protease may be Paneibacillus polymyxa neutral protease.
  • the neutral protease concentration may be about 2 U/ml to about 21 U/ml.
  • the neutral protease concentration may be about 2 U/ml to about 7 U/ml, about 2 U/ml to about 12 U/ml, about 2 U/ml to about 17 U/ml, about 2 U/ml to about 21 U/ml, about 7 U/ml to about 12 U/ml, about 7 U/ml to about 17 U/ml, about 7 U/ml to about 21 U/ml, about 12 U/ml to about 17 U/ml, about 12 U/ml to about 21 U/ml, or about 17 U/ml to about 21 U/ml.
  • the neutral protease concentration may be about 2 U/ml, about 7 U/ml, about 12 U/ml, about 17 U/ml, or about 21 U/ml. In some embodiments, the neutral protease concentration may be at least about 2 U/ml, about 7 U/ml, about 12 U/ml, or about 17 U/ml. In some embodiments, the neutral protease concentration may be at most about 7 U/ml, about 12 U/ml, about 17 U/ml, or about 21 U/ml. In some embodiments, the digestion solution may comprise the neutral protease at an activity of about 19.6 U/ml.
  • the collagenase concentration is about 0.05 U/ml to about 1.6 U/ml. In some embodiments, the collagenase concentration is about 0.05 U/ml to about 0.1 U/ml, about 0.05 U/ml to about 0.15 U/ml, about 0.05 U/ml to about 0.2 U/ml, about 0.05 U/ml to about 0.25 U/ml, about 0.05 U/ml to about 0.3 U/ml, about 0.05 U/ml to about 0.35 U/ml, about 0.05 U/ml to about 0.4 U/ml, about 0.05 U/ml to about 0.8 U/ml, about 0.05 U/ml to about 1.2 U/ml, about 0.05 U/ml to about 1.6 U/ml, about 0.1 U/ml to about 0.15 U/ml, about 0.1 U/ml to about 0.2 U/ml, about 0.1 U/ml to about 0.25 U/ml, about 0.1
  • the collagenase concentration is about 0.05 U/ml, about 0.1 U/ml, about 0.15 U/ml, about 0.2 U/ml, about 0.25 U/ml, about 0.3 U/ml, about 0.35 U/ml, about 0.4 U/ml, about 0.8 U/ml, about 1.2 U/ml, or about 1.6 U/ml.
  • the collagenase concentration is at least about 0.05 U/ml, about 0.1 U/ml, about 0.15 U/ml, about 0.2 U/ml, about 0.25 U/ml, about 0.3 U/ml, about 0.35 U/ml, about 0.4 U/ml, about 0.8 U/ml, or about 1.2 U/ml. In some embodiments, the collagenase concentration is at most about 0.1 U/ml, about 0.15 U/ml, about 0.2 U/ml, about 0.25 U/ml, about 0.3 U/ml, about 0.35 U/ml, about 0.4 U/ml, about 0.8 U/ml, about 1.2 U/ml, or about 1.6 U/ml.
  • neutral protease concentration and collagenase concentrations (Cl and C2 collagenase) and ratio of solution volume (mis) to bone fragment weight (mgs) are determined.
  • the total collagenase concentrations are about 25 pg/ml to about 100 pg/ml. In some embodiments, the total collagenase concentrations are about 25 pg/ml to about 32.5 pg/ml, about 25 pg/ml to about 47.5 pg/ml, about 25 pg/ml to about 42.5 pg/ml, about 25 pg/ml to about 50 pg/ml, about 25 pg/ml to about 65 pg/ml, about 25 pg/ml to about 77.5 pg/ml, about 25 pg/ml to about 85 pg/ml, about 25 pg/ml to about 100 pg/ml, about 32.5 pg/ml to about 47.5 pg/ml, about 32.5 pg/ml to about
  • the total collagenase concentrations are about 25 gg/ml, about 32.5 gg/ml, about 47.5 gg/ml, about 42.5 gg/ml, about 50 gg/ml, about 65 gg/ml, about 77.5 gg/ml, about 85 gg/ml, or about 100 gg/ml. In some embodiments, the total collagenase concentrations are at least about 25 gg/ml, about 32.5 gg/ml, about 47.5 gg/ml, about 42.5 gg/ml, about 50 gg/ml, about 65 gg/ml, about 77.5 gg/ml, or about 85 gg/ml. In some embodiments, the total collagenase concentrations are at most about
  • the mass ratio of Cl and C2 for each concentration are 70:30, 54:46, 37:63, 82: 18 and 90: 10, respectively.
  • the volume to weight ratio of digestion solution to captured ground bone is about 1 : 1 to about 15: 1, e.g., about 5: 1. In some embodiments, the ratio may be 1 : 1, 2.5:1, 5:1, 7.5: 1, 10: 1 and 15: 1 (volume:weight).
  • the incubation period is about 1 hour to about 4 hours. In some embodiments, the incubation period is about 1 hour to about 1.5 hours, about 1 hour to about 2 hours, about 1 hour to about 2.5 hours, about 1 hour to about 3 hours, about 1.5 hours to about 2 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 3 hours, about 2 hours to about 2.5 hours, about 2 hours to about 3 hours, or about 2.5 hours to about 3 hours.
  • the incubation period is about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. In some embodiments, the incubation period is at least about 1 hour, about 1.5 hours, about 2 hours, or about 2.5 hours. In some embodiments, the incubation period is at most about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, or about 4 hours. In some cases, the digestion solution is contacted with the captured ground bone for up to about 4 hours.
  • the optimal volume-to- weight ratio has been found to be 5:1 at an optimal incubation time of 2.5 hours.
  • the optimal protease produced neutral protease activity of
  • fragments of VB bone are placed in cryoprotectant solution comprised of PLASMA-LYTETM, 2.5% human serum albumin and 10% dimethyl sulfoxide (DMSO) and incubated for 1 hour at 4°C.
  • the incubation period is about 1 hour to about 3 hours.
  • the incubation period is about 1 hour to about 1.5 hours, about 1 hour to about 2 hours, about 1 hour to about 2.5 hours, about 1 hour to about 3 hours, about 1.5 hours to about 2 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 3 hours, about 2 hours to about 2.5 hours, about 2 hours to about 3 hours, or about 2.5 hours to about 3 hours.
  • the incubation period is about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. In some embodiments, the incubation period is at least about 1 hour, about 1.5 hours, about 2 hours, or about 2.5 hours. In some embodiments, the incubation period is at most about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours.
  • the solution is removed and the bone fragments cooled at a rate of ⁇ l°/min to -86°C and then plunged into liquid nitrogen. After 24-48 hours in liquid nitrogen, the bone fragments are thawed rapidly in a water bath set at 37°C and then washed in saline and digested using the collagenase/protease solution described above.
  • the volume-to-weight ratio was 5: 1 at an incubation time of 2.5 hours.
  • the protease produced neutral protease activity of 19.6 U/ml.
  • the mixture of cells liberated by digesting VB bone fragment is cultured on tissue- coated plastic in the presence of Mesencult medium to select proliferative vBA-MSC. Freshly digested preparations as well as different passages of VB-MSC can be characterized by flow cytometry, colony forming unit-fibroblast (CFU-F) potential, population doubling time (PDT) and trilineage (adipogenic, chondrogenic, and osteogenic) differentiation in vitro.
  • CFU-F colony forming unit-fibroblast
  • PDT population doubling time
  • trilineage adipogenic, chondrogenic, and osteogenic
  • the method of human MSC extraction disclosed herein may be capable of extracting quantities of about 10 million to about 10 billion.
  • human MSCs may be administered in quantities of about 10 million to about 100 million, about 10 million to about 1 billion, about 10 million to about 10 billion, about 100 million to about 1 billion, about 100 million to about 10 billion, or about 1 billion to about 10 billion.
  • human MSCs may be administered in quantities of about 10 million, about 100 million, about 1 billion, or about 10 billion.
  • human MSCs may be administered in quantities of at least about 10 million, about 100 million, or about 1 billion.
  • human MSCs may be administered in quantities of at most about 100 million, about 1 billion, or about 10 billion.
  • extracted MSCs may be (e.g., vBA-MSCs and/or vBM-MSCs) cultured and passaged to realize clinical scale MSC preparation having a desired number of MSCs with the antigen profiles taught herein.
  • a clinical scale preparation may be obtained by serial passage expansion where each passage includes a step of splitting the previous culture into a plurality of cultures at a given ratio. Each passaging step increases the number of concurrent cultures in the preparation.
  • clinical scale preparations having the instant preparation profiles e.g. antigen profile, TNFRI profile, cryopreservation profile, differentiation profile, and/or sterility (with respect to pathogens) are successfully produced.
  • MSCs for clinical use are derived from the continuous manufacturing process of obtained bone marrow from deceased-donor vertebral bodies to selection of MSC for further cell culture. MSCs are cultured out of the bone marrow units collected. The overall manufacturing process to derive the final MSC product is illustrated in FIG 1. As shown in FIG. 1, MSCs are cultured out of primary bone marrow units using a tiered culture system.
  • Passage 0 to Passage 1 Primary Cell Culture and Master Cell Bank (MCB) Preparation
  • MSC Primary Cell Culture and Master Cell Bank
  • MSC Culture Media may comprise Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM) supplemented with human platelet lysate (hPL) and basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).
  • hPL human platelet lysate
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • no heparin is included in the MSC Culture Media.
  • the MSC Culture Media comprises no bovine or porcine components.
  • no antibiotics and/or antimycotics are used in the MSC Culture Media. During culturing of the P0 cells, fresh media changes occur about every three to about four days.
  • Pl cells are cultured for about five to about six days and until they have reached greater than about 75% confluence.
  • Pl cells When the Pl cells are to be cryofrozen, the cells are detached, resuspended in PlasmaLyte-A which may be supplemented with human serum albumin (HSA) and dimethyl sulfoxide (DMSO). About 13 million cells/mL of Pl cells can be packaged in 2-mL CellSeal® closed-system cryovials at 2 mL per cryovial using an automatic filler. The cryovials can be cryopreserved and placed into the vapor phase above LN2 in alarm monitored cryogenic tanks for storage at ⁇ -140°C. These cells constitute the Master Cell Bank (MCB).
  • MCA Master Cell Bank
  • the Pl cells are detached and further cultured into passage 2 cells.
  • Pl cells One vial of Pl cells, if cryofrozen, is thawed and plated in MSC Culture Media for about five to about six days. These are now termed Passage 2 or P2 cells. When the Pl cells reach greater than 75% confluence, the P2 cells are detached and immediately replated in MSC Culture Media. These cells are considered Passage 3 or P3 cells.
  • the P3 cells are cultured for about five to about 6 days. Once the culturing container have reached greater than 75% confluence, cells are detached, resuspended in PlasmaLyte-A, optionally supplemented with HSA and DMSO. About 6 million cells/mL of P3 cells can be packaged in cryobags at about 410 million P3 cells in 65 mL per bag. The bags are then cryopreserved and placed into the vapor phase above LN2 in alarm monitored cryogenic tanks for storage at ⁇ -140°C. These cells now constitute the Working Cell Bank (WCB).
  • WBC Working Cell Bank
  • the P3 cells are detached and further cultured into passage 4 cells.
  • P4 cells are cultured for about four days to about five days. Once the culturing container has reached greater than 75% confluence, P4 cells are detached and resuspended in PlasmaLyte-A, optionally supplemented with HSA and DMSO. About 20 million cells/mL of P4 cells can packaged in 5-mL CellSeal closed-system cryovials at 5 mL per cryovial using an automatic filler. Cells are then cryopreserved and placed into the vapor phase above LN2 in alarm monitored cryogenic tanks for storage at ⁇ -140°C. These cells constitute the End of Production Bank (EOP).
  • EOP End of Production Bank
  • the P4 cells are detached and then further cultured to become a cell composition for administration to a subject in need.
  • one vial from the EOP bank is thawed, if cryofrozen, and plated in MSC Culture Media at a density from about 32,000 to about 40,000 cells/cm 2 . These cells are cultured briefly (from about 20 hours to about 28 hours) to allow cell recovery only and prior to entering logarithmic phase. After this brief culturing, cells are detached, resuspended in PlasmaLyte-A, optionally supplemented with HSA. About 10 million cells/mL can be packaged in 5 mL vials (e.g., CellSeal closed-system cryovials). A clinical dose equates to two (2) 5 mL vials.
  • 5 mL vials e.g., CellSeal closed-system cryovials
  • Cells can be used at the site where this final culturing occurred. Alternately, cells can be shipped to a treatment site under cold, non-freezing temperatures, e.g., from about 2 to about 8°C. Up to 10 clinical doses may be prepared at one time, depending on the treatment schedule.
  • P4 cells are detached and plated in MSC Culture Media at a density from about 32,000 to about 40,000 cells/cm 2 . These cells are cultured briefly (from about 20 hours to about 28 hours) to allow cell recovery only (e.g., to allow metabolic activity to resume post-thaw) and prior to entering logarithmic phase. After this brief culturing, cells are detached, resuspended in PlasmaLyte-A, optionally supplemented with HSA. About 10 million cells/mL can be packaged in 5 mL vials (e.g., CellSeal closed-system cryovials). A clinical dose equates to two (2) 5 mL vials, e.g., about 100 million cells.
  • 5 mL vials e.g., CellSeal closed-system cryovials
  • Cells can be used at the site where this final culturing occurred. Alternately, cells can be shipped to a treatment site under cold, non-freezing temperatures, e.g., from about 2 to about 8°C. Up to 10 clinical doses may be prepared at one time, depending on the treatment schedule.
  • the primary MSCs may be further passaged to non-primary cells (e.g. removed from the culture surface and expanded into additional area) by seeding at a density of about 1,000 to about one million nucleated cells/cm 2 of culture dish (e.g. about 5,900 cells/cm 2 plus and minus about 1,200), and then culturing for additional days, e.g. about 14 ⁇ about 2 days.
  • non-primary cells e.g. removed from the culture surface and expanded into additional area
  • the primary cells may be grown to confluence, and in some instances may be passaged to a second culture of nonprimary cells by seeding the primary cells from a confluent primary cell culture in the second culture surface in an amount below confluence and growing the non-primary culture to confluence. This method can be repeated for additional passages.
  • the MSCs in the treatment composition may originate from sequential generation number (i.e., they are within about 1 or about 2 or about 3 or about 4 cell doublings of each other).
  • the average number of cell doublings in the present composition treatment composition may be about 20 to about 25 doublings.
  • the average number of cell doublings in the present treatment composition may be about 9 to about 13 (e.g., about 11 or about 11.2) doublings arising from the primary culture, plus about 1, about 2, about 3, or about 4 doublings per passage (for example, about 2.5 doublings per passage).
  • Exemplary average cell doublings in present preparations may be of about 13.5, about 16, about 18.5, about 21, about 23.5, about 26, about 28.5, about 31, about 33.5, or about 36 when produced by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passages, respectively.
  • the MSCs in the treatment composition may originate from MSCs that were cultured through about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passages.
  • Confluence refers to the percentage of the surface of a culture dish that is covered by adherent cells. For example, 50 percent confluence means roughly half of the surface is covered, while 100 percent confluence means the surface is completely covered by the cells, and no more room is left for the cells to grow as a monolayer.
  • 75% confluence or greater is meant about 75% confluence, about 76% confluence, about 77% confluence, about 78% confluence, about 79% confluence, about 80% confluence, about 81% confluence, about 82% confluence, about 83% confluence, about 84% confluence, about 85% confluence, about 86% confluence, about 87% confluence, about 88% confluence, about 89% confluence, about 90% confluence, about 91% confluence, about 92% confluence, about 93% confluence, about 94% confluence, about 95% confluence, about 96% confluence, about 97% confluence, about 98% confluence, about 99% confluence, or about 100% confluence.
  • Confluence can be determined by any standard method used in the field. See, e.g., Haenel, Frauke, and Norbert Garbow. "Cell counting and confluency analysis as quality controls in cell-based assays.” Multimode Detection (2014): 1-5.
  • no heparin is included in the MSC Culture Media.
  • the MSC Culture Media comprises no bovine or porcine components.
  • no antibiotics and/or antimycotics are used in the MSC Culture Media.
  • extracted MSCs are cultured in a MSC Culture Medium wherein the medium is configured to generate MSCs having the instant preparation profiles, e.g. antigen profile, TNFRI profile, cryopreservation profile, differentiation profile, and/or sterility (with respect to pathogens).
  • the medium comprises minimal essential medium (MEM).
  • the medium comprises alpha MEM.
  • the medium comprises human platelet lysate (hPL), e.g., StemulateTM.
  • the medium comprises fibroblast growth factor (FGF) e.g., carrier free FGF and/or FGF-2).
  • FGF fibroblast growth factor
  • the medium comprises epidermal growth factor (EGF, e.g., carrier free EGF).
  • EGF epidermal growth factor
  • the medium comprises alpha MEM, hPL, FGF, EGF, or any combination thereof.
  • the medium comprises alpha MEM, hPL, FGF, and EGF.
  • the medium does not further require heparin.
  • hPL is present in the MSC Culture Medium at about 1 % to about 21 %. In some embodiments, hPL is present in the medium at about 1 % to about 3 %, about 1 % to about 5 %, about 1 % to about 7 %, about 1 % to about 9 %, about 1 % to about
  • hPL is present in the medium at about 1 %, about 3 %, about 5 %, about 7 %, about 9 %, about 10 %, about 11 %, about 13 %, about 15 %, about 17 %, about 19 %, or about 21 %. In some embodiments, hPL is present in the medium at least about 1 %, about 3 %, about 5 %, about 7 %, about 9 %, about 10 %, about 11 %, about 13 %, about 15 %, about 17 %, or about 19 %.
  • hPL is present in the medium at most about 3 %, about 5 %, about 7 %, about 9 %, about 10 %, about 11 %, about 13 %, about 15 %, about 17 %, about 19 %, or about 21 %.
  • FGF is present in the medium at about 0.5 ng/mL to about 5 ng/mL.
  • FGF is present in the MSC Culture Medium at about 0.5 ng/mL to about 1 ng/mL, about 0.5 ng/mL to about 1.5 ng/mL, about 0.5 ng/mL to about 2 ng/mL, about 0.5 ng/mL to about 2.5 ng/mL, about 0.5 ng/mL to about 3 ng/mL, about 0.5 ng/mL to about 3.5 ng/mL, about 0.5 ng/mL to about 4 ng/mL, about 0.5 ng/mL to about 4.5 ng/mL, about 0.5 ng/mL to about 5 ng/mL, about 1 ng/mL to about 1.5 ng/mL, about 1 ng/mL to about 2 ng/mL, about 1 ng/mL to about 2.5 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 3.5 ng/mL
  • FGF is present in the medium at about 0.5 ng/mL, about 1 ng/mL, about 1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, about 4.5 ng/mL, or about 5 ng/mL. In some embodiments, FGF is present in the medium at least about 0.5 ng/mL, about 1 ng/mL, about
  • FGF is present in the medium at most about 1 ng/mL, about 1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, or about 5 ng/mL.
  • EGF is present in the MSC Culture Medium at about 0.5 ng/mL to about 5 ng/mL. In some embodiments, EGF is present in the medium at about 0.5 ng/mL to about 1 ng/mL, about 0.5 ng/mL to about 1.5 ng/mL, about 0.5 ng/mL to about 2 ng/mL, about 0.5 ng/mL to about 2.5 ng/mL, about 0.5 ng/mL to about 3 ng/mL, about 0.5 ng/mL to about 3.5 ng/mL, about 0.5 ng/mL to about 4 ng/mL, about 0.5 ng/mL to about 4.5 ng/mL, about 0.5 ng/mL to about 5 ng/mL, about 1 ng/mL to about 1.5 ng/mL, about 1 ng/mL to about 2 ng/mL, about 1 ng/mL to about 2.5 ng/mL, about 1
  • EGF is present in the medium at about 0.5 ng/mL, about 1 ng/mL, about 1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, about 4.5 ng/mL, or about 5 ng/mL.
  • EGF is present in the medium at least about 0.5 ng/mL, about 1 ng/mL, about 1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, or about 4.5 ng/mL. In some embodiments, EGF is present in the medium at most about 1 ng/mL, about 1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, about 4.5 ng/mL, or about 5 ng/mL.
  • the MSC Culture Medium comprises a modified alpha MEM.
  • the modified alpha MEM comprises one or more inorganic salts, one or more amino acids, one or more vitamins, glucose, lipoic acid, sodium bicarbonate, sodium pyruvate, or any combination thereof.
  • the one or more inorganic salts comprise calcium chloride (dihydrate), magnesium sulfate (heptahydrate), potassium chloride, sodium chloride, sodium phosphate monobasic (dehydrate), or any combination thereof.
  • each inorganic salt present in the MSC Culture Medium is present at about 100 mg/Liter to about 800 mg/Liter.
  • each inorganic salt present in the medium is present at about 100 mg/Liter to about 200 mg/Liter, about 100 mg/Liter to about 300 mg/Liter, about 100 mg/Liter to about 400 mg/Liter, about 100 mg/Liter to about 500 mg/Liter, about 100 mg/Liter to about 600 mg/Liter, about 100 mg/Liter to about 700 mg/Liter, about 100 mg/Liter to about 800 mg/Liter, about 200 mg/Liter to about 300 mg/Liter, about 200 mg/Liter to about 400 mg/Liter, about 200 mg/Liter to about 500 mg/Liter, about 200 mg/Liter to about 600 mg/Liter, about 200 mg/Liter to about 700 mg/Liter, about 200 mg/Liter to about 800 mg/Liter, about 300 mg/Liter to about 400 mg/Liter, about 300 mg/Liter to about 500 mg/Liter, about 300 mg/Liter to about 600 mg/Liter, about 300 mg/Liter to about 700 mg/Liter, about 200 mg/
  • each inorganic salt present in the medium is present at about 100 mg/Liter, about 200 mg/Liter, about 300 mg/Liter, about 400 mg/Liter, about 500 mg/Liter, about 600 mg/Liter, about 700 mg/Liter, or about 800 mg/Liter. In some embodiments, each inorganic salt present in the medium is present at least about 100 mg/Liter, about 200 mg/Liter, about 300 mg/Liter, about 400 mg/Liter, about 500 mg/Liter, about 600 mg/Liter, or about 700 mg/Liter.
  • each inorganic salt present in the medium is present at most about 200 mg/Liter, about 300 mg/Liter, about 400 mg/Liter, about 500 mg/Liter, about 600 mg/Liter, about 700 mg/Liter, or about 800 mg/Liter.
  • the one or more amino acids comprise glycine, alanine, alanyl-glutamine, arginine (HC1), asparagine (monohydrate), aspartic acid, cysteine (HC1) (monohydrate), cystine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or any combination thereof.
  • the one or more amino acids are present in the L isoform.
  • the one or more amino acids are present in the D isoform.
  • each amino acid present in the medium is present at about 10 mg/Liter to about 100 mg/Liter.
  • each amino acid present in the MSC Culture Medium is present at about 10 mg/Liter to about 20 mg/Liter, about 10 mg/Liter to about 30 mg/Liter, about 10 mg/Liter to about 40 mg/Liter, about 10 mg/Liter to about 50 mg/Liter, about 10 mg/Liter to about 60 mg/Liter, about 10 mg/Liter to about 70 mg/Liter, about 10 mg/Liter to about 80 mg/Liter, about 10 mg/Liter to about 90 mg/Liter, about 10 mg/Liter to about 100 mg/Liter, about 20 mg/Liter to about 30 mg/Liter, about 20 mg/Liter to about 40 mg/Liter, about 20 mg/Liter to about 50 mg/Liter, about 20 mg/Liter to about 60 mg/Liter, about 20 mg/Liter, about 10 mg/Liter to about 40 mg/Liter, about 20 mg/Liter to about 50 mg/Lite
  • each amino acid present in the medium is present at about 10 mg/Liter, about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, or about 100 mg/Liter. In some embodiments, each amino acid present in the medium is present at least about 10 mg/Liter, about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, or about 90 mg/Liter.
  • each amino acid present in the medium is present at most about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, or about 100 mg/Liter. In some embodiments, each amino acid present in the medium is present at about 100 mg/Liter to about 500 mg/Liter.
  • each amino acid present in the medium is present at about 100 mg/Liter to about 200 mg/Liter, about 100 mg/Liter to about 300 mg/Liter, about 100 mg/Liter to about 400 mg/Liter, about 100 mg/Liter to about 500 mg/Liter, about 200 mg/Liter to about 300 mg/Liter, about 200 mg/Liter to about 400 mg/Liter, about 200 mg/Liter to about 500 mg/Liter, about 300 mg/Liter to about 400 mg/Liter, about 300 mg/Liter to about 500 mg/Liter, or about 400 mg/Liter to about 500 mg/Liter.
  • each amino acid present in the medium is present at about 100 mg/Liter, about 200 mg/Liter, about 300 mg/Liter, about 400 mg/Liter, or about 500 mg/Liter. In some embodiments, each amino acid present in the medium is present at least about 100 mg/Liter, about 200 mg/Liter, about 300 mg/Liter, or about 400 mg/Liter. In some embodiments, each amino acid present in the medium is present at most about 200 mg/Liter, about 300 mg/Liter, about 400 mg/Liter, or about 500 mg/Liter.
  • the one or more vitamins comprise ascorbic acid, biotin, choline chloride, calcium pantothenate, folic acid, myo-inositol, niacinamide, pyridoxal (HC1), pyruvic acid (sodium salt), riboflavin, thiamine (HC1), vitamin B12, or any combination thereof.
  • the one or more vitamins are present in the L isoform.
  • the one or more vitamins are present in the D isoform.
  • the one or more vitamins are present in both isoforms.
  • each vitamin present in the MSC Culture Medium is present at about 0.1 mg/Liter to about 2 mg/Liter.
  • each vitamin present in the medium is present at about 0.1 mg/Liter to about 0.3 mg/Liter, about 0.1 mg/Liter to about 0.5 mg/Liter, about 0.1 mg/Liter to about 0.7 mg/Liter, about 0.1 mg/Liter to about 0.9 mg/Liter, about 0.1 mg/Liter to about 1.1 mg/Liter, about 0.1 mg/Liter to about 1.3 mg/Liter, about 0.1 mg/Liter to about 1.5 mg/Liter, about 0.1 mg/Liter to about 1.7 mg/Liter, about 0.1 mg/Liter to about 1.9 mg/Liter, about 0.1 mg/Liter to about 2 mg/Liter, about 0.3 mg/Liter to about 0.5 mg/Liter, about 0.3 mg/Liter to about 0.7 mg/Liter, about 0.3 mg/Liter to about 0.9 mg/Liter, about 0.3 mg/Liter to about 1.1 mg/Liter, about 0.3 mg/Liter to about 1.3 mg/Liter, about 0.3 mg/Liter
  • each vitamin present in the medium is present at about 0.1 mg/Liter, about 0.3 mg/Liter, about 0.5 mg/Liter, about 0.7 mg/Liter, about 0.9 mg/Liter, about 1.1 mg/Liter, about 1.3 mg/Liter, about 1.5 mg/Liter, about 1.7 mg/Liter, about 1.9 mg/Liter, or about 2 mg/Liter.
  • each vitamin present in the medium is present at least about 0.1 mg/Liter, about 0.3 mg/Liter, about 0.5 mg/Liter, about 0.7 mg/Liter, about 0.9 mg/Liter, about 1.1 mg/Liter, about 1.3 mg/Liter, about 1.5 mg/Liter, about 1.7 mg/Liter, or about 1.9 mg/Liter.
  • each vitamin present in the medium is present at most about 0.3 mg/Liter, about 0.5 mg/Liter, about 0.7 mg/Liter, about 0.9 mg/Liter, about 1.1 mg/Liter, about 1.3 mg/Liter, about 1.5 mg/Liter, about 1.7 mg/Liter, about 1.9 mg/Liter, or about 2 mg/Liter.
  • each vitamin present in the medium is present at about 10 mg/Liter to about 120 mg/Liter. In some embodiments, each vitamin present in the medium is present at about 10 mg/Liter to about 20 mg/Liter, about 10 mg/Liter to about 30 mg/Liter, about 10 mg/Liter to about 40 mg/Liter, about 10 mg/Liter to about 50 mg/Liter, about 10 mg/Liter to about 60 mg/Liter, about 10 mg/Liter to about 70 mg/Liter, about 10 mg/Liter to about 80 mg/Liter, about 10 mg/Liter to about 90 mg/Liter, about 10 mg/Liter to about 100 mg/Liter, about 10 mg/Liter to about 110 mg/Liter, about 10 mg/Liter to about 120 mg/Liter, about 20 mg/Liter to about 30 mg/Liter, about 20 mg/Liter to about 40 mg/Liter, about 20 mg/Liter to about 50 mg/Liter, about 20 mg/Liter to about 60 mg/Liter, about 20 mg
  • each vitamin present in the medium is present at about 10 mg/Liter, about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, about 110 mg/Liter, or about 120 mg/Liter.
  • each vitamin present in the medium is present at least about 10 mg/Liter, about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, or about 110 mg/Liter.
  • each vitamin present in the medium is present at most about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, about 110 mg/Liter, or about 120 mg/Liter.
  • the glucose comprised in the medium is anhydrous. In some embodiments, the glucose is present in the L isoform. In some embodiments, the glucose is present in the D isoform. In some embodiments, the glucose is present in both isoforms. In some embodiments, glucose present in the MSC Culture Medium is present at about 500 mg/Liter to about 1,600 mg/Liter.
  • glucose present in the medium is present at about 500 mg/Liter to about 600 mg/Liter, about 500 mg/Liter to about 700 mg/Liter, about 500 mg/Liter to about 800 mg/Liter, about 500 mg/Liter to about 900 mg/Liter, about 500 mg/Liter to about 1,000 mg/Liter, about 500 mg/Liter to about 1,100 mg/Liter, about 500 mg/Liter to about 1,200 mg/Liter, about 500 mg/Liter to about 1,300 mg/Liter, about 500 mg/Liter to about 1,400 mg/Liter, about 500 mg/Liter to about 1,500 mg/Liter, about 500 mg/Liter to about 1,600 mg/Liter, about 600 mg/Liter to about 700 mg/Liter, about 600 mg/Liter to about 800 mg/Liter, about 600 mg/Liter to about 900 mg/Liter, about 600 mg/Liter to about 1,000 mg/Liter, about 600 mg/Liter to about 1,100 mg/Liter, about 600 mg/Liter to about 1,
  • glucose present in the medium is present at about 500 mg/Liter, about 600 mg/Liter, about 700 mg/Liter, about 800 mg/Liter, about 900 mg/Liter, about 1,000 mg/Liter, about 1,100 mg/Liter, about 1,200 mg/Liter, about 1,300 mg/Liter, about 1,400 mg/Liter, about 1,500 mg/Liter, or about 1,600 mg/Liter.
  • glucose present in the medium is present at least about 500 mg/Liter, about 600 mg/Liter, about 700 mg/Liter, about 800 mg/Liter, about 900 mg/Liter, about 1,000 mg/Liter, about 1,100 mg/Liter, about 1,200 mg/Liter, about 1,300 mg/Liter, about 1,400 mg/Liter, or about 1,500 mg/Liter.
  • glucose present in the medium is present at most about 600 mg/Liter, about 700 mg/Liter, about 800 mg/Liter, about 900 mg/Liter, about 1,000 mg/Liter, about 1,100 mg/Liter, about 1,200 mg/Liter, about 1,300 mg/Liter, about 1,400 mg/Liter, about 1,500 mg/Liter, or about 1,600 mg/Liter.
  • lipoic acid present in the MSC Culture Medium is present at about 0.05 mg/Liter to about 0.5 mg/Liter. In some embodiments, the lipoic acid is present in the medium in the form of DL-thiotic acid.
  • lipoic acid present in the medium is present at about 0.05 mg/Liter to about 0.1 mg/Liter, about 0.05 mg/Liter to about 0.15 mg/Liter, about 0.05 mg/Liter to about 0.2 mg/Liter, about 0.05 mg/Liter to about 0.25 mg/Liter, about 0.05 mg/Liter to about 0.3 mg/Liter, about 0.05 mg/Liter to about 0.35 mg/Liter, about 0.05 mg/Liter to about 0.4 mg/Liter, about 0.05 mg/Liter to about 0.45 mg/Liter, about 0.05 mg/Liter to about 0.5 mg/Liter, about 0.1 mg/Liter to about 0.15 mg/Liter, about 0.1 mg/Liter to about 0.2 mg/Liter, about 0.1 mg/Liter to about 0.25 mg/Liter, about 0.1 mg/Liter to about 0.3 mg/Liter, about 0.1 mg/Liter to about 0.35 mg/Liter, about 0.1 mg/Liter to about 0.4 mg/Liter
  • lipoic acid present in the medium is present at about 0.05 mg/Liter, about 0.1 mg/Liter, about 0.15 mg/Liter, about 0.2 mg/Liter, about 0.25 mg/Liter, about 0.3 mg/Liter, about 0.35 mg/Liter, about 0.4 mg/Liter, about 0.45 mg/Liter, or about 0.5 mg/Liter. In some embodiments, lipoic acid present in the medium is present at least about 0.05 mg/Liter, about 0.1 mg/Liter, about 0.15 mg/Liter, about 0.2 mg/Liter, about 0.25 mg/Liter, about 0.3 mg/Liter, about 0.35 mg/Liter, about 0.4 mg/Liter, or about 0.45 mg/Liter.
  • lipoic acid present in the medium is present at most about 0.1 mg/Liter, about 0.15 mg/Liter, about 0.2 mg/Liter, about 0.25 mg/Liter, about 0.3 mg/Liter, about 0.35 mg/Liter, about 0.4 mg/Liter, about 0.45 mg/Liter, or about 0.5 mg/Liter.
  • sodium bicarbonate present in the MSC Culture Medium is present at about 250 mg/Liter to about 2,000 mg/Liter. In some embodiments, sodium bicarbonate present in the medium is present at about 250 mg/Liter to about 500 mg/Liter, about 250 mg/Liter to about 750 mg/Liter, about 250 mg/Liter to about 1,000 mg/Liter, about
  • sodium bicarbonate present in the medium is present at about 250 mg/Liter, about 500 mg/Liter, about 750 mg/Liter, about 1,000 mg/Liter, about 1,250 mg/Liter, about 1,500 mg/Liter, about 1,750 mg/Liter, or about 2,000 mg/Liter. In some embodiments, sodium bicarbonate present in the medium is present at least about 250 mg/Liter, about 500 mg/Liter, about 750 mg/Liter, about 1,000 mg/Liter, about 1,250 mg/Liter, about 1,500 mg/Liter, or about 1,750 mg/Liter.
  • sodium bicarbonate present in the medium is present at most about 500 mg/Liter, about 750 mg/Liter, about 1,000 mg/Liter, about 1,250 mg/Liter, about 1,500 mg/Liter, about 1,750 mg/Liter, or about 2,000 mg/Liter.
  • sodium pyruvate present in the MSC Culture Medium is present at about 50 mg/Liter to about 160 mg/Liter. In some embodiments, sodium pyruvate present in the medium is present at about 50 mg/Liter to about 60 mg/Liter, about 50 mg/Liter to about 70 mg/Liter, about 50 mg/Liter to about 80 mg/Liter, about 50 mg/Liter to about 90 mg/Liter, about 50 mg/Liter to about 100 mg/Liter, about 50 mg/Liter to about 110 mg/Liter, about 50 mg/Liter to about 120 mg/Liter, about 50 mg/Liter to about 130 mg/Liter, about 50 mg/Liter to about 140 mg/Liter, about 50 mg/Liter to about 150 mg/Liter, about 50 mg/Liter to about 160 mg/Liter, about 60 mg/Liter to about 70 mg/Liter, about 60 mg/Liter to about 80 mg/Liter, about 60 mg/Liter to about 90 mg/Liter, about 60
  • sodium pyruvate present in the medium is present at about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, about 110 mg/Liter, about 120 mg/Liter, about 130 mg/Liter, about 140 mg/Liter, about 150 mg/Liter, or about 160 mg/Liter.
  • sodium pyruvate present in the medium is present at least about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, about 110 mg/Liter, about 120 mg/Liter, about 130 mg/Liter, about 140 mg/Liter, or about 150 mg/Liter.
  • sodium pyruvate present in the medium is present at most about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, about 110 mg/Liter, about 120 mg/Liter, about 130 mg/Liter, about 140 mg/Liter, about 150 mg/Liter, or about 160 mg/Liter.
  • the pH of the alpha MEM is from about 7.0 to about 7.4.
  • the alpha MEM comprises the ingredients are presented in
  • the vBA-MSCs and/or vBM-MSCs are cultured in a MSC Culture Medium comprising alpha MEM as described in Table 5, 10% hPL (e.g., StemulateTM, 2 ng/mL recombinant, FGF (e.g., basic fibroblast growth factor (FGF-2) and/or carrier free FGF), and 2 ng/mL recombinant, epidermal growth factor (EGF, e.g., carrier free EGF).
  • hPL e.g., StemulateTM
  • FGF e.g., basic fibroblast growth factor (FGF-2) and/or carrier free FGF
  • EGF epidermal growth factor
  • the following reagents may be used during the manufacture of an MSC composition, as described above, and at the described concentrations: Phosphate Buffered Saline (PBS) at 100% Dimethyl Sulfoxide (DMSO) at 5%; Plasma Lyte-A Injection pH 7.4 at 100%; Human Serum Albumin, 25% at 2.5%; MSC Growth Media (Alpha MEM) at 100%; FGF2 at 2ng/mL; EGF at 2ng/mL; TrypLETM Select at 100%; and Human Platelet Lysate at 10%.
  • PBS Phosphate Buffered Saline
  • DMSO Dimethyl Sulfoxide
  • Plasma Lyte-A Injection pH 7.4 at 100%
  • Human Serum Albumin 25% at 2.5%
  • MSC Growth Media Alpha MEM
  • FGF2 at 2ng/mL
  • EGF at 2ng/mL
  • TrypLETM Select at 100%
  • Human Platelet Lysate at 10%.
  • said cell culture was cryopreserved in a cry opreservation media, wherein said cry opreservation media comprises an electrolyte formulation, human serum albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof.
  • HSA human serum albumin
  • DMSO dimethyl sulfoxide
  • cryovials are cryopreserved in a method comprising a precooling step or equilibration at 4 °C followed by passive cry opreservation at a rate of -1 °C per minute to about - 80 °C or less; then the cryovials are placed into the vapor phase above liquid nitrogen in cryogenic tanks for storage at less than or equal to -140 °C.
  • said cryopreservation media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 3% to about 4%, about
  • said cry opreservation media comprises about 1% to about 5% HSA. In some embodiments, said cryopreservation media comprises about 2.5% HSA.
  • said cryopreservation media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 3% to about 4%, about
  • said cry opreservation media comprises about 1% to about 10% DMSO. In some embodiments, said cryopreservation media comprises about 5% DMSO.
  • said electrolyte formulation is Plasmalyte A.
  • the method further comprises, prior to (c), resuspending said cell culture in a rinse media, wherein said rinse media comprises an electrolyte formulation, human serum albumin (HSA), or both.
  • the method further comprises, prior to (c), resuspending said cell culture in a rinse media, wherein said rinse media comprises an electrolyte formulation, human serum albumin (HSA), or both.
  • the rinse media is fresh.
  • said rinse media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%
  • the systems and methods described herein have the ability to generate about 220 cryovials (26 million cells/vial) of passage 1 (Pl) master cell bank (MCB), which can generate approximately 193,600 vials (100 million cells/vial) of passage 4 (P4) marrow-derived mesenchymal stem cell final product with the potential to treat about 193,600 subjects at a dose of 100 million cells/dose.
  • the systems and methods described herein generate between about 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, or more than 300 cryovials of passage 1 master cell bank.
  • each cryovial comprises about 10 million, about 11 million, about 12 million, about 13 million, about 14 million, about 15 million, about 16 million, about 17 million, about 18 million, about 19 million, about 20 million, about 21 million, about 22 million, about 23 million, about24 million, about 25 million, about 26 million, about 27 million, about 28 million, about 29 million, about 30 million, about 32 million, about 32 million, about 33 million, about 34 million, about 35 million, about 36 million, about 37 million, about 38 million, about 48 million, or 40 million cells per vial of passage 1 master bank cells. In some embodiments, this allows the generation of about 100,000, about 110,000, about 120,000, about 130,000, about
  • (P4) cells In some embodiments, this allows the generation of at least about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about
  • each vial of P4 cells comprises about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140, 150 million, about 160 million, about 170 million, about 180 million, about 290 million, about 200 million or more than 200 million cells.
  • each vial of P4 cells comprises at least about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140, 150 million, about 160 million, about 170 million, about 180 million, about 290 million, about 200 million or more than 200 million cells.
  • this allows the treatment of about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about 260,000, about 270,000, about 280,000, about 290,000, about or 300,000 patients.
  • this allows the treatment of at least about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about 260,000, about 270,000, about 280,000, about 290,000, about or 300,000 patients.
  • MSCs and compositions comprising the same are provided to an end user (e.g., treatment facility) in a condition where they can be immediately be used (i.e., injected into a subject) and with minimal processing.
  • end user e.g., treatment facility
  • MSC sample preparation for immediate injection by the end user and where the MSCs do not require further processing steps upon arrival to the treatment facility.
  • the methods and systems disclosed herein comprise sample preparation of MSCs to including thawing cryopreserved MSCs and maintaining the MSCs under specific conditions (e.g. specific temperature(s)) for a period of time.
  • the methods and systems disclosed herein also comprise sample preparation of MSCs including thawing cryopreserved MSCs, maintaining the MSCs for a period of time under specific conditions (e.g. a specific/first temperature), and then maintaining the MSCs under different conditions (e.g. at a different temperature than the temperature under which the MSCs were maintained immediately post-thaw).
  • this change in temperature is a cooling.
  • the MSCs are maintained in hypothermic conditions post-thaw until direct infusion into a subject.
  • the first temperature that the cryopreserved population of stem cells is warmed to is greater than about 0 °C. In some embodiments, the first temperature is greater than about 20 °C.
  • the second temperature that the stem cells are stored at is a hypothermic temperature.
  • a frozen cryovial comprising cryopreserved cells e.g., P3 cells
  • the vial will be kept in the water bath until approximately 80% of ice has melted (which takes about two to three minutes).
  • a cryovial may then be sprayed with sterile 70% ethanol (EtOH), wiped with sterile wipes and transferred into a biosafety cabinet.
  • Cells in the thawed vial can then transferred to a sterile conical tube, the cells diluted with PlasmaLyte-A + 0.5% HSA, centrifuged (e.g., at room temperature) to form a cell pellet, and the supernatant is removed and the pellet is resuspended in a culturing medium, e.g., MSC Culture Media, for further plating and culturing, e.g., as Passage 4 (P4).
  • a culturing medium e.g., MSC Culture Media
  • a method for preparing stem cells for infusion comprising: (a) providing a cryopreserved population of cells comprising said stem cells; (b) warming said stem cells to a first temperature and holding said stem cells at said first temperature for a first period of time; and (c) changing said first temperature to a second temperature and maintaining said stem cells at said second temperature for a second period of time.
  • said first temperature is greater than about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C
  • said first temperature is about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15
  • said first temperature is greater than 0 °C.
  • said second temperature is less than about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C
  • said second temperature is about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22
  • said second temperature is hypothermic. In some embodiments, said second temperature is less than 40 °C.
  • said first time period is less than about one week. In some embodiments, said time period is less than about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In some embodiments, said time period is less than about 5 days. In some embodiments, said time period is less than about 2 days. In some embodiments, said time period is less than about 1 day.
  • said time period is less than about 24 hours, about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour.
  • said time period is less than about 12 hours. In some embodiments, said time period is less than about 6 hours. In some embodiments, said time period is less than about 2 hours.
  • said time period is less than about 60 minutes, about 59 minutes, about 58 minutes, about 57 minutes, about 56 minutes, about 55 minutes, about 54 minutes, about 53 minutes, about 52 minutes, about 51 minutes, about 50 minutes, about 49 minutes, about 48 minutes, about 47 minutes, about 46 minutes, about 45 minutes, about 44 minutes, about 43 minutes, about 42 minutes, about 41 minutes, about 40 minutes, about 39 minutes, about 38 minutes, about 37 minutes, about 36 minutes, about 35 minutes, about 34 minutes, about 33 minutes, about 32 minutes, about 31 minutes, about 30 minutes, about 29 minutes, about 28 minutes, about 27 minutes, about 26 minutes, about 25 minutes, about 24 minutes, about 23 minutes, about 22 minutes, about 21 minutes, about 20 minutes, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute.
  • the stem cells are maintained after they are warmed/thawed. In some embodiments, this maintenance is not accounted for in the first period of time described herein. In some embodiments, this maintenance is accounted for in the first period of time. In some embodiments, this maintenance follows similar methods as the MSC culturing methods described herein. In some embodiments, the post-thaw maintenance methods comprise not allowing the MSCs to double in population. In some embodiments, the MSCs are recovered and/or packaged prior to doubling. In some embodiments, the post-thaw culturing maintenance methods comprise culturing plating said stem cells at about 500 cells/cm 2 to about 4,000 cells/cm 2 .
  • the post-thaw culturing maintenance methods comprise culturing plating said stem cells at about 500 cells/cm 2 to about 1,000 cells/cm 2 , about 500 cells/cm 2 to about 1,500 cells/cm 2 , about 500 cells/cm 2 to about 2,000 cells/cm 2 , about 500 cells/cm 2 to about 2,500 cells/cm 2 , about 500 cells/cm 2 to about 3,000 cells/cm 2 , about 500 cells/cm 2 to about 3,500 cells/cm 2 , about 500 cells/cm 2 to about 4,000 cells/cm 2 , about 1,000 cells/cm 2 to about 1,500 cells/cm 2 , about 1,000 cells/cm 2 to about 2 ,000 cells/cm 2 , about 1,000 cells/cm 2 to about 2,500 cells/cm 2 , about 1,000 cells/cm 2 to about 3,000 cells/cm 2 , about 1,000 cells/cm 2 to about 3,500 cells/cm 2 , about 1,000 cells/cm 2 to about 3,500 cells/c
  • the post-thaw culturing maintenance methods comprise culturing plating said stem cells at about 500 cells/cm 2 , about 1,000 cells/cm 2 , about 1,500 cells/cm 2 , about 2,000 cells/cm 2 , about 2,500 cells/cm 2 , about 3,000 cells/cm 2 , about 3,500 cells/cm 2 , or about 4,000 cells/cm 2 .
  • the post-thaw culturing maintenance methods comprise culturing plating said stem cells at least about 500 cells/cm 2 , about 1,000 cells/cm 2 , about 1,500 cells/cm 2 , about 2,000 cells/cm 2 , about
  • the post-thaw culturing maintenance methods comprise culturing plating said stem cells at most about 1,000 cells/cm 2 , about 1,500 cells/cm 2 , about 2,000 cells/cm 2 , about 2,500 cells/cm 2 , about 3,000 cells/cm 2 , about 3,500 cells/cm 2 , or about 4,000 cells/cm 2 .
  • the post-thaw maintenance methods comprise plating said stem cells at about 3,000 to about 10,000, about 3,000 to about 9,000, about 3,000 to about 8,000 about, about 3,000 to about 7,000, about 3,000 to about 6,000, about 3,000 to about 5,000, about 3,000 to about 4,000, about 10,000 to about 50,000, about 10,000 to about 40,000, about 10,000 to about 30,000, about 10,000 to about 20,000, about 20,000 to about 50,000, about 20,000 to about 40,000, about 20,000 to about 30,000, about 30,000 to about 50,000, about 30,000 to about 40,000, or about 40,000 to about 50,000 cells/cm 2 at said first temperature.
  • the method further comprises, prior to (c), maintaining said stem cells at about 3,000 cells/cm 2 to about 50,000 cells/cm 2 at said first temperature.
  • the post-thaw culturing maintenance methods comprise culturing plating said stem cells at about 10,000 cells/cm 2 to about 50,000 cells/cm 2 .
  • the postthaw culturing maintenance methods comprise culturing plating said stem cells at about 10,000 cells/cm 2 to about 15,000 cells/cm 2 , about 10,000 cells/cm 2 to about 20,000 cells/cm 2 , about 10,000 cells/cm 2 to about 25,000 cells/cm 2 , about 10,000 cells/cm 2 to about 30,000 cells/cm 2 , about 10,000 cells/cm 2 to about 35,000 cells/cm 2 , about 10,000 cells/cm 2 to about 40,000 cells/cm 2 , about 10,000 cells/cm 2 to about 45,000 cells/cm 2 , about 10,000 cells/cm 2 to about 50,000 cells/cm 2 , about 15,000 cells/cm 2 to about 20,000 cells/cm 2 , about 15,000 cells/cm 2 to about 25,000 cells/cm 2 , about 15,000 cells/cm 2 to about 30,000 cells/cm 2 , about 15,000 cells/cm 2 to about 35,000 cells/cm 2 , about 15,000 cells/cm 2 , about 1
  • the post-thaw culturing maintenance methods comprise culturing plating said stem cells at about 10,000 cells/cm 2 , about 15,000 cells/cm 2 , about 20,000 cells/cm 2 , about 25,000 cells/cm 2 , about 30,000 cells/cm 2 , about 35,000 cells/cm 2 , about 40,000 cells/cm 2 , about 45,000 cells/cm 2 , or about 50,000 cells/cm 2 .
  • the post-thaw culturing maintenance methods comprise culturing plating said stem cells at least about 10,000 cells/cm 2 , about 15,000 cells/cm 2 , about 20,000 cells/cm 2 , about 25,000 cells/cm 2 , about 30,000 cells/cm 2 , about 35,000 cells/cm 2 , about 40,000 cells/cm 2 , or about 45,000 cells/cm 2 .
  • the post-thaw culturing maintenance methods comprise culturing plating said stem cells at most about 15,000 cells/cm 2 , about 20,000 cells/cm 2 , about 25,000 cells/cm 2 , about 30,000 cells/cm 2 , about 35,000 cells/cm 2 , about 40,000 cells/cm 2 , about 45,000 cells/cm 2 , or about 50,000 cells/cm 2 .
  • the method further comprises, prior to (c), maintaining said stem cells at about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, 10,000, about 20,000, about 30,000, about 40,000, or about 50,000 cells/cm 2 at said first temperature. In some embodiments, for a method provided herein, the method further comprises, prior to (c), maintaining said stem cells at about 32,000 cells/cm 2 at said first temperature. In some embodiments, the post-thaw maintaining takes place in a T25 flask. In some embodiments, the post-thaw maintaining takes place in a T75 flask.
  • the post-thaw maintaining takes place in another sized flasks.
  • the post-thaw culturing maintenance methods increases the number of cells in a culture vessel from about 5% to about 500%.
  • the post-thaw culturing maintenance methods may increase the number of cells in a culture vessel by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200%, about 220%, about 240%, about 260%, about 280%, about 300%, about 320%, about 340%, about 360%, about 380%, about 400%, about 420%, about 440%, about 460%, about 480%, or about 500%.
  • the post-thaw culturing maintenance methods may increase the number of cells in a culture vessel by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 200%, about 200% to about 300%, about 300% to about 400%, or about 400% to about 500%.
  • the stem cells are packaged in 5 mL volumes of 10 * 10 6 live cells/mL in Plasma-Lyte A + 2.5% HSA (Rinse Media). In some embodiments, the stem cells are packaged in volumes of about 1 x 10 6 live cells/mL to about 12 x 10 6 live cells/mL. In some embodiments, the stem cells are packaged in volumes of about 1 x 10 A 6 live cells/mL to about 2 x 10 6 live cells/mL, about 1 x 10 6 live cells/mL to about 3 x 10 6 live cells/mL, about
  • the stem cells are packaged in volumes of about 1 x 10 6 live cells/mL, about 2 x 10 6 live cells/mL, about 3 x 10 6 live cells/mL, about 4 x 10 6 live cells/mL, about 5 x 10 6 live cells/mL, about 6 x 10 6 live cells/mL, about 7 x 10 6 live cells/mL, about 8 x 10 6 live cells/mL, about 9 x 10 6 live cells/mL, about 10 x 10 6 live cells/mL, about 11 x 10 6 live cells/mL, or about 12 x 10 6 live cells/mL.
  • the stem cells are packaged in volumes of at least about 1 x 10 6 live cells/mL, about 2 x 10 6 live cells/mL, about 3 x 10 6 live cells/mL, about 4 x 10 6 live cells/mL, about 5 x 10 6 live cells/mL, about 6 x 10 6 live cells/mL, about 7 x 10 6 live cells/mL, about 8 x 10 6 live cells/mL, about 9 x 10 6 live cells/mL, about 10 x 10 6 live cells/mL, or about 11 x 10 6 live cells/mL.
  • the stem cells are packaged in volumes of at most about 2 x 10 6 live cells/mL, about 3 x 10 6 live cells/mL, about 4 x 10 6 live cells/mL, about 5 x 10 6 live cells/mL, about 6 x 10 6 live cells/mL, about 7 x 10 6 live cells/mL, about 8 x 10 6 live cells/mL, about 9 x 10 6 live cells/mL, about 10 x 10 6 live cells/mL, about 11 x 10 6 live cells/mL, or about 12 x 10 6 live cells/mL.
  • the stem cells are put under the second temperature for the second time period.
  • the second temperature is less than about 40 °C, about 39 °C, about 38 °C, about 37 °C, about 36 °C, about 35 °C, about 34 °C, about 33 °C, about 32 °C, about 31 °C, about 30 °C, about 29 °C, about 28 °C, about 27 °C, about 26 °C, about 25 °C, about 24 °C, about 23 °C, about 22 °C, about 21 °C, about 20 °C, about 19 °C, about 18 °C, about 17 °C, about 16 °C, about 15 °C, about 14 °C, about 13 °C, about 12 °C, about 11 °C, about 10 °C, about 9 °C, about 8 °C, about 7 °C, about 6 °C, about 5 °C, about 4
  • said cell culture is maintained at about 40 °C, about 39 °C, about 38 °C, about 37 °C, about 36 °C, about 35 °C, about 34 °C, about 33 °C, about 32 °C, about 31 °C, about 30 °C, about 29 °C, about 28 °C, about 27 °C, about 26 °C, about 25 °C, about 24 °C, about 23 °C, about 22 °C, about 21 °C, about 20 °C, about 19 °C, about 18 °C, about 17 °C, about 16 °C, about 15 °C, about 14 °C, about 13 °C, about 12 °C, about 11 °C, about 10 °C, about 9 °C, about 8 °C, about 7 °C, about 6 °C, about 5 °C, about 4 °C, about 3 °C, about 2 °C, or about 1 °C.
  • the second temperature is less than 37 °C. In some embodiments, the second temperature is less than 35 °C. In some embodiments, the second temperature is less than 30 °C. In some embodiments, the second temperature is less than 25 °C. In some embodiments, said the second temperature is less than 20 °C. In some embodiments, the second temperature is about 2 °C to about 8 °C.
  • said second time period is less than about one week. In some embodiments, said time period is less than about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In some embodiments, said time period is less than about 5 days. In some embodiments, said time period is less than about 2 days. In some embodiments, said time period is less than about 1 day.
  • said time period is less than about 24 hours, about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour.
  • said time period is less than about 12 hours. In some embodiments, said time period is less than about 6 hours. In some embodiments, said time period is less than about 2 hours.
  • said time period is less than about 60 minutes, about 59 minutes, about 58 minutes, about 57 minutes, about 56 minutes, about 55 minutes, about 54 minutes, about 53 minutes, about 52 minutes, about 51 minutes, about 50 minutes, about 49 minutes, about 48 minutes, about 47 minutes, about 46 minutes, about 45 minutes, about 44 minutes, about 43 minutes, about 42 minutes, about 41 minutes, about 40 minutes, about 39 minutes, about 38 minutes, about 37 minutes, about 36 minutes, about 35 minutes, about 34 minutes, about 33 minutes, about 32 minutes, about 31 minutes, about 30 minutes, about 29 minutes, about 28 minutes, about 27 minutes, about 26 minutes, about 25 minutes, about 24 minutes, about 23 minutes, about 22 minutes, about 21 minutes, about 20 minutes, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute.
  • Cultured P4 cells or the equivalent may be briefly cultured for about 20 to about 28 hours to allow metabolic activity to resume post-thaw.
  • P4 or the equivalent cells are detached from the culturing vessel, rinsed, and washed using PBS via centrifugation, e.g., at 500xg. After centrifugation, the supernatant is aspirated, and cells are resuspended in PBS. An aliquot of this preparation is taken for a viable cell count via trypan blue or an equivalent assay using acridine orange/propidium iodide. This count dictates final concentration requirements for final formulation and packaging.
  • the cells are centrifuged, supernatant is aspirated, and the pellet is resuspended in PlasmaLyte-A + 2.5% HSA at about 10 million cells/mL.
  • the cells are then packaged in 5 mL vials, e.g., CellSeal closed-system cryovials. Two 5 ml vials at 10 million cells/mL equate to one dose (, e.g., of about 100 million cells) for a patient in need, e.g., having a perianal fistula.
  • the preparations and compositions of the present disclosure may comprise at least about 100 million vBA-MSCs and/or vBM-MSCs having an antigen profile of more than about 1.75% CD45+ cells, at least about 95% CD105+ cells, and at least about 95% CD 166+ cells and the cells may be expanded ex vivo from passage 2 until passage 4 while maintaining population uniformity based upon the antigen profile (i.e. more than about 1.75% CD45+ cells, at least about 95% CD105+ cells, and at least about 95% CD166+ cells).
  • the preparations and compositions of the present disclosure may comprise vBA-MSCs and/or vBM-MSCs having an antigen profile of reduced expression of one or more senescent cell markers, as compared to bone marrow-derived MSCs prepared according to known MSC culturing techniques.
  • the one or more senescent cell markers comprise MIC-A, MIC-B, ULBP2, or any combination thereof.
  • NK cell-mediated immune responses are stimulated by MIC-A, MIC-B, and/or ULBP2.
  • an MSC preparation or compositions for administration to a subject in need comprises MSC cells suspended in PlasmaLyte- A with 2.5% Human Serum Albumin (HSA).
  • HSA Human Serum Albumin
  • the vBA-MSCs and/or vBM-MSCs preparations and compositions described herein comprise an amount of cells that express one or more senescent cell markers of about 1 % less than bone marrow-derived MSCs to about 100 % less than bone marrow-derived MSCs.
  • the vBA-MSCs and/or vBM-MSCs preparations and compositions described herein comprise an amount of cells that express one or more senescent cell markers of about 100 % less than bone marrow-derived MSCs to about 90 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 80 % less than bone marrow-derived MSCs, about 100 % less than bone marrow- derived MSCs to about 70 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 60 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 50 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 40 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs, about 100
  • the vBA-MSC preparations and compositions described herein comprise an amount of cells that express one or more senescent cell markers of about 100 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs, about 60 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs, about 40 % less than bone marrow-derived MSCs, about 30 % less than bone marrow-derived MSCs, about 20 % less than bone marrow- derived MSCs, about 10 % less than bone marrow-derived MSCs, about 5 % less than bone marrow-derived MSCs, or about 1 % less than bone marrow-derived MSCs.
  • the vBA-MSCs and/or vBM-MSCs preparations and compositions described herein comprise an amount of cells that express one or more senescent cell markers of at least about 100 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs, about 70 % less than bone marrow- derived MSCs, about 60 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs, about 40 % less than bone marrow-derived MSCs, about 30 % less than bone marrow-derived MSCs, about 20 % less than bone marrow-derived MSCs, about 10 % less than bone marrow-derived MSCs, or about 5 % less than bone marrow-derived MSCs.
  • the vBA-MSCs and/or vBM-MSCs preparations and compositions described herein comprise an amount of cells that express one or more senescent cell markers of at most about 90 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs, about 60 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs, about 40 % less than bone marrow-derived MSCs, about 30 % less than bone marrow-derived MSCs, about 20 % less than bone marrow-derived MSCs, about 10 % less than bone marrow-derived MSCs, about 5 % less than bone marrow-derived MSCs, or about 1 % less than bone marrow- derived MSCs.
  • the preparations and compositions of the present disclosure generate a lessened NK cell-mediated immune response upon administration to a subject comprising mis-matched MHC molecules (e.g. mis-matched human leukocyte antigens when the subject is a human), as compared to administration of a composition comprising bone marrow-derived MSCs.
  • the preparations and compositions of the present disclosure do not generate a NK cell-mediated immune response upon administration to a subject comprising mis-matched MHC molecules (e.g. mis-matched human leukocyte antigens when the subject is a human).
  • described herein is a method of administering a composition comprising MSCs to a subject with a perianal fistula.
  • described here is a method of using GMP grade MSC product in patients with refractory fistulizing perianal CD.
  • the method of using allogeneic bone marrow derived mesenchymal stem cells (MSCs) produces regenerative signals.
  • the compositions described herein are comprised of culture- expanded mesenchymal stromal cells (MSCs) isolated from the bone marrow of consented and disease-screened deceased organ and tissue donors. Treating a subject with an autoimmune disorder such as Crohn’s disease with allogenic cells can provide improved treatment outcomes by providing a balance of healthy cells within the subject.
  • the final product is composed of MSCs formulated at 20M cells/mL in Plasma Lyte A® with 2.5% human serum albumin (HSA) and 5% DMSO, and manufactured in 5mL cryopreserved units.
  • MSCs for administration to subject having a perianal fistula are derived from the continuous manufacturing process of obtained bone marrow from deceased-donor vertebral bodies to selection of MSC for further cell culture. MSCs are cultured out of the bone marrow units collected. The overall manufacturing process to derive the final MSC product is illustrated in FIG 1. As shown in FIG. 1, MSCs are cultured out of primary bone marrow units using a tiered culture system.
  • compositions and methods of the present disclosure include intentional HLA mis-matching between a donor and a recipient.
  • transplantation of HLA mis-matching bone marrow products of the present invention following NMA conditioning improves a patient’s outcome yet avoids GVHD.
  • the improved outcome and avoided GVHD occurs in the absence of an immunosuppressive prophylaxis.
  • a mis-matching occurs when a donor and recipient differ by at least one HLA marker, e.g., at least two, three, four, five, six, seven, eight, nine, ten, or eleven HLA markers.
  • a mis-matching occurs when a donor and a recipient have fewer than six HLA matches out of eight HLA markers, fewer than four HLA matches out of six HLA markers, or fewer than five HLA matches out of ten HLA markers. In some embodiments, a mis-matching occurs when a donor and a recipient have fewer than half of the characterized HLA markers matching. In some embodiments, there is HLA mis-matching between the HSCs and the patient. In some embodiments, there is HLA mis-matching between the human cadaver and the patient.
  • MSCs may administered prophylactically, perioperatively or postoperatively with SOT or other VCA procedures.
  • MSCs administered may comprise human MSCs derived from bone marrow, adherent vertebral body MSCs (vBA-MSCs), or both.
  • the MSCs are administered to a subject with a perianal fistula.
  • the MSCs are injected into the perianal fistula.
  • the composition of human MSCs provided for inhibiting an immune response may be administered in quantities of about 10 million to about 10 billion.
  • human MSCs may be administered in quantities of about 10 million to about 100 million, about 10 million to about 1 billion, about 10 million to about 10 billion, about 100 million to about 1 billion, about 100 million to about 10 billion, or about 1 billion to about 10 billion.
  • human MSCs may be administered in quantities of about 10 million, about 100 million, about 1 billion, or about 10 billion.
  • human MSCs may be administered in quantities of at least about 10 million, about 100 million, or about 1 billion.
  • human MSCs may be administered in quantities of at most about 100 million, about 1 billion, or about 10 billion.
  • the MSCs are administered to a subject with a perianal fistula.
  • the MSCs are injected into the perianal fistula.
  • the composition of MSCs may be comprised of less than 5% CD45+. In some embodiments, the composition of MSCs may be comprised of less than about 0.5 % CD45+ to about 10 % CD45+. In some embodiments, the composition of MSCs may be comprised of less than about 10 % CD45+ to about 9 % CD45+, about 10 % CD45+ to about 8 % CD45+, about 10 % CD45+ to about 7 % CD45+, about 10 % CD45+ to about 6 % CD45+, about 10 % CD45+ to about 5 % CD45+, about 10 % CD45+ to about 4 % CD45+, about 10 % CD45+ to about 3 % CD45+, about 10 % CD45+ to about 2 % CD45+, about 10 % CD45+ to about 1 % CD45+, about 10 % CD45+ to about 0.5 % CD45+, about 9 % CD45+ to about 8 % CD
  • the composition of MSCs may be comprised of less than at least about 10 % CD45+, about 9 % CD45+, about 8 % CD45+, about 7 % CD45+, about 6 % CD45+, about 5 % CD45+, about 4 % CD45+, about 3 % CD45+, about 2 % CD45+, about 1 % CD45+, or about 0.5 % CD45+.
  • the composition of MSCs may be comprised of less than at least about 10 % CD45+, about 9 % CD45+, about 8 % CD45+, about 7 % CD45+, about 6 % CD45+, about 5 % CD45+, about 4 % CD45+, about 3 % CD45+, about 2 % CD45+, or about 1 % CD45+.
  • the composition of MSCs may be comprised of less than at most about 9 % CD45+, about 8 % CD45+, about 7 % CD45+, about 6 % CD45+, about 5 % CD45+, about 4 % CD45+, about 3 % CD45+, about 2 % CD45+, about 1 % CD45+, or about 0.5 % CD45+.
  • the composition of MSCs may comprise more than 1% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.1% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.2% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.3% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.4% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.5% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.6% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.7% CD45+ cells.
  • the composition of MSCs may comprise more than 1.8% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.9% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 2% CD45+ cells. [0318] In some embodiments, the composition of MSCs may be comprised of at least 90% CD105+ cells. In some embodiments, the composition of MSCs may be comprised of at least about 70 % CD 105+ cells to about 100 % CD 105+ cells.
  • the composition of MSCs may be comprised of at least about 100 % CD 105+ cells to about 95 % CD 105+ cells, about 100 % CD 105+ cells to about 94 % CD 105+ cells, about 100 % CD 105+ cells to about 93 % CD 105+ cells, about 100 % CD 105+ cells to about 92 % CD 105+ cells, about 100 % CD 105+ cells to about 91 % CD 105+ cells, about 100 % CD 105+ cells to about 90 % CD 105+ cells, about 100 % CD 105+ cells to about 85 % CD 105+ cells, about 100 % CD105+ cells to about 80 % CD105+ cells, about 100 % CD105+ cells to about 75 % CD105+ cells, about 100 % CD 105+ cells to about 70 % CD 105+ cells, about 95 % CD 105+ cells to about 94 % CD105+ cells, about 95 % CD105+ cells to about 93 % CD105+
  • the composition of MSCs may be comprised of at least about 100 % CD105+ cells, about 95 % CD105+ cells, about 94 % CD105+ cells, about 93 % CD105+ cells, about 92 % CD105+ cells, about 91 % CD 105+ cells, about 90 % CD 105+ cells, about 85 % CD 105+ cells, about 80 % CD105+ cells, about 75 % CD105+ cells, or about 70 % CD105+ cells.
  • the composition of MSCs may be comprised of at least at least about 100 % CD105+ cells, about 95 % CD 105+ cells, about 94 % CD 105+ cells, about 93 % CD 105+ cells, about 92 % CD105+ cells, about 91 % CD105+ cells, about 90 % CD105+ cells, about 85 % CD105+ cells, about 80 % CD105+ cells, or about 75 % CD105+ cells.
  • the composition of MSCs may be comprised of at least at most about 95 % CD 105+ cells, about 94 % CD 105+ cells, about 93 % CD 105+ cells, about 92 % CD 105+ cells, about 91 % CD 105+ cells, about 90 % CD105+ cells, about 85 % CD105+ cells, about 80 % CD105+ cells, about 75 % CD105+ cells, or about 70 % CD 105+ cells.
  • the composition of MSCs may be comprised of at least 90% CD166+ cells. In some embodiments, the composition of MSCs may be comprised of at least about 70 % CD 166+ cells to about 100 % CD 166+ cells.
  • the composition of MSCs may be comprised of at least about 100 % CD 166+ cells to about 95 % CD 166+ cells, about 100 % CD 166+ cells to about 94 % CD 166+ cells, about 100 % CD 166+ cells to about 93 % CD 166+ cells, about 100 % CD 166+ cells to about 92 % CD 166+ cells, about 100 % CD 166+ cells to about 91 % CD 166+ cells, about 100 % CD 166+ cells to about 90 % CD 166+ cells, about 100 % CD 166+ cells to about 85 % CD 166+ cells, about 100 % CD166+ cells to about 80 % CD166+ cells, about 100 % CD166+ cells to about 75 % CD166+ cells, about 100 % CD 166+ cells to about 70 % CD 166+ cells, about 95 % CD 166+ cells to about 94 % CD166+ cells, about 95 % CD166+ cells to about 93 % CD166+
  • the composition of MSCs may be comprised of at least about 100 % CD166+ cells, about 95 % CD 166+ cells, about 94 % CD 166+ cells, about 93 % CD 166+ cells, about 92 % CD 166+ cells, about 91 % CD 166+ cells, about 90 % CD 166+ cells, about 85 % CD 166+ cells, about 80 % CD166+ cells, about 75 % CD166+ cells, or about 70 % CD166+ cells.
  • the composition of MSCs may be comprised of at least at least about 100 % CD166+ cells, about 95 % CD 166+ cells, about 94 % CD 166+ cells, about 93 % CD 166+ cells, about 92 % CD166+ cells, about 91 % CD166+ cells, about 90 % CD166+ cells, about 85 % CD166+ cells, about 80 % CD166+ cells, or about 75 % CD166+ cells.
  • the composition of MSCs may be comprised of at least at most about 95 % CD 166+ cells, about 94 % CD 166+ cells, about 93 % CD 166+ cells, about 92 % CD 166+ cells, about 91 % CD 166+ cells, about 90 % CD 166+ cells, about 85 % CD 166+ cells, about 80 % CD 166+ cells, about 75 % CD 166+ cells, or about 70 % CD 166+ cells.
  • the compositions described herein are administered to treat a medical condition.
  • the medical condition comprises a perianal fistula.
  • the composition of human MSCs provided for treating a medical condition may be administered in quantities of about 10 million to about 10 billion.
  • human MSCs may be administered in quantities of about 10 million to about 100 million, about 10 million to about 1 billion, about 10 million to about 10 billion, about 100 million to about 1 billion, about 100 million to about 10 billion, or about 1 billion to about 10 billion.
  • the human MSCs may be administered in quantities of about 10 million, about 100 million, about 1 billion, or about 10 billion.
  • human MSCs may be administered in quantities of at least about 10 million, about 100 million, or about 1 billion. In some embodiments, human MSCs may be administered in quantities of at most about 100 million, about 1 billion, or about 10 billion. In some embodiments, the human MSCs are isolated and processed as described herein.
  • the cell composition may aid in producing a state of mixed chimerism.
  • the administered cell composition within a subject of human MSCs may generate CD45-huCD73+huCD90+ cells within said subject.
  • the administered human MSCs may be derived from bone marrow (vBM-MSC), adherent vertebral body MSCs (vBA-MSCs), or both.
  • a method and composition are provided for treating a medical condition by administering a composition of at least 10 million human MSCs and at least 500,000 nucleated BM cells or derivatives thereof.
  • the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof into a subject may result in successful engraftment of at least a subset of the nucleated BM cells within the subject.
  • the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof into a subject may generate hematopoietic cells of a particular HLA haploid in a background of hematopoietic cells of another particular HLA haploid wherein the other particular HLA haploid is mis-matched with the former HLA haploid.
  • the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof into a subject may generate hematopoietic cells of a particular HLA haploid specific to the donor of the human MSCs and nucleated BM cells or derivatives thereof in a background of hematopoietic cells of a particular HLA haploid specific to the subject.
  • the hematopoietic cells generated in the subject following administration of human MSCs and nucleated BM cells or derivatives thereof are CD45+ cells.
  • the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof may generate CD45+H2d+ cells in a background of CD45+H2b+ cells. In some embodiments, the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof may generate CD45-huCD73+huCD90+ cells. In some embodiments, the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof may generate a mixed chimerism within the subject. In some embodiments, the generated mixed chimerism within the subject may be maintained for at least 120 days from administration of the nucleated BM cells.
  • the generated mixed chimerism within the subject may be maintained for about 1 month to about 12 months. In some embodiments, the generated mixed chimerism within the subject may be maintained for about 1 month to about 2 months, about 1 month to about 3 months, about 1 month to about 4 months, about 1 month to about 5 months, about 1 month to about 6 months, about 1 month to about 7 months, about 1 month to about 8 months, about 1 month to about 9 months, about
  • the generated mixed chimerism within the subject may be maintained for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months. In some embodiments, the generated mixed chimerism within the subject may be maintained for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 11 months.
  • the generated mixed chimerism within the subject may be maintained for at most about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
  • the generated mixed chimerism within the subject may be maintained for about 1 year to about 10 years. In some embodiments, the generated mixed chimerism within the subject may be maintained for about 1 year to about 2 years, about 1 year to about 3 years, about 1 year to about 4 years, about 1 year to about 5 years, about 1 year to about 6 years, about 1 year to about 7 years, about 1 year to about 8 years, about 1 year to about 9 years, about 1 year to about 10 years, about 2 years to about 3 years, about 2 years to about 4 years, about 2 years to about 5 years, about 2 years to about 6 years, about 2 years to about 7 years, about 2 years to about 8 years, about 2 years to about 9 years, about 2 years to about 10 years, about 3 years to about 4 years, about 3 years to about 5 years, about 3 years to about 6 years, about 3 years to about 7 years, about 3 years to about 8 years, about 3 years to about 9 years, about 3 years to about 10 years, about 4 years to about 5 years, about 3 years to about 6 years, about 3 years to about
  • the generated mixed chimerism within the subject may be maintained for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years. In some embodiments, the generated mixed chimerism within the subject may be maintained for at least about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, or about 9 years. In some embodiments, the generated mixed chimerism within the subject may be maintained for at most about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about
  • the method of first administrating a composition of human MSCs and nucleated BM cells or derivatives thereof may further comprise a second administration of at least 10 million human MSCs to the subject in need thereof 1 day, 2 days, 3 days, 4 days, or any combination thereof after the first administration.
  • the treated medical condition may be an immune response.
  • the medical condition may be a perianal fistula.
  • the treated medical condition may be an autoimmune disease.
  • the autoimmune disease may be Crohn’s Disease.
  • the composition of human MSCs and nucleated BM cells or derivatives thereof may include human MSCs derived from BM (vBM-MSCs), adherent vertebral body MSCs (vBA-MSCs), or both. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include MSCs in quantities of about
  • the composition of human MSCs and nucleated BM cells or derivatives thereof may include MSCs in quantities of about 10 million to about 100 million, about 10 million to about 1 billion, about 10 million to about 10 billion, about 100 million to about 1 billion, about 100 million to about 10 billion, or about 1 billion to about 10 billion. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include MSCs in quantities of about 10 million, about 100 million, about 1 billion, or about 10 billion. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include MSCs in quantities of at least about 10 million, about 100 million, or about 1 billion.
  • the composition of human MSCs and nucleated BM cells or derivatives thereof may include nucleated BM in quantities about 1 million to about 2 million. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include nucleated BM in quantities about 1 million to about 1.5 million, about 1 million to about 2 million, or about 1.5 million to about 2 million. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include nucleated BM in quantities about 1 million, about 1.5 million, or about 2 million. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include nucleated BM in quantities at least about 1 million, or about 1.5 million. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include nucleated BM in quantities at most about 1.5 million, or about 2 million.
  • the human MSCs may be derived from the same origin as the nucleated BM cells. In some embodiments, the human MSCs may be derived from a different origin as the nucleated BM cells. In some embodiments, the composition may further comprise a human organ. In some embodiments, the human organ may be a heart, kidney, liver, lung, pancreases, intestine, thymus, or uterus. In some embodiments, the composition comprised of human MSCs and nucleated BM cells or derivative thereof may include MSCs with a matched human leukocyte antigen (HLA) haploid type as the human organ.
  • HLA human leukocyte antigen
  • the composition comprised of human MSCs and nucleated BM cells or derivative thereof may include MSCs with mis-matched HLA haploid type as the human organ. In some embodiments, the composition comprised of human MSCs and nucleated BM cells or derivative thereof may include MSCs and nucleated BM cells or derivatives thereof with a mis-matched HLA haploid type as the human organ. In some embodiments, the composition comprised of human MSCs and nucleated BM cells or derivative thereof may include MSCs and nucleated BM cells or derivatives thereof with a matched HLA haploid type as the human organ.
  • the nucleated BM cells or derivatives thereof may comprise hematopoietic stem cells (HSC).
  • HSC hematopoietic stem cells
  • the human MSCs may comprise a mismatched HLA haploid type as the nucleated BM cells or derivatives thereof.
  • compositions comprising one or more of the cell types described herein.
  • described herein is a composition comprising vBA-MSCs.
  • the vBA-MSCs are .
  • the vBA-MSCs are isolated and processed as described herein.
  • the compositions described herein comprise at least about 500,000 vBA-MSCs to about 1,000,000,000 vBA-MSCs.
  • compositions described herein comprise at least about 500,000 vBA-MSCs to about 1,000,000 vBA-MSCs, about 500,000 vBA-MSCs to about 10,000,000 vBA-MSCs, about 500,000 vBA-MSCs to about 100,000,000 vBA-MSCs, about 500,000 vBA-MSCs to about 1,000,000,000 vBA-MSCs, about 1,000,000 vBA-MSCs to about 10,000,000 vBA-MSCs, about 1,000,000 vBA-MSCs to about 100,000,000 vBA-MSCs, about 1,000,000 vBA-MSCs to about 1,000,000,000 vBA-MSCs, about 10,000,000 vBA-MSCs to about 100,000,000 vBA-MSCs, about 10,000,000 vBA-MSCs to about 1,000,000,000 vBA-MSCs, or about 100,000,000 vBA- MSCs to about 1,000,000,000 vBA-MSCs.
  • the compositions described herein comprise at least about 500,000 vBA-MSCs, about 1,000,000 vBA-MSCs, about 10,000,000 vBA-MSCs, about 100,000,000 vBA-MSCs, or about 1,000,000,000 vBA-MSCs. In some embodiments, the compositions described herein comprise at least at least about 500,000 vBA-MSCs, about 1,000,000 vBA-MSCs, about 10,000,000 vBA-MSCs, or about 100,000,000 vBA-MSCs. In some embodiments, the compositions described herein comprise at least at most about 1,000,000 vBA-MSCs, about 10,000,000 vBA-MSCs, about 100,000,000 vBA-MSCs, or about 1,000,000,000 vBA-MSCs. In some embodiments, the compositions described herein comprise vBA-MSCs and bone marrow-derived MSCs.
  • described herein is a composition comprising Nucleated BM cells.
  • the nucleated BM cells are isolated and processed as described herein.
  • the compositions described herein comprise at least about 500,000 nucleated BM cells to about 2,000,000 nucleated BM cells.
  • the compositions described herein comprise at least about 500,000 nucleated BM cells to about 1,000,000 nucleated BM cells, about 500,000 nucleated BM cells to about 1,500,000 nucleated BM cells, about 500,000 nucleated BM cells to about 2,000,000 nucleated BM cells, about 1,000,000 nucleated BM cells to about 1,500,000 nucleated BM cells, about 1,000,000 nucleated BM cells to about 2,000,000 nucleated BM cells, or about 1,500,000 nucleated BM cells to about 2,000,000 nucleated BM cells. In some embodiments, the compositions described herein comprise at least about 500,000 nucleated BM cells, about 1,000,000 nucleated BM cells, about 1,500,000 nucleated BM cells, or about 2,000,000 nucleated BM cells.
  • compositions described herein comprise at least at least about 500,000 nucleated BM cells, about 1,000,000 nucleated BM cells, or about 1,500,000 nucleated BM cells. In some embodiments, the compositions described herein comprise at least at most about 1,000,000 nucleated BM cells, about 1,500,000 nucleated BM cells, or about 2,000,000 nucleated BM cells.
  • the compositions described herein comprise both vBA- MSCs and nucleated BM cells.
  • the vBA-MSCs and nucleated BM cells of a single composition are derived from a single donor.
  • the compositions described herein comprise both vBM-MSCs and nucleated BM cells.
  • the vBM-MSCs and nucleated BM cells of a single composition are derived from a single donor.
  • the compositions described herein comprise both vBM-MSCs and vBA-MSCs.
  • the vBM-MSCs and vBA-MSCs of a single composition are derived from a single donor.
  • compositions described herein comprise vBM-MSCs, vBA-MSCs, and nucleated BM cells.
  • the vBM-MSCs, vBA-MSCs, and nucleated BM cells of a single composition are derived from a single donor.
  • the nucleated bone marrow cells or derivatives thereof comprise hematopoietic stem cells (HSCs).
  • HSCs hematopoietic stem cells
  • the HSCs comprise CD34+ cells.
  • described herein is a method for treating a perianal fistula in a subject, the method comprising injecting into the perianal fistula a first dose of a composition comprising more than about 10 million human mesenchymal stem cells (MSCs) obtained from a non-living source.
  • MSCs human mesenchymal stem cells
  • described herein are methods of administering a composition comprising the MSCs described herein to a subject with a perianal fistula.
  • MSCs in perianal fistulas is based upon 1) their anti-inflammatory properties; 2) published experience of MSC in this condition and perianal Crohn’s fistula demonstrating efficacy and safety; 3) existence of cGMP methods for their isolation and growth.
  • the subject has an autoimmune disease. In some embodiments, the subject has Crohn’s disease. In some embodiments, the perianal fistula is refractory. In some embodiments, the subject is at least 18 years of age. In some embodiments, the subject has failed at least one medical treatment to treat the fistula. In some embodiments, the medical treatment to treat the fistula comprises treatment with azarthiprine, methotrexate, 6-mercaptopurine, anti-TNF, anti-integrin, or anti-interleukin. In some embodiments, the subject has undergone surgical repair to treat the fistula. In some embodiments, the surgical repair is selected from the list consisting of seton placement, glue or plug insertion, local tissue flaps, and ligation of intersphincteric fistula tract repair
  • the perianal fistula has been present for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5, about months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months. In some embodiments, the perianal fistula has been present for about 1 to 12 months, about 2 to 11 months, about 3 to 10 months, about 4 to 9 months, about 5 to 8 months, or about 6 to 7 months.
  • the method comprises administering a first dose.
  • the method comprises administering a first dose of at least about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140 million, about 150 million, about 160 million, about 170 million, about 180 million, about 190 million, about 200 million or more than 200 million MSCs.
  • the method comprise administering a dose of from about 60 million to 200 million, 70 million to 200 million, 80 million to 200 million, 90 million to 200 million, 100 million to 200 million, 110 million to 200 million, 120 million to 200 million, 130 million to 200 million, 140 million to 200 million, 150 million to 200 million, 160 million to 200 million, 170 million to 200 million, 180 million to 200 million, or 190 million to 200 million MSCs.
  • the MSCs are administered by injecting into the perianal fistula.
  • the MSCs are obtained from a non-living source.
  • the methods described herein comprise administering a first dose of MSCs. In some embodiments, the methods described herein comprise administering a second dose of MSCs. In some embodiments, the MSCs are administered at least one time, two times, three times, four times, five times or more than five times. In some embodiments, the MSCs are administered no more than one time, two times, three times, four times, or five times. In some embodiments, at least one month, two months, three months, four months, five months, six month, seven months, eight months, nine months, ten months, eleven months or a year separate the time between administration. In some embodiments, the MSCs are administered by injecting into the perianal fistula.
  • the MSCs are obtained from a non-living source. In some embodiments, the MSCs are obtained using the methods described herein. In some embodiments, the MSCs comprise vBA-MSCs. In some embodiments, the MSCs comprises vBM-MSCs.
  • the method comprises administering a second dose of MSCs. In some embodiments, the method comprises administering a second dose of at least about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140 million, about 150 million, about 160 million, about 170 million, about 180 million, about 190 million, about 200 million or more than 200 million MSCs.
  • the method comprise administering a second dose of from about 60 million to 200 million, 70 million to 200 million, 80 million to 200 million, 90 million to 200 million, 100 million to 200 million, 110 million to 200 million, 120 million to 200 million, 130 million to 200 million, 140 million to 200 million, 150 million to 200 million, 160 million to 200 million, 170 million to 200 million, 180 million to 200 million, or 190 million to 200 million MSCs.
  • the MSCs are administered by injecting into the perianal fistula.
  • the first and the second dose independently comprise more than about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140 million, about 150 million, about 160 million, about 170 million, about 180 million, about 190 million, about 200 million or more than 200 million MSCs.
  • the method comprise administering a dose of from about 60 million to 200 million, 70 million to 200 million, 80 million to 200 million, 90 million to 200 million, 100 million to 200 million, 110 million to 200 million, 120 million to 200 million, 130 million to 200 million, 140 million to 200 million, 150 million to 200 million, 160 million to 200 million, 170 million to 200 million, 180 million to 200 million, or 190 million to 200 million MSCs.
  • the MSCs are administered by injecting into the perianal fistula.
  • the methods described herein comprise injecting a composition comprising MSCs into a perianal fistula. In some embodiments, the injection comprising injecting a composition comprising MSCs into the submucosal layer of the bowel wall. In some embodiments, the injection comprises injecting a composition comprising MSCs along the length or the fistula tract. In some embodiments, the methods comprise injecting a composition comprising MSCs in parallel of the wall of the fistula tract. In some embodiments, the methods comprise injecting a composition comprising MSCs no deeper than 2 mm from the fistula tract wall.
  • each fistula is injected at least one, two, three, four, five, or more than five times. In some embodiments, each half of the fistula is injected. In some embodiments, each quadrant of the fistula is injected.
  • the MSCs are administered via injection. In some embodiments, the MSCs are administered via injection to the perianal fistula. In some embodiments, about at least about 1 mL, about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL, about 1.5 mL, about 1.6 mL, about 1.7 mL, about 1.8 mL, about 1.9.
  • each fistula tract will be injected.
  • a single fistula will be injected at least 1, 2, 3, 4, 5, 6 7, 8, 9, or 10 times.
  • the subject has at least a second perianal fistula.
  • the second perianal fistula is administered a first dose of a composition comprising MSCs.
  • the at least second perianal fistula is administered a dose of dose of at least about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140 million, about 150 million, about 160 million, about 170 million, about 180 million, about 190 million, about 200 million or more than 200 million MSCs.
  • the at least second perianal fistula is administered a second dose of from about 60 million to 200 million, 70 million to 200 million, 80 million to 200 million, 90 million to 200 million, 100 million to 200 million, 110 million to 200 million, 120 million to 200 million, 130 million to 200 million, 140 million to 200 million, 150 million to 200 million, 160 million to 200 million, 170 million to 200 million, 180 million to 200 million, or 190 million to 200 million MSCs.
  • the MSCs are administered by injecting into the perianal fistula.
  • the subject has at least a third perianal fistula.
  • the third perianal fistula is administered a first dose of a composition comprising MSCs.
  • the at least third perianal fistula is administered a dose of dose of at least about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140 million, about 150 million, about 160 million, about 170 million, about 180 million, about 190 million, about 200 million or more than 200 million MSCs.
  • the at least third perianal fistula is administered a second dose of from about 60 million to 200 million, 70 million to 200 million, 80 million to 200 million, 90 million to 200 million, 100 million to 200 million, 110 million to 200 million, 120 million to 200 million, 130 million to 200 million, 140 million to 200 million, 150 million to 200 million, 160 million to 200 million, 170 million to 200 million, 180 million to 200 million, or 190 million to 200 million MSCs.
  • the MSCs are administered by injecting into the perianal fistula.
  • the MSCs are allogeneic to the subject. In some embodiments, the MSCs are HLA-matched to the subject. In some embodiments, the MSCS are HLA-mismatched to the subject. In some embodiments, the MSCs and the subject have fewer than seven HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have fewer than six HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have fewer than five HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have fewer than four HLA matches out of eight HLA markers.
  • the MSCs and the subject have fewer than three HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have fewer than two HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have no HLA matches out of eight HLA markers. In some embodiments, the MSCs are obtained from a non-living source.
  • the MSCs have been cultured for at least one passage prior to administration to the subject. In some embodiments the MSCs have been cultured for at least one, two, three, four, five, six, or seven passages before administration to the subject.
  • the MSCs have been cryopreserved before administration to the subject using the methods described herein. In some embodiments, the MSCs have been thawed before administration. In some embodiments, the MSCs have been thawed to at least about 0°C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C or 10 °C.
  • the MSCs have been thawed to from about 0 °C to 10 °C, 0 °C to 9 °C, 0 °C to 8 °C, 0 °C to 7 °C,0 °C to 6 °C, 0 °C to 5 °C, 0 °C to 4 °C, 0 °C to 3°C, 0 °C to 2 °C or 0 °C to 1 °C.
  • the MSCs have been thawed at least about 1 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 19 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 or 24 hours prior to administration to the subject.
  • the MSCs have been thawed for at least about 1 days, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 6 days, 6.5 days, 7 days, or more than 7 days prior to administration to the subject.
  • the MSCs are warmed to room temperature before administration to a subject using the methods described herein. In some embodiments, the MSCs are warmed to about body temperature before administration to a subject. In some embodiments, the MSCs are warmed to about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, or 38°C before administration to a subject.
  • administration of the MSCs results in a reduction in size of the fistula, absence of fluid collection, or a lack of edema. In some embodiments, administration of the MSCs result in greater than or equal to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or complete cessation of drainage of the fistula. In some embodiments, administration of the MSCs results in epithelization of the external fistula opening.
  • the subject experiences a complete healing of the perianal fistula. In some embodiments, the subject experiences a complete healing of the perianal fistula after the first injection. In some embodiments the subject experiences a complete healing of the perianal fistula after the second injection. In some embodiments, complete healing is assessed b radiographic healing, clinical healing, or a combination thereof. In some embodiments, radiographic healing comprises a MRI with an absence of fluid collection greater than 2 cm in 2 of 3 dimensions, a lack of edema, lack of inflammation, or lack of signs of active inflammatory response, or a combination thereof. In some embodiments, clinical healing comprises 100% cessation of draining on both clinical exam with deep palpation and as per patient report, epithelization of the external fistula opening, or a combination thereof.
  • the subject experiences a partial healing of the perianal fistula. In some embodiments, the subject experiences a partial healing of the perianal fistula after the first injection. In some embodiments the subject experiences a partial healing of the perianal fistula after the second injection. In some embodiments, partial health is assessed by radiographic healing, clinical healing, or a combination thereof. In some embodiments, radiographic healing comprises a MRI with an absence of fluid collection greater than 2 cm in 2 of 3 dimensions, a lack of edema, lack of inflammation, or lack of signs of active inflammatory response, or a combination thereof. In some embodiments, clinical healing comprises greater than or equal to 50% cessation of drainage on both clinical exam with deep palpation and as per patient report, epithelization of the external fistula opening, or a combination thereof.
  • a method of reducing the size of a perianal fistula comprising administering a composition comprising MSCs to a subject.
  • administration of the MSCs result in greater than or equal to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% reduction in size of the fistula.
  • the method comprises injecting the composition into the perianal fistula.
  • the method comprises injecting a first dose of a composition comprising more than about 10 million MSCs obtained from a non-living source.
  • a method of reducing the amount of fluid collection in a perianal fistula comprising administering a composition comprising MSCs to a subject.
  • administration of the MSCs result in greater than or equal to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% reduction in fluid collection of the fistula.
  • the method comprises injecting the composition into the perianal fistula.
  • the method comprises injecting a first dose of a composition comprising more than about 10 million MSCs obtained from a non-living source.
  • the product administered comprises MSC formulated Plasma-Lyte ®.
  • the product administered comprises Human Serum Albumin (HSA).
  • HSA Human Serum Albumin
  • the product administered comprises at least about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% HSA.
  • the product administered comprise DMSO. In some somebody, the product administered comprise at least about 1%, about 2%, about 3%. 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% DMSO.
  • the product administered comprises at least about 5M, about 10M, about 15M, about 20M, about 25M, about 30M, about 40M, about 45M, or about 50M cells/mL.
  • the product administered comprises MSC formulated Plasma-Lyte® A, 2.5% Human Serum Albumin (HSA), and 5% DMSO at 20M cells/mL.
  • the final product has an endotoxin dose of less than about 1, 2, 3, 4, or 5 Endotoxin Units (EU)/mL.
  • Endotoxin testing is performed using the Endosafe®-PTSTM system according to SOP EPIC Limulus Amebocyte Lysate (LAL) Assay for Endotoxin Detection Using the Endosafe® - PTSTM.
  • the Endosafe®-PTSTM is a rapid, point-of-use test system that provides quantitative LAL results within 15 minutes after specimen preparation.
  • the PTSTM utilizes LAL reagents in an FDA-licensed disposable test cartridge with a handheld reader for a completely contained, real-time endotoxin testing system.
  • the PTSTM can be used to get a quick read on raw materials and STAT samples that require immediate analysis.
  • the flexibility of the PTSTM allows it to be used in conventional, quality control testing laboratories as well as at the point of sample collection.
  • Preferably cells for clinical use have ⁇ 2.5 EU/mL for release.
  • From as in “from 1 to 10” includes the initial and final number recited. Therefore, “from 1 to 10” includes the whole numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and includes fractions thereof, (e.g., about .1, .2, .3, .4, .5, .6, .7, .8, and about .9).
  • each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.
  • “or” may refer to “and”, “or,” or “and/or” and may be used both exclusively and inclusively.
  • the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.
  • determining means determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
  • the terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount.
  • the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase from about 10 to about 100% as compared to a reference level, standard, or control.
  • Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.
  • “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount.
  • “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease from about 10 to about 100% as compared to a reference level.
  • a “subject” can be a biological entity containing expressed genetic materials.
  • the biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa.
  • the subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro.
  • the subject can be a mammal.
  • the mammal can be a human.
  • the subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.
  • zzz vivo is used to describe an event that takes place in a subject’s body.
  • ex vivo is used to describe an event that takes place outside of a subject’s body.
  • An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.
  • An example of an ex vivo assay performed on a sample is an “in vitro” assay.
  • z z vitro is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained.
  • In vitro assays can encompass cell-based assays in which living or dead cells are employed.
  • In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
  • MSCs [that] were ultimately obtained from a human cadaver relate to the original source of the MSCs.
  • MSCs were directly obtained from deceased human donors, i.e., cadavers.
  • MSCs directly obtained from deceased human donors are cultured for one or more passage; in these embodiments, a fraction of the MSCs present after one passage, will no have been obtained from a deceased from a donor.
  • the MSCs even after culturing
  • the ultimate source of a MSC was not a living donor.
  • treatment or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated.
  • a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • a prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • a subj ect at risk of developing a particular disease, or to a subj ect reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
  • an "effective amount” or “therapeutically-effective amount” refers to that amount of a bone marrow product and/or HSCs contained in a bone marrow product as described herein which, when administered to a subject (e.g., human), that sufficient to promote treating a disease, e.g., a hematologic cancer.
  • a subject e.g., human
  • the amount of a bone marrow product and/or HSCs contained in the bone marrow product that constitutes a “therapeutically-effective amount” will vary depending on the cell preparations, the condition and its severity, the manner of administration, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
  • CD34 is used in reference to an antigen present on immature hematopoietic precursor cells and all hematopoietic colony-forming cells in bone marrow and blood. Certain populations of non-hematopoietic (i.e., CD45 negative) cells also express CD34. Of hematopoietic (i.e., CD45+ cells), the CD34 antigen expression is highest on early progenitor cells and decreases with the maturation of cells. The CD34 antigen is absent on fully differentiated hematopoietic cells. Normal peripheral blood lymphocytes, monocytes, granulocytes, and platelets do not express the CD34 antigen.
  • tissue processing protocol Described herein is an exemplary tissue processing protocol.
  • the tissue being processed can be vertebral bodies.
  • the tissue processing protocol can yield the bone marrow cells described herein.
  • I l l - remove outer nonsterile bag and dispose. Open inner bag and dispose of bag.
  • Unwrap specimen from blue towel and lap sponges Record presence of packing materials and condition of the spine for: minimum 2 layers of sterile bas; blue towel; lap sponges; tissue moisture maintenance; and presence of pedicles.
  • Spray balance (CS-5000 model) with 70% IPA and place in a clean area inside the Biosafety cabinet (BSC). Tare balance with the sterile bag. Place VBs that will be processed further into the sterile bag, and record mass. Record the # of VBs used for BM extraction.
  • BM Extract Total Mass (g) (D.3 [B]), Empty Mass (g) (D.l [A]), Empty Mass of all TRANSFER-PACKS (g) (A x 6), and Total Mass of BM Extract (g) (B-C).
  • centrifuge 1. Set the centrifuge to 500 x g for 15 minutes at room temperature, with a brake setting of 4. Centrifuge post-fat intermediate bags with tubing up. Use volume compensating plates to prevent creasing of bags during centrifugation.
  • vials for research use may be prepared. Label the required number of 5 mL cryovials and fill each one with 5 mL of BM by syringe or pipette.
  • Described herein is a protocol for isolating cells expressing CD34 from fresh or thawed bone marrow (BM) from deceased donors. .
  • Cell Prep Bag 1 (can be more than 1 bag)
  • Protocol for fresh bone marrow product
  • Optional step If clumps are present, insert standard blood filter, filter the cells and transfer to the second Cell Prep Bag.
  • the calculated volume of IVIG added should be included in the final labeling weight, not to exceed 95 g or 190 g, depending on scale of preparation (Table 6).
  • the volume of the selected cells at the end is expected to be -40-50 ml for the standard selection tubing set and -75-80 ml for large selection.
  • BM thawing cryopreserved bone marrow
  • the procedure can be for thawing cryopreserved BM bags and vials for testing.
  • thawing bags or vials e.g. clinical process validations, clinical development, etc.
  • it can be used to thawing of bone marrow product or thawing of bone marrow vials.
  • BSC biosafety cabinet
  • Sysmex XP-300 Hematology Analyzer to obtain counts on the Sysmex XP-300.
  • Cadaver- derived MSCs have been compared in a series of standard, accepted assays to MSCs from a standard, live-donor bone marrow source.
  • Cadaver- derived MSCs are bioequivalent to this reference source of MSCs, and are expected to have the same, remarkably safe safety profile as the numerous other sources of MSCs that have been administered to patients.
  • MSC from all 3 sources demonstrated essentially identical immunophenotypic cell surface marker profiles, with very low numbers of cells that expressed CD14, CD19, CD34, CD45 and HLA-DR, and, conversely, nearly all cells expressed CD73, CD90 and CD 105 (FIG. 2).
  • MSC from each source grew rapidly in culture through 5 passages (the longest period examined) with no differences in population doubling times (PDTs) between passages 4 and 5 (FIG. 3).
  • the CFU-F potential at passage 3 of BM-MSC freshly isolated from living and deceased donors was the same, whereas significantly lower (P ⁇ 0.05) CFU-F were present in Ex LD BM-MSC (FIG. 4).
  • trilineage differentiation potentials were compared and it was found that each MSC population formed adipocytes, chondrocytes and osteocytes in vitro at qualitatively the same frequencies (FIG. 5).
  • Example 5 Illustrative methods for preparing a final MSC Product
  • Bone marrow cells, CD34+ isolates, or isolated MSCs are obtained.
  • cryopreserved bone marrow is utilized for primary cell culture.
  • the cryopreserved bag is taken out of inventory and an MSC Batch Record is initiated. A unit is thawed as per the batch record instruction.
  • Approximately 4.5 billion mononuclear cells are plated in six CellBIND® 10-chamber CellSTACKS® and cultured for 10-14 days in MSC Culture Media (Alpha MEM with 10% human platelet lysate (hPL) and 2ng/mL each FGF-2 and EGF). No bovine or porcine components and no antibiotics and antimycotics are used in production.
  • the cells are cultured for about 14 days with fresh media changes every 3 to 4 days. These are termed passage 0 (P0) cells.
  • a MCB cell population will have the following preferable features described in Table 7 and as shown in the column “Result” actual results have been obtained from MCB cells prepared according to the methods of the present disclosure.
  • P2 cells are cultured for about 4 to about 5 days and once the flasks have reached greater than 75% confluence, cells are detached using TrypLE.
  • P3 cells are passage 3 cells. The P3 cells are cultured for about 4 to about 5 days and once the flasks reached greater than 75% confluence, cells are detached using TrypLE.
  • cryopreservation comprises precooling/equilibration at 4°C followed by passive cry opreservation at a rate of -l°C/minute to ⁇ -80 °C and then moving into vapor phase above LN2 for storage at ⁇ -140 °C. This constitutes the WCB.
  • a WCB cell population will have the following preferable features described in Table 8 and as shown in the column “Result” actual results have been obtained from WCB cells prepared according to the methods of the present disclosure.
  • P4 passage 4
  • the P4 cells are cultured for about 4 to about 5 days and once the flasks reached greater than 75% confluence, cells are detached, resuspended in PlasmaLyte-A + 2.5% HSA + 5% DMSO at about 20 million cells/mL and packaged in 5 mL CellSeal closed-system cryovials at 5 mL per cryovial using an automatic filler. Cells are then cryopreserved and placed into the vapor phase above LN2 in alarm monitored cryogenic tanks for storage at ⁇ -140 °C.
  • EOP cell population will have the following preferable features described in Table 9 and as shown in the column “Result” actual results have been obtained from EOP cells prepared according to the methods of the present disclosure.
  • P4 EOP aliquots will be thawed and plated in CellBIND 5-chamber or 10-chamber CellSTACK(s) in MSC Culture Media for cryo-recovery.
  • the P4 cells are briefly cultured for about 20 to about 28 hours to allow metabolic activity to resume post-thaw. After this brief culturing, cells are detached using TrypLE, flasks are rinsed, and the pooled cells are then washed using PBS via centrifugation at 500xg. After centrifugation, the supernatant is aspirated, and cells are resuspended in PBS. An aliquot of this preparation is taken for a viable cell count via trypan blue or an equivalent assay using acridine orange/propidium iodide. This count dictates final concentration requirements for final formulation and packaging.
  • the time elapsed through each culture step is variable, with greater than 75% confluence generally achieved in about 4 to about 5 days for passaged cells.
  • Time elapsed from a culture harvest to the start of the cryopreservation is preferably less than or about 6 hours. Upon cryopreservation cells are theoretically stable indefinitely.
  • the cells are then centrifuged, supernatant is aspirated, and the pellet is resuspended in PlasmaLyte-A + 2.5% HSA at about 10 million cells/mL.
  • the cells are then packaged in 5 mL CellSeal closed-system cryovials at 5 mL per cryovial.
  • Two CellSeal vials at 10 million cells/mL equate to one dose for a patient , e.g., about 100 million cells. Cells may be shipped to clinical site at 2-8°C.
  • the vials can be kept in the 2-8°C shipper until use.
  • the cells will be administered to a subject in need less than 48 hours after receipt; in most cases, cells should not administered more than 72 hours after receipt.
  • a product suitable for administration to a subject in need e.g., having a perianal fistula, will have the following preferable features described in Table 10.
  • all post-thaw testing will consist of viability (Cellometer, AO/PI); CFU- F (potency); and sterility.
  • Cells should be greater than or equal to 70% viable; greater than or equal to 20,000 colonies per million cells plated; and no growth on sterility testing.
  • Example 6 Methods for treating a fistula
  • a clinical phase IB/IIA trial is being performed to assess treating perianal fistulas with MSCs.
  • Patients with a single or multi tract fistula arising from the rectum or anal canal that travels through the internal/extemal sphincter complex to the perianal skin will be treated by direct injection of 1-2 doses of 100 million MSCs.
  • the rationale for this dose comes from several other studies reporting safety and efficacy with 60-200 million cells injected.
  • the product administered is derived from a single, deceased healthy donor. There is evidence demonstrating safety in perianal Crohn’s fistula.
  • the primary endpoint of this study is to determine the safety and feasibility of 1-2 treatments of 100 million allogeneic bone marrow derived MSCs for the treatment of perianal fistulizing Crohn’s disease.
  • the secondary objectives of this study include assessing complete or partial fistula healing induced by the delivery of 1-2 doses of 100 million allogeneic bone marrow derived MSCs on perianal fistulizing Crohn’s disease. Patients will be assessed as having complete healing; partial healing; lack of response; or worsening disease. Complete healing is assessed by radiographic healing (MRI with an absence of a fluid collection >2 cm in 3 of 3 dimensions, lack of edema, lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain) or clinical healing (100% cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening).
  • radiographic healing MRI with an absence of a fluid collection >2 cm in 3 of 3 dimensions, lack of edema, lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain
  • clinical healing (100% cessation of drainage on both clinical
  • Partial healing is assessed by radiographic healing (MRI with an absence of a fluid collection >2 cm in 2 of 3 dimensions, lack of edema, lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain) or clinical healing (greater than or equal to 50 % cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening).
  • Lack of Response is assessed by radiographic and clinical healing which does not meet the threshold for Partial Healing.
  • Worsening disease is assessed by radiographic (MRI with a fluid collection >2 cm in 2 of 3 dimensions, edema, inflammation or sign of active inflammatory response. An increased number of tracts may be seen, or increased branching from the primary tract) or clinical (increased drainage per patient report and on clinical exam) signs.
  • the patients have a diagnosis of CD for at least 6 months.
  • the patients enrolled in this study have perianal fistulas in the setting of Crohn’s disease and have failed at least one previous medical treatment and have had at least one surgical repair(s).
  • Failure of conventional medical therapy is defined as a lack of response to systemic immune suppression (e.g. azathioprine, methotrexate, 6-mercaptopurine) or biologic (e.g. anti-TNF, anti-integrin, anti-interleukin) therapies to treat fistulizing CD for at least 3 months.
  • Failure of surgical repair includes seton placement, glue or plug insertion, local tissue flaps, and/or ligation of intersphincteric fistula tract repair.
  • Exclusion criteria include inability to give informed consent; clinically significant medical conditions within the six months before administration of MSCs: e.g. myocardial infarction, active angina, congestive heart failure or other conditions that would, in the opinion of the investigators, compromise the safety of the patient; having Hepatitis B or C, HIV, abnormal AST or ALT at screening; a history of cancer including melanoma (with the exception of localized skin cancers) in the past five years; investigational drug within one month of treatment; pregnant or breast feeding or trying to become pregnant; presence of a rectovaginal or perineal body fistula; a change in Crohn’s immunosuppressive regimen within the 2 months prior to enrollment; uncontrolled intestinal Crohn’s disease which will require escalation for medical therapy or surgery within 2 months of enrollment; severe anal canal disease that is stenotic and requires dilation; currently taking corticosteroids; or allergy to DMSO.
  • medical conditions within the six months before administration of MSCs e.g. myo
  • the investigational agent is comprised of culture-expanded mesenchymal stromal cells (MSC) isolated from the bone marrow of vertebral bodies of consented and disease- screened deceased organ and tissue donors.
  • MSC mesenchymal stromal cells
  • the final product is composed of MSC formulated Plasma-Lyte® A, 2.5% Human Serum Albumin (HSA), and 5% DMSO at 20M cells/mL.
  • the final product is certified to have an endotoxin level of ⁇ 2 Endotoxin Units (EU)/mL.
  • EU Endotoxin Units
  • the total volume for injection is lOmL, for a maximum endotoxin of 20EU/dose.
  • the vials may be transferred to cryogenic storage or kept in the dry shipper for up to 24 hours if preparation for administration is to occur within that time frame.
  • the cells must be stored at ⁇ -140 °C until they are thawed.
  • cryogenic vials were thawed, contacted with a growth medium at a sufficient temperature and sufficient time, and shipped to the lab on wet ice.
  • the methods described in the section entitled “cryorecovery” will be employed.
  • MSC must be maintained in vapor phase liquid nitrogen at or colder than -135°C during storage Temperature readings must be monitored daily (excluding weekends and holidays) by either attaching a printout from the freezer’s electronic monitoring system or recording in the “LN2 Freezer Temperature Log”. Either log must be completed to document the method of temperature monitoring.
  • a vial is immersed directly in a 37°C water bath with continuous, gentle agitation achieved through swirling the vial until the last of the ice just melts.
  • the vial is then removed from the bath and accessed through the needle port and transferred to a 50 cc conical tube.
  • 5 mL of rinse medium (one volume) consisting of Plasmalyte-A + 2.5% HSA is added to the cells at a rate of 0.5 mL per minute, gently agitating the tube by swirling after each addition. Once the last 0.5mL addition has taken place, another 5mL of Plasmalyte-A + 2.5% HSA is added abruptly, resulting in a final volume of 15 mL.
  • Cryo-recovered P4 vBM-MSC cells are produced from human organ and tissue donors and packaged at 5 mL volumes of 10 * 10 6 viable P4 vBM-MSCs/mL in Plasma-Lyte A + 2.5% HSA (Rinse Media) in CellSeal® 5 mL cryovials (Sexton Biotechnologies, Indianapolis, IN). This product and packaging configuration is designed for direct injection with no further manipulation required at the clinical site. After receipt at clinical site, store Cryo-recovered P4 vBM-MSC vials at hypothermic temperatures (2-8°C) in the Cryoport C3 shipper until ready for patient administration.
  • hypothermic temperatures (2-8°C
  • the CellSTACK is removed from the incubator, the media is removed, and the 125mL of TrypLE is added.
  • Viable cells will be counted and resuspended in Plasmalyte + 2.5% HSA to 10M cells per mL concentration, and dispensed into 5 mL plastic syringes.
  • the cells will be transported in a validated container at 20-25°C by trained personnel to the operating room where the Primary Investigator, Amy Lightner or designee will receive the cells.
  • the exam under anesthesia will proceed with removal of setons and debridement and cleansing of the fistula tract.
  • the internal opening of the fistula tract(s) will be ligated closed.
  • the MSCs will delivered via direct injection through a 22G needle.
  • the internal opening (on the anal canal/rectal wall) will be closed with suture ligation (a stitch of the mucosa and submucosa) using absorbable 2-0 PDS suture.
  • suture ligation a stitch of the mucosa and submucosa
  • absorbable 2-0 PDS suture A total of 100 million cells in 10 mL of Plasma-Lyte® A supplemented with HSA (2.5%) will be injected via a 22 gauge needle will then be used for cell injection. 1.4 mL will be injected through the 22 gauge needle just deep to the suture ligation of the internal opening, into the submucosal layer of the bowel wall.
  • the external opening of the fistula tract will be identified and 2.5 mL will be injected along the length of the fistula tract (in parallel to the wall of the tract no deeper than 2 mm from the fistula tract wall) in each of the four quadrants for a total of 10 mL injected around, and in parallel to the wall of the tract). If there are multiple tracts, these volumes will be equally divided between the fistula tracts. Patients may possibly be treated again after 3 months if the fistula is not completely healed and they did not have an allergic reaction after the first treatment.
  • Subjects will be screened at outpatient clinic visits in the colorectal and/or gastroenterology departments and interested qualified subjects will be offered participation in the trial and consented.
  • patients After consent, patients will undergo general health assessment with laboratory workup including a CBC with differential and C-Reactive protein, AST/ALT, Acute Hepatitis Panel, HIV, and creatinine. All blood products and concomitant medications will be reviewed and recorded. If there are indications from the exam and MRI of active abscess at the time of consent, as part of standard of care for fistulizing perianal Crohn’s disease the patient would undergo an exam under anesthesia (EUA) by a colorectal surgeon for drainage of any abscess and placement of seton. If the patient has a seton already in place and there is no sign of abscess on MRI or exam in the prior 30 days, the patient will be offered the MSC injection procedure (Visit 2).
  • Randomization to treatment with MSC versus control with normal saline will be assigned by an electronic data capture system. Fifteen patients with no active abscess and/or who have had successful alleviation of abscess and epithelialization of the fistula tract after seton placement will undergo EUA for placement of 100 million bone marrow derived allogeneic MSCs.
  • the dominant tract is the tract with the largest diameter opening which is assessed at the time of exam under anesthesia with a fistula probe, or if the diameter is equivalent, the longest fistula tract length.
  • the internal opening (on the anal canal/rectal side) will be closed with suture ligation (a stitch of the mucosa and submucosa) using absorbable 2- 0 PDS suture.
  • suture ligation a stitch of the mucosa and submucosa
  • absorbable 2- 0 PDS suture A total of 100 million cells in 10 mL of Plasma-Lyte® A supplemented with human serum albumin (2.5%) and dimethyl sulfoxide (2.5%) will be prepared per MSC Instructions for Use (IFU).
  • the solution will be drawn into three separate 5 mL syringes using a 14-gauge needle. A 20-gauge needle will then be used for cell injection.
  • 1.4 mL will be injected through the 20-gauge needle just deep to the suture ligation of the internal opening, into the submucosal layer of the bowel wall.
  • the external opening of the fistula tract will be identified and 2.5 mL will be injected along the length of the fistula tract (in parallel to the wall of the tract no deeper than 2 mm from the fistula tract wall) in each of the four quadrants for a total of 10 mL injected around, and in parallel to the wall of the tract). If there are multiple tracts, these volumes will be equally divided between the fistula tracts. If the patient is randomized into the control arm, the patients will still have a suture ligation of the internal opening and injection of normal saline rather than the MSC product.
  • Follow up visits may include collecting medical surgery history; a general and perianal exam with vital signs, including assessing clinical healing by physical exam of the perianal area and compression of the external opening of the fistula tract to assess for drainage; PDAI score; Wexner Incontinence Score; EQ-5D; IBD-PRTI; IBDQ, SF-36; laboratory studies including CBC with differential and CRP; and concomitant medications; and recording any adverse events.

Abstract

Provided are methods and compositions for the treatment of perianal fistula in Crohn's disease. Said compositions and methods comprise injecting at least 10 million human mesenchymal stem cells (MSCs) into the fistula, wherein the MSCs are obtained from a non-living source such as the vertebral bodies of human cadaveric donors.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF PERIANAL FISTULA IN CROHN’S DISEASE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/130,273, filed December 23, 2020. The entire contents the priority application is expressly incorporated herein by reference.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with the support of the United States government under Contract number 5R44AI129444 from the National Institute of Allergy and Infectious Diseases, 2R44HL142418 from the National Heart, Lung and Blood Institute, and 1U01 AI138334 from the National Institute of Allergy and Infectious Diseases.
BACKGROUND
[0003] Crohn’s disease (CD), a chronic inflammatory disease of the gastrointestinal tract. At least 26% of patients with CD will develop perianal fistulas in the first two decades following diagnosis particularly those with colonic and rectal involvement. These patients experience significant pain, persistent drainage, recurrent perianal abscess, and ongoing need to access medical care. There exists an unmet need for therapeutic methods that treat perianal fistulas associated with Crohn’s Disease.
SUMMARY
[0004] An aspect of the present disclosure is a method for treating a perianal fistula in a subject. Th method comprising injecting into the perianal fistula a first dose of a composition comprising more than about 10 million human mesenchymal stem cells (MSCs) obtained from a non-living source.
[0005] In some embodiments, the subject is afflicted with an autoimmune disease, e.g., associated with Crohn’s Disease.
[0006] In various embodiments, the method further comprises injecting into the fistula an at least second dose of a composition comprising more than about 10 million MSCs. In some cases, the first dose and/or the at least second dose independently comprise more than about 50 million MSCs. In some cases, the first dose and/or the at least second dose independently comprise more than about 100 million MSCs. [0007] In embodiments, the at least second dose is administered about one month, two months, three months, four months, five months, or six months after the first dose.
[0008] In some embodiments, the subject has an at least second perianal fistula. The at least second perianal fistula may be injected with a first dose of a composition comprising more than about 10 million MSCs. The at least second perianal fistula may be injected with an at least second dose of a composition comprising more than about 10 million MSCs. The at least second fistula may be injected with a first dose of a composition comprising more than about 50 million MSCs and/or an at least second dose of a composition comprising more than about 50 million MSCs.
[0009] In various embodiments, the MSCs are allogenic to the subject.
[0010] In embodiments, the MSCs are HLA matched or mis-matched to the subject. The MSCs and subject may have fewer than six HLA matches out of eight HLA markers, fewer than four HLA matches out of six HLA markers, or fewer than five HLA matches out of ten HLA markers.
[0011] In some embodiments, the MSCs are obtained from one or more vertebral bodies. [0012] In various embodiments, the MSCs are obtained from a single donor.
[0013] In embodiments, the composition comprises from about 0.5 % CD45+ cells to about 10 % CD45+ cells, e.g., the composition comprises less than about 5% CD45+ cells.
[0014] In some embodiments, the composition comprises from about 70 % CD105+ cells to about 100 % CD105+ cells, e.g., the composition comprises at least 90% CD105+ cells.
[0015] In embodiments, the composition comprises from about 70 % CD 166+ cells to about 100 % CD166+ cells, e.g., the composition comprises at least about 90% CD166+ cells. [0016] In embodiments, the MSCs are vertebral body bone marrow MSCs (vBM-MSCs).
[0017] In some embodiments, the MSCs are vertebral body bone adherent MSCs (vBA- MSCs). In some cases, the vBA-MSCs are separated from a vertebral body or a fragment thereof by incubation of the vertebral body or the fragment thereof in a collagenase solution. [0018] In embodiments, the MSCs are vertebral body bone adherent MSCs (vBA-MSCs). [0019] In embodiments, the MSCs are both vBA-MSCs and vBM-MSCs.
[0020] In some embodiments, the MSCs have been cultured for at least one passage, at least two passages, at least three passages, at least four passages, or at least five passages.
[0021] In various embodiments, the MSCs have been cryopreserved before injecting into the perianal fistula. In some cases, the MSCs have been warmed to a temperature of about 0 °C to about 4 °C at least 12 hours before injecting into the perianal fistula. The MSCs may have been warmed to a temperature of about 0 °C to about 4 °C at least 24 hours before inj ecting into the perianal fistula. In some cases, after being cryopreserved, the MSCs are contacted with a growth medium at a suitable temperature and suitable time sufficient to provide an at least 25% to at least 250% increase in cell number. In embodiments, the MSCs have been warmed to a temperature of about 0 °C to about 4 °C at least two days, three days, four days, five days, six days, or a week before injecting into the perianal fistula.
[0022] In embodiments, the MSCs are warmed to about room temperature before injecting into the perianal fistula.
[0023] In some embodiments, the MSCs are warmed to about body temperature before injecting into the perianal fistula.
[0024] In various embodiments, the composition comprising the MSCs includes two or more of Plasma-Lyte® A, Human Serum Albumin (HSA), and DMSO, e.g., the composition comprising the MSCs includes Plasma-Lyte® A, HSA, and DMSO.
[0025] In embodiments, the composition comprising the MSCs includes at least about 2 million cells per ml, e.g., the composition comprising the MSCs includes at least about 3 million cells per ml, 4 million cells per ml, 5 million cells per ml, 6 million cells per ml, 7 million cells per ml, 8 million cells per ml, 9 million cells per ml, 10 million cells per ml, 15 million cells per ml, 20 million cells per ml, 30 million cells per ml, 40 million cells per ml, or 50 million cells per ml.
[0026] In some embodiments, the composition comprising the MSCs includes at least about 2 million MSCs per ml, e.g., the composition comprising the MSCs includes at least about 3 million MSCs per ml, 4 million MSCs per ml, 5 million MSCs per ml, 6 million MSCs per ml, 7 million MSCs per ml, 8 million MSCs per ml, 9 million MSCs per ml, 10 million MSCs per ml, 15 million MSCs per ml, 20 million MSCs per ml, 30 million MSCs per ml, 40 million MSCs per ml, or 50 million MSCs per ml.
[0027] In various embodiments, the composition has an endotoxin level of less than about 1, 2, 3, 4, or 5 Endotoxin Units (EU)/ml, e.g., the composition has an endotoxin level of less than about 2 EU/ml.
[0028] In embodiments, the injecting is via a 22G needle.
[0029] In some embodiments, each dose comprises at least about 1 ml of the composition, e.g., each dose comprises at least about 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml of the composition.
[0030] In embodiments, the injecting is into the submucosal layer of the bowel wall.
[0031] In embodiments, the injecting is along the length of the fistula tract and/or in parallel to the wall of the tract and/or no deeper than 2 mm from the fistula tract wall.
[0032] In some embodiments, the subject has received a previous medical and/or surgical therapy for treating perianal fistula. In some cases, the subject has not responded fully to the previous medical and/or surgical therapy for treating perianal fistula. The previous medical therapy may comprise one or more of systemic immune suppression (e.g. azathioprine, methotrexate, 6- mercaptopurine) or biologic (e.g. anti-TNF, anti-integrin, anti-interleukin) therapies. The previous surgical therapy may comprise a surgical repair including seton placement, glue or plug insertion, local tissue flaps, and/or ligation of intersphincteric fistula tract repair.
[0033] In various embodiments, the non-living source from which the MSCs were ultimately obtained from a human cadaver.
[0034] In embodiments, the subject experiences a complete healing as assessed by radiographic healing (MRI with an absence of a fluid collection >2 cm in 3 of 3 dimensions, lack of edema, a lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain) and/or clinical healing (100% cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening).
[0035] In some embodiments, the subject experiences a partial healing as assessed by radiographic healing (MRI with an absence of a fluid collection >2 cm in 2 of 3 dimensions, lack of edema, lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain) and/or clinical healing (Greater than or equal to 50 % cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening).
[0036] Another aspect of the present disclosure is a composition for use in any herein- disclosed method.
[0037] Yet another aspect of the present disclosure is a composition, comprising about at least 10 million human mesenchymal stem cells (MSCs) obtained from a non-living source. The composition is capable of treating a perianal fistula in a subject.
[0038] In various embodiments, the composition comprises less than 5% CD45+ cells.
[0039] In embodiments, the composition comprises at least 90% CD 105+ cells.
[0040] In some embodiments, the composition comprises at least 90% CD 166+ cells.
[0041] In various embodiments, the human MSCs comprise vertebral body bone marrow MSCs (vBM-MSCs), adherent vertebral body MSCs (vBA-MSCs), or both.
[0042] In embodiments, the MSCs have been cultured for at least one passage, at least two passages, at least three passages, at least four passages, or at least five passages.
[0043] In some embodiments, the MSCs are obtained from one or more vertebral bodies. [0044] In various embodiments, the MSCs are obtained from a single donor.
[0045] In embodiments, the MSCs have been cryopreserved. In some cases, the MSCs have been warmed to a temperature of about 0 °C to about 4 °C at least 12 hour, 24 hours, two days, three days, four days, five days, six days, or a week before use in treating a perianal fistula in a subject. The MSCs may be warmed to about room temperature before use in treating a perianal fistula in a subject. The MSCs may be warmed to about body temperature before use in treating a perianal fistula in a subject.
[0046] In some embodiments, the composition comprises at least about 2 million cells per ml, 3 million cells per ml, 4 million cells per ml, 5 million cells per ml, 6 million cells per ml, 7 million cells per ml, 8 million cells per ml, 9 million cells per ml, 10 million cells per ml, 15 million cells per ml, 20 million cells per ml, 30 million cells per ml, 40 million cells per ml, or 50 million cells per ml. In some cases, the at least about 2 million cells, 3 million cells, 4 million cells, 5 million cells, 6 million cells, 7 million cells, 8 million cells, 9 million cells, 10 million cells, 15 million cells, 20 million cells, 30 million cells, 40 million cells, or 50 million cells are MSCs.
[0047] In embodiments, the MSCs were ultimately obtained from a human cadaver.
INCORPORATION BY REFERENCE
[0048] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1 is a schematic showing an illustrative manufacturing process for mesenchymal stromal cells (MSCs).
[0050] FIG. 2 depicts surface marker expression of cells derived from previously expanded live donor BM-MSC (Ex LD BM-MSC), freshly isolated live donor BM-MSC (LD BM-MSC) and deceased donor BM-MSC (DD BM-MSC).
[0051] FIG. 3 depicts the population double times of cells derived from previously expanded live donor BM-MSC (Ex LD BM-MSC), freshly isolated live donor BM-MSC (LD BM-MSC) and deceased donor BM-MSC (DD BM-MSC). [0052] FIG. 4 depicts the CFU-F potential of cells derived from previously expanded live donor BM-MSC (Ex LD BM-MSC), freshly isolated live donor BM-MSC (LD BM-MSC) and deceased donor BM-MS. (DD BM-MSC).
[0053] FIG. 5 depicts the ability of cells derived from previously expanded live donor BM- MSC (Ex LD BM-MSC), freshly isolated live donor BM-MSC (LD BM-MSC) and deceased donor BM-MSC (DD BM-MSC) to form adipocytes, chrondrocyes and osetocytes.
DETAILED DESCRIPTION
[0054] Provided herein are methods and compositions for treating a perianal fistula in a subject. The methods comprise injecting into the perianal fistula a first dose of a composition comprising more than about 10 million human mesenchymal stem cells (MSCs) obtained from a non-living source.
INTRODUCTION
[0055] Thirty-seven percent of patients with perianal fistulizing Crohn’s disease experience refractory disease. As a result, patients cycle through numerous immunosuppressive medications that can have significant side effects, and >90% undergo multiple surgical interventions putting them at risk of incontinence. While up to 56% of patients can achieve healing with infliximab and seton placement and 64% with optimized tissue flaps, 40% of patients are left with active disease, facing a lifetime of debilitating morbidity or, alternatively, a proctectomy.
[0056] The successful use of mesenchymal stem cells (MSCs) for the treatment of a refractory rectovaginal fistula in the setting of CD was first reported in 2003. These promising results generated a wave of phase I, phase II, and phase III trials to study the safety and efficacy of using MSCs to treat perianal CD. Despite the heterogeneity in protocols using allogeneic or autologous MSCs, derived from both bone marrow or adipose tissue, administered at various doses, delivered as a singular or repeat injection, delivered with or without scaffolding, the results of all completed trials have been encouraging with regard to both safety and efficacy. However, expansion of clinical trials into clinical practice is limited by the required institutional infrastructure for cell-based therapeutics, limited stability of cells on transfer from a GMP grade lab to the treating physician, and prohibitive cost in treating large numbers of patients. Therefore, the expansion of regenerative products with anti-inflammatory and angiogenic properties for the treatment of perianal fistulizing CD offers a promising alternative to drug based therapy.
[0057] Human bone marrow contains a population of nonhematopoietic progenitor cells, referred to as mesenchymal stem/stromal cells (MSCs). Bone marrow (BM)-MSCs are multipotent cells that reside in the perivascular space in bone marrow, and play key roles in supporting hematopoiesis and mediating tissue homeostasis and repair. Traditionally BM-MSC have been obtained via percutaneous aspiration of living donor BM from the iliac crest. Deceased human donor vertebral bodies are a much more plentiful source of MSC. MSC within the medullary cavity reside both within the BM and trabecular bone compartments.
[0058] Culture-expanded MSCs are clonogenic and multipotent, with the capacity to differentiate into mesodermal cell types, including bone, cartilage, and fat under defined conditions. MSCs exhibit low immunogenicity: MSCs express low levels of major histocompatibility complex (MHC) class I antigens, and are negative for MHC class II molecule, HLA-DR, and for the co-stimulatory molecules CD40, CD80, and CD86 essential for immune recognition. Functionally, MSCs do not elicit a proliferative response to T cells and do not stimulate clinically significant immune responses following allogeneic transplantation in vivo. These immune characteristics allow MSCs to be used in the allogeneic setting between unrelated, human leukocyte antigen- unmatched donor and recipient.
[0059] MSCs have been shown to attenuate inflammatory and immunological processes relevant to GVHD. MSCs demonstrate immunosuppressive activity in T cell-driven immune responses in animal models of allogenic skin graft rejection and GVHD. In vitro, MSCs suppress T-cell proliferation in response to allo-antigenic and mitogenic stimulation, and stimulate an increase in the regulatory T cell (Treg) population. Data suggest that Tregs play an important role in inhibiting allogeneic T cell response. In co-culture systems, MSCs alter the cytokine secretion profile of immune cells (dendritic cells, naive and effector T cells, natural killer cells), decreasing expression of pro-inflammatory cytokines (e.g. IFNy, TNFa) and increasing secretion of anti-inflammatory cytokines (e.g. IL-4, IL- 10). The immunomodulatory effects of MSCs are attributable, at least in part, to secretion of soluble factors such as PGE2. In addition, MSCs may mediate tissue protection and repair at sites of injury by secretion of soluble factors that are known to mediate processes such as inhibition of apoptotic cell death, recruitment of endogenous stem cell populations and angiogenesis.
Preparation of Bone for Marrow extraction
Processing of Bone [0060] In various embodiments, an Organ Procurement Organization (OPOs) performs evaluation and donor recoveries according to 21 CFR § 1271. A donor screening and monitoring process may include completion and assessment of a Uniform Donor Risk Assessment Interview (UDRAI). The UDRAI comprises of flowcharts, guidance documents, and questionnaires which are used to screen potential donors related to medical history, behavioral history, travel history and social history. Serology testing may be completed in a CLIA/CMS-approved laboratory using FDA-cleared test kits to rule out viral pathogens. Donors should be negative or non-reactive for HIV-1, HIV-2, hepatitis B virus (HBV, surface and core antigen), hepatitis C virus (HCV), Syphilis (Treponema pallidum), human T- lymphotropic virus types 1 and 2 (HTLV-1, HTLV-2), West Nile Virus, Chagas (Trypanosoma cruzi). Toxoplasmosis, and Epstein-Barr Virus (EBV). Donors may be screened for Cytomegalovirus (CMV). In some embodiments, a donor is under 30 years old, non-smoker, and confirmed CMV negative.
[0061] In some embodiments, the methods and composition described herein comprises mesenchymal stem cells (MSCs) obtained from a non-living source. In some embodiments, the MSCs are obtained from a donor bone. In some embodiments, the donor bone is vertebral bodies. However, it is understood that the methods described herein can be used on the ilium, a combination of the vertebral bodies and ilium, or other bones suitable for extraction of bone marrow and cells from the marrow, even donor bones with lower expected yields.
[0062] It is understood that the donor bones can be procured according to fixed protocols for clinical recovery. Bones can be recovered by surgeons or by personnel at a trained OPO (organ procurement organization) using an osteotome and mallet from consented organ and tissue donors. Unprocessed bones are preferably wrapped in sponges and towels soaked in saline to ensure moisture retention during hypothermic shipment on wet ice at a temperature of 0 to 10 °F to a processing facility.
[0063] The process for preparing the donor bone can occur soon after the bone is obtained from the deceased donor or can occur after the donor bone has been shipped in a hypothermic environment to a processing facility. Since the donor bone can experience prolonged periods of ischemia during recovery and shipment to the processing facility, care must be taken to track the length and type of ischemia — e.g., warm ischemia and cold ischemia. As described in more detail herein, bone subject to predetermined periods of warm ischemia and/or cold ischemia are suitable for obtaining meaningful quantities of viable bone marrow cells.
[0064] During the processing of the donor bone, the bone is debrided in an ISO-5 (class 100) environment (biosafety cabinet) with an ISO-7 (class 10,000) background (clean room), with special care taken to sterilize the bag containing the donor bone, such as by spraying with 70% isopropanol. In one embodiment, the debridement is conducted manually using scalpels, osteotomes and gouges. In processing vertebrae, typically a spinal segment including multiple vertebral levels will be provided. In a typical case, the spine segment runs from T8 to L5, for ten vertebral bodies. During initial debridement of the spinal segment, when enough soft tissue has been removed to visualize the pedicles, the pedicles are removed using either a tissue processing band saw or a bone saw, such as the Stryker System 6 Saw (Stryker, Kalamazoo, MI), or with a hand tool. Special care is taken to avoid breaching the cortical bone which would expose the cancellous bone, to ensure that the hypoxic cancellous bone marrow remains protected throughout the entire debriding process. The anterior element of the vertebral bodies remain, while the pedicles and posterior elements are discarded.
[0065] Using a boning knife or tissue processing band saw, the vertebral bodies (VB) are separated at the intervertebral discs. The intervertebral disc and soft tissue remaining on each vertebral body is removed with a scalpel, scissors and/or osteotomes, leaving clean, separated VBs. In the case of donor ilium, the soft tissue can be removed with gouges and a scalpel, with special care again taken to ensure that the cortical bone is not breached. Any anatomical pathologies or injuries of the bone are noted and recorded as part of the batch record for the marrow ultimately obtained from the bones. Bones damaged during the recovery process are discarded.
[0066] In some cases, cadaver bones undergo a “pre-processing” to reduce contaminants carried by the cadaver bone and which risk transferring the contamination to the facility that the bone is processed. In these cases, two technicians perform different aspects of the pre-processing. A first technician opens a package containing harvested cadaver bones, preferably contained in a sealed, inner bag. The second technician, wearing sterile gloves, removes the cadaver bone from the package and places the tissue in a first (rinse) basin. The second technician scrubs all surfaces of cadaver bone vigorously with an about 4% chi orhexi dine gluconate solution for about 3 minutes while in or above the rinse basin. The first technician, wearing sterile gloves, pours sterile saline onto the scrubbed cadaver bone, with the runoff being captured in the rinse basin. A sufficient amount of saline is poured onto the cadaver bone to rinse all of its surfaces. The rinsed cadaver bone is then placed on a sterile cloth adjacent to the rinse basin. The saline rinse may be repeated as necessary. Alcohol, e.g., 70% isopropyl alcohol, is poured over the cadaver bone. A sufficient amount of alcohol is poured onto the cadaver bone to contact all of its surfaces. The alcohol runoff is captured in the rinse basin. The cadaver bone is placed in an open container which is sprayed with alcohol and, then the open container and bone is transferred to a hood, where further processing of the bone can take place.
[0067] An aspect of the present disclosure comprises a method for processing bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone from a deceased donor; contacting the bone with a bleach solution for at least about 10 minutes to at least about 25 minutes, wherein the bone is submerged in the bleach solution; extracting the bone marrow or the derivative thereof from the bone, wherein at least 90% of CD34+ cells comprised in the bone marrow or the derivative thereof are viable. In some embodiments, the bone marrow or derivative thereof is contacted with the bleach solution for at least about 25 minutes. In some embodiments, the bleach solution comprises 10% bleach. In some embodiments, the bone is a vertebral body. In some embodiments, the hydrogen peroxide is a 3% hydrogen peroxide solution. In some embodiments, the method further comprises transferring the bleached bone product from a container comprising the bleach solution to a container containing the hydrogen peroxide solution. In some embodiments, the method further comprises a step of agitating the bleached bone product within the hydrogen peroxide solution. In some embodiments, the submerging the bleached bone product in a solution comprising hydrogen peroxide comprises: submerging the bleached bone product in a container containing the hydrogen peroxide solution; detecting foam or froth associated with the bleached bone product; and repeating the submerging until no foam or froth is detected. In some embodiments, the method further comprises manually removing soft tissue from a bleached bone product that is associated with foam or froth. In some embodiments, an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance visibility of any foam or froth associated with the bleached bone product.
[0068] In some embodiments, the bone in the bleach solution are agitated (e.g., shaken).
[0069] The VBs are placed into a sterile bag and submerged in a 10% bleach solution, yielding a concentration of 5,000 ppm free chlorine, for a predetermined period, typically 5 or more minutes. Bleach has a broad spectrum of anti-microbial activity, does not leave a toxic residue, is unaffected by water hardness and is fast acting.
[0070] Bone marrow from each group of VBs processed at different duration of bleach treatment can be tested by flow cytometry to assess the viability of the cells isolated from the bone marrow. Soaking the VBs for more than 10 minutes yields no significant difference in cell viability compared to when the VBs are soaked for up to 25 minutes. However, without wishing to be bound by theory, an increase in bleaching time improves the ultimate product. For example, increasing the soaking of the VBs in bleach for longer period of time allows the bleach to fill the cavity or crevice of the VBs to further decontaminate or sterilize the VBs. [0071] Bone marrow from each group of VBs processed at different duration of bleach treatment can be tested by flow cytometry to assess the viability of the cells isolated from the bone marrow (Table 1) As seen from Table, soaking the VBs for more than 10 minutes yields no significant difference in cell viability compared to when the VBs are soaked for up to 25 minutes.
Table 1. Bleach Soak of Vertebral Bodies
Figure imgf000012_0001
[0072] In some embodiments, the bleach treatment comprises using 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or higher percentage of bleach. In some embodiments, the bleach treatment comprises contacting the VBs with bleach for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11, minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, or longer duration. In some embodiments, the viability of the bone marrow cells isolated from the VBs treated with the bleach treatment is not decreased at any duration of bleach treatment described herein compared to bone marrow cells isolated from the VBs without the bleach treatment. In some embodiments, the viability of the bone marrow cells isolated from the VBs treated with 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, or longer duration of the bleach treatment is not decreased or is decreased by less than 3% compared to the viability of the bone marrow cells isolated from the VBs treated with the 10 minutes bleach treatment. In some embodiments, the viability of the bone marrow cells isolated from the VBs treated with more than 10 minutes decreased by less than 2% compared to the viability of the bone marrow cells isolated from the VBs treated with the 10 minutes bleach treatment. In some embodiments, the viability of the bone marrow cells isolated from the VBs treated with more than 10 minutes decreased by less than 1% compared to the viability of the bone marrow cells isolated from the VBs treated with the 10 minutes bleach treatment.
[0073] Interestingly, bleach treatment provides surface sterilization of the bone, but does not penetrate the BM-containing compartment. Therefore, the bleach treatments disclosed herein do not substantially reduce the yield of viable cells obtained from BM.
[0074] In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least about 80% to about 95%. In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95%. In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least at least about 80%, about 85%, or about 90%. In some embodiments, the percentage of viable CD34+ cells comprised in the bone marrow or derivative thereof extracted from the bone submerged in bleach is at least at most about 85%, about 90%, or about 95%.
[0075] At the end of the bleaching period, the bones are transferred to another sterile bag and submerged in a 3% hydrogen peroxide (H2O2) solution. In some cases, the H2O2 solution comprises PLASMA-LYTE™ (a multiple electrolyte injection that is a sterile, nonpyrogenic isotonic solution that is a base source of water and electrolyte-balanced crystalloids for the cells, obtained from Baxter Healthcare, Ltd.). In some cases, the H2O2 solution comprises PLASMA- LYTE™ and Human Serum Albumin (HSA) which is a stabilizing reagent and storage agent (it may be diluted in the H2O2 solution to achieve 2.5% HSA). The bag is closed and shaken briefly to ensure that the entire surface of the bone is in contact with the solution. Most living cells include catalase, which is an enzyme that catalyzes the breakdown of H2O2 into H2O and O2. This breakdown manifests as foam or froth when the H2O2 solution contacts soft tissue but not bone. The foam level can be observed as an indication of the amount of soft tissue remaining on the bone. This observation can be performed manually by a human processor or, in another embodiment, by an automated processor. The automated processor incorporates a visualization device, such as a camera, and object recognition software that can determine foam levels within the bag. The addition of an inert contrast dye can help the human or automated processor detect the foam level. If any foam or froth is observed, the bone is returned for further processing to remove all of the remaining soft tissue from the bone. Once the VBs or ilium has been cleaned of all soft tissue, the bones are transferred to a new sterile bag. The bag is filled with IL of PLASMA-LYTE™ (multiple electrolytes injection obtained from Baxter Healthcare, Ltd.), or other suitable sterile, nonpyrogenic isotonic solution. The bag is closed and shaken briefly to ensure that the entire bone is contacted with the PLASMA-LYTE™.
[0076] In some embodiments, the method further comprises a step of agitating the bleached bone product within the hydrogen peroxide solution. In some embodiments, the submerging the bleached bone product in a solution comprising hydrogen peroxide comprises: submerging the bleached bone product in a container containing the hydrogen peroxide solution; detecting foam or froth associated with the bleached bone product; and repeating the submerging until no foam or froth is detected. In some embodiments, the method further comprises manually removing soft tissue from a bleached bone product that is associated with foam or froth. In some embodiments, an inert contrast dye is added to the solution comprising hydrogen peroxide to enhance visibility of any foam or froth associated with the bleached bone product. [0077] The bleaching step and the hydrogen peroxide steps may be repeated multiple times. [0078] Without wishing to be bound by theory, it is believed that the H2O2 solution not only helps surface sterilize the bone, it helps break down any residual bleach into salt, oxygen, and water. [0079] After the surface sterilization, the cadaver bone may be rinsed with water, a saline, or with a cryoprotectant solution. Then the surface sterilized cadaver bone may be placed in a closed container comprising a cryoprotectant solution and the pressure is reduced.
Predicting Cell Viability Based on Ischemia Time
[0080] Ischemia time of the donor bone impacts the viability of the cells extracted using the processes described herein. According to the present disclosure, total ischemia is defined as the interval starting at time of death (the point at which the donor's arterial system was cross-clamped and circulation ceased) and ending with the start of the recovery of cells from the bone. For purposes of statistical modeling, this total interval can be separated into three successive and mutually exclusive time components: (a) Warm Ischemia Time (WIT) - beginning at time of death and ending either when bones are recovered and packed on ice or when the body is placed in a cooler; (b) Body Cooling Time (BCT) - beginning when the body is placed in the cooler and ending when bones are packed on ice; and (c) Cold Ischemia Time (CIT) - beginning when bones are packed on ice and ending when processing begins for extraction of cells, such as HSPCs. Thus, Total Ischemia Time = (WIT) + (BCT) + (CIT). For cases where whole-body cooling is not used, BCT is zero and Total Ischemia Time = (WIT) + (CIT).
[0081] In addition to Total Ischemia Time, a variable corresponding to processing experience can be incorporated into the viability determination. It is known that learning curves exert significant effects on outcomes, so to control for this fact a variable EXP can be defined as the number of donors processed prior to the current donor — i.e., for the 1th donor, EXP = i — 1. Other variables can include bone type (such as vertebral bodies and ilia), donor sex and donor age.
[0082] In one aspect, the outcome variables are: the proportion of a particular cell population, such as CD34+ cells, that are viable, the total number of colony forming units (CFUs) per 105 nucleated cells detected following cell processing, and the number of CFU granulocyte macrophages (CFU-GM) detected per 105 nucleated cells.
[0083] According to the present disclosure, an ordinary least squares (OLS) beta regression model can be used to predict the outcome variables, with linear regression models used for CFU and CFU-GM and a beta regression model used for the proportion of viable CD34+ cells, or %CD34+, where 0 < (%CD34+) < 1. The beta regression equation for predicting %CD34+ is:
[0084] The regression models are based on un-adjusted models that only account for the ischemia-based variables and not the experience, bone type, donor sex and donor age variables. A fully adjusted model for %CD34+ that accounts for all of the variables. The results of these models are depicted in Tables 2-4.
Table 2. %CD34+ values for the coefficients
Figure imgf000015_0001
Table 3. CFU values for the coefficients
Figure imgf000015_0002
Figure imgf000016_0001
[0085] The coefficient Pi attempts to quantify the effect of the number of donors processed (i.e., experience) on cell quantity and viability. In the fully adjusted CFU model, coefficient P2 corresponds to the experience at a particular facility based on the assumption that facilities can have different learning trajectories. Either or both of these coefficients may be modified or even eliminated.
Table 4. CFU -GM values for the coefficients
Figure imgf000016_0002
[0086] Applying these models to observed data can be used to determine the effect of ischemia time variables on %CD34+ on the amount of CFU-GM. The data in these tables can be used to decide whether a particular donor bone can yield sufficient cells to warrant further processing of the donor bone. In other words, the predictive models can be used to establish ischemia tolerance limits and HSPC quality acceptance criteria. For instance, with respect to the %CD34+ outcome variable, predicted values of over 80% may be required in order to consider the particular donor bone.
[0087] The models described above suggest that acceptable levels of HSPC quality are achievable despite the prolonged ischemia times that are inevitable when bones must be procured by geographically-dispersed OPOs and shipped long distances to processing centers. Even under such conditions, favorable combinations of warm- and cold-ischemia times can be achieved, enabling %CD34+ viabilities in the range of 80-90%. The models also suggest that refrigerating the body prior to bone recovery, a practice that is common in the recovery of tissues, is less beneficial in the context of bone marrow recovery. For instance, when wholebody cooling was used, CD34+ viability averaged 72.75%, whereas when body cooling was not used, the average was just under 90%. These models suggest that an optimal practice would be to dispense with body cooling and move recovered bone as quickly as possible to a cold ischemic environment. The models further suggest that limiting WIT (warm ischemia time) to less than eight (8) hours and CIT (cold ischemia time) to less than 40 hours optimizes the opportunity to recover meaningful quantities of viable cells from donor bone. In some embodiments, more than of 40 hours of CIT are acceptable, e.g., 50 or 60 hours.
[0088] The models disclosed herein predict viability in which an 80% CD34+ (or at least 70%) cell viability threshold is determined to be acceptable.
[0089] Further details of the method for predicting cell viability of the present disclosure are found in Woods et al., “Ischemia considerations for the development of an organ and tissue donor derived bone marrow bank.” J Transl Med 18, 300 (2020). https://doi.Org/10.l 186/s 12967- 020-02470-1, the entire disclosure of which is incorporated herein by reference. See, in particular, the table disclosed therein which identify specific combinations of WIT and CIT and BCT which provide satisfactory cell yields.
Cryoprotecting a bone
[0090] Surprisingly, by immersing a cadaver bone in a closed container of cryoprotectant and applying an intermittent vacuum to the closed container, the cryoprotectant infiltrates the cadaver bone significantly more rapidly that would occur by passive diffusion. Such effective infiltration of cryoprotectant contributes to reduced ice crystal formation during freezing of the cadaver bone and, ultimately, extraction of viable bone marrow cells that have replicative potential.
[0091] Cadaver bone can be contacted with a cryoprotectant solution for a length of time and under conditions sufficient to allow infiltration of a cryoprotectant solution into the cadaver bone. Methods for cry opreserving bone is described below and elsewhere. In some cases, the conditions sufficient to allow infiltration of a cryoprotectant solution involve use of vacuum-assisted infiltration of a cryoprotectant into a cadaver bone as disclosed in PCT/US2021/042064, the contents of which are incorporated by reference in its entirety. In other cases, cadaver bone is submerged in a cryoprotectant solution and without use of a vacuum.
[0092] An aspect of the present disclosure is a method for cry opreserving a cadaver bone using vacuum to assist infiltration of a cryoprotectant into the cadaver bone. The method comprises steps of: (a) placing a cadaver bone in a closed container comprising a cryoprotectant solution; (b) reducing the pressure in the closed container, and optionally, holding the closed container at reduced pressure, to remove at least a portion of the water present in the cadaver bone; (c) raising the pressure in the closed container and holding the closed container at a raised pressure to allow infiltration of the cryoprotectant solution into the cadaver bone; (d) removing the cadaver bone from the closed container; and (e) chilling the cadaver bone to a temperature at least below 0 °C, thereby cryopreserving the cadaver bone. [0093] Surprisingly, by immersing a cadaver bone in a closed container of cryoprotectant and applying an intermittent vacuum to the closed container, the cryoprotectant infiltrates the cadaver bone significantly more rapidly that would occur by passive diffusion. With respect to PCT/US2021/042064, compare FIG. 2 with FIG. 4A and FIG. 4B and FIG. 3 with FIG. 5. Such effective infiltration of cryoprotectant contributes to reduced ice crystal formation during freezing of the cadaver bone and, ultimately, extraction of viable bone marrow cells that have replicative potential.
[0094] Steps (b) and (c) may occur only once or steps (b) and (c) may be repeated at least once, at least twice, at least four times, at least five times, or at least six times. In some embodiments, repeating the reduced pressure and the raised pressure may increase infiltration of a cryoprotectant into a cadaver bone. See, e.g., FIG. 5 of PCT/US2021/042064. In other embodiments, there is sufficient infiltration of cryoprotectant into a cadaver bone after a single cycle of reduced pressure and raised pressure.
[0095] In various embodiments, a cadaver bone (e.g., vertebral body) is bisected, cut into quarters, or more extensively divided prior to vacuum-assisted infiltration of the cryoprotectant.
[0096] The reduced pressure in the closed container may any pressure value from about -400 mmHg to about -800 mmHg. The pressure requirement should be sufficient to remove at least a portion of the water present in the cadaver bone. The reduced pressure in the closed container may have a value of about -400 mmHg, -425 mmHg, -450 mmHg, -475 mmHg, -500 mmHg, - 525 mmHg, -550 mmHg, -575 mmHg, -600 mmHg, -625 mmHg, -650 mmHg, -675 mmHg, - 700 mmHg, -725 mmHg, -750 mmHg, -775 mmHg, or - 800 mmHg. In some embodiments, the reduced pressure in the closed container is from about -400 mmHg to -500 mmHg.
[0097] In some embodiments, it takes from about one minute to about 10 minutes for the closed container to reach a desired reduced pressure once the pressure in the closed container begins reducing. As examples, the closed container is may take less than 1 minute, about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or about 10 minutes and any length of time in between (e.g., a fraction of a minute, e.g., about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds, and any number of seconds therebetween) to reach the desired reduced pressure. In some embodiments, the cadaver bone reaches the desired reduced pressure rapidly, e.g., from about one second to about one minute.
[0098] In some embodiments, the cadaver bone is held at the reduced pressure once the reduced pressure has been reached. The cadaver bone may be held for from less than one minute to about 50 minutes. As examples, the closed container is held at reduced pressure for less than one minute, about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 minutes, 49 minutes, or about 50 minutes and any length of time in between (e.g., a fraction of a minute, e.g., about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds, and any number of seconds therebetween). In some embodiments, the cadaver bone is not held at reduced pressure for any measurable time and instead, the method progresses to step (c) of raising the pressure in the closed container.
[0099] In step (c), the pressure of the closed container is raised until the pressure is from about 0 mmHg to about 760 mmHg. In other words, the pressure is raised to up to standard atmospheric temperature. The exact raised pressure may be any amount within the specified range, e.g., 0 mmHg, 50 mmHg, 100 mmHg, 150 mmHg, 200 mmHg, 250 mmHg, 300 mmHg, 350 mmHg, 400 mmHg, 450 mmHg, 500 mmHg, 550 mmHg, 600 mmHg, 650 mmHg, 700 mmHg, or 750 mmHg. However, the raised pressure must be high enough to allow infiltration of the cryoprotectant solution into the cadaver bone.
[0100] The closed container may be held at the raised pressure for less than about two hours. As examples, for less than one hour, less than one-half hour, about one-half hour, or less time. In some embodiments, the closed container is held at the raised pressure for ten minutes. The duration that the closed container is held at the raised pressure must be long enough to allow infiltration of the cryoprotectant solution into the cadaver bone. As examples, the closed container is held at the raised pressure for about, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, or about 30 minutes, and any length of time in between (e.g., a fraction of a minute, e.g., about 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, about 50 seconds, and any number of seconds therebetween).
[0101] The closed container and the cryoprotectant contained therein may be at room temperature. Alternately, the closed container and the cryoprotectant contained therein may be below room temperature, e.g., as low as 4 °C. The closed container and the cryoprotectant contained therein may be above room temperature, e.g., as high as 37 °C.
[0102] Any suitable cryoprotectant may be used in a cryoprotectant solution. Examples of cryoprotectant include dimethyl sulfoxide (also known as DMSO, C2H6OS, and ME2SO); 1, 2 propane diol; propylene glycol; ethylene glycol; glycerol; foramamide; ethanediol or butane 2, 3 diol; hydroxyethyl starch (HES); dextran; sucrose; trehalose; lactose; raffinose; ribotol; mannitol; and polyvinylpyrrolidone (PVP). In some embodiments, the cryoprotectant is DMSO. The cryoprotectant solution may comprise from about 10% DMSO to about 30% DMSO, e.g., about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or about 30% DMSO. The cryoprotectant solution may comprise about 20% DMSO. In some embodiments, a higher percentage of cryoprotectant is preferred, e.g., percentages that are two times higher than equivalent cell suspension values to help drive osmotic penetration.
[0103] The cryoprotectant solution may have water or a saline as base. In some embodiments, the saline is isotonic to human tissues. In embodiments the saline is a 0.9% saline solution. Any commercially-available saline solution may be used: sodium chloride solution, PBS, HEPES, Ringers or Lactate. The saline may be 0.9% sodium chloride.
[0104] The cryoprotectant solution may further comprise a protein. As examples, the protein may be a human albumin (e.g., HSA) or a constituent of a human platelet lysate. An example of a commercially-available human platelet lysate product is Stemulate (from Cook Regentec).
[0105] In some cases, the cryoprotectant solutions comprises about 10% protein, e.g., 10% human platelet lysate or 10% albumin.
[0106] In one case, the cryoprotectant solution comprises about 20% DMSO and about 10% human platelet lysate in 0.9% NaCl.
[0107] In another case, the cryoprotectant solution comprises about 40% DMSO and about 10% human platelet lysate in 0.9% NaCl.
[0108] In yet another case, the cryoprotectant solution comprises about 60% DMSO and about 10% human platelet lysate in 0.9% NaCl.
[0109] In a further case, the cryoprotectant solution comprises about 80% DMSO and about 10% human platelet lysate in 0.9% NaCl.
[0110] In a case, the cryoprotectant solution comprises about 100% DMSO in 0.9% NaCl.
[0111] In any of the above aspects, the method may comprise a step of increasing the pressure in the closed container comprising a cryoprotectant to above 760 mmHg by introducing a compressed gas (e.g., nitrogen, xenon, CO2, argon, H2S, or helium), a gas released by sublimination (e.g., CO2 via dry ice), or a gas provided by evaporation (e.g., nitrogen via liquid nitrogen), thereby permeating gas into the cadaver bone. In embodiments, the gas is CO2 , e.g., compressed CO2. In some embodiments, the gas is nitrogen, e.g., compressed nitrogen. The time required for gas infiltration into a vertebral body is less when the gas is compressed versus a gas obtained by sublimination.
[0112] Alternately, in any of the above-mentioned aspects, rather than placing a cadaver bone in closed container comprising a cryoprotectant solution, the cadaver bone is placed in a closed container that lacks a cryoprotectant solution. In these alternate aspects, the method comprises a step of increasing the pressure in the closed container (which lacks a cryoprotectant solution) to above 760 mmHg by introducing a compressed gas (e.g., nitrogen, xenon, CO2, argon, H2S, or helium), a gas released by sublimination (e.g., CO2 via dry ice), or a gas provided by evaporation (e.g., nitrogen via liquid nitrogen), thereby permeating gas into the cadaver bone. Any method disclosed herein may be adapted by comprising initial steps of placing a cadaver bone in closed container that lacks a cryoprotectant solution and increasing the pressure in the closed container to above 760 mmHg by introducing a compressed gas, a gas released by sublimination, or a gas provided by evaporation; in a later step, a cryoprotectant solution is added to the closed container. In embodiments, the gas is CO2 , e.g., compressed CO2. In some embodiments, the gas is nitrogen, e.g., compressed nitrogen.
[0113] Without wishing to be bound by theory, increasing the pressure in a closed container by introducing a compressed gas (e.g. , nitrogen, xenon, CO2, argon, H2S, or helium) , a gas released by sublimination (e.g., CO2 via dry ice), or a gas provided by evaporation (e.g., nitrogen via liquid nitrogen), promotes infiltration of the cryoprotectant solution into the cadaver bone.
[0114] In some cases, the closed container comprises solid materials, e.g., metal, plastic, or other polymers. In some cases, the closed container comprises a foam material, e.g., Styrofoam.
[0115] In alternate aspects, a cadaver bone is infiltrated with a cryoprotectant without use of a vacuum. Here, an intact vertebral body, a vertebral body that has been bisected, cut into quarters, or more extensively divided is submerged into a cryoprotectant solution for a length of time and under conditions sufficient to allow infiltration of the cryoprotectant solution into the cadaver bone.
[0116] The bone or bone fragment is placed, e.g., submerged, in a cryoprotectant solution and incubated for 1 hour at about 4°C. In some embodiments, the incubation period is about 1 hour to about 3 hours. In some embodiments, the incubation period is about 1 hour to about 1.5 hours, about 1 hour to about 2 hours, about 1 hour to about 2.5 hours, about 1 hour to about 3 hours, about 1.5 hours to about 2 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 3 hours, about 2 hours to about 2.5 hours, about 2 hours to about 3 hours, or about 2.5 hours to about 3 hours. In some embodiments, the incubation period is about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. In some embodiments, the incubation period is at least about 1 hour, about 1.5 hours, about 2 hours, or about 2.5 hours. In some embodiments, the incubation period is at most about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours.
[0117] Any suitable cryoprotectant may be used in a cryoprotectant solution. Examples of cryoprotectant include dimethyl sulfoxide (also known as DMSO, C2H6OS, and ME2SO); 1, 2 propane diol (also known as propylene glycol); ethylene glycol; glycerol; foramamide; ethanediol, butane 2, 3 diol; hydroxyethyl starch (HES); dextran; sucrose; trehalose; lactose; raffinose; ribotol; mannitol; and polyvinylpyrrolidone (PVP). In some embodiments, the cryoprotectant is DMSO. The cryoprotectant solution may comprise from about 5% DMSO to about 100% DMSO, e.g, about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,
33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,
49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or about 100% DMSO. The cryoprotectant solution may comprise about 20% DMSO. In some embodiments, the cryoprotectant solution may comprise about 40% DMSO or 60% DMSO. In some embodiments, a higher percentage of cryoprotectant is preferred, e.g., percentages that are two times higher than equivalent cell suspension values to help drive osmotic penetration.
[0118] The cryoprotectant solution may have water or a saline as base. In some embodiments, the saline is isotonic to human tissues. In embodiments the saline is a 0.9% saline solution. Any commercially available saline solution may be used: sodium chloride solution, PBS, HEPES, Ringers or Lactate. The saline may be 0.9% sodium chloride.
[0119] The cryoprotectant solution may further comprise a protein. As examples, the protein may be a human albumin (e.g., HSA) or a constituent of a human platelet lysate. An example of a commercially available human platelet lysate product is Stemulate™ (from Cook® Regentec).
[0120] In some embodiments, the cryoprotectant solutions comprises about 10% protein, e.g., 10% human platelet lysate or 10% albumin. [0121] In one case, the cryoprotectant solution comprises about 20% DMSO and about 10% human platelet lysate in 0.9% NaCl.
[0122] In another case, the cryoprotectant solution comprises about 40% DMSO and about 10% human platelet lysate in 0.9% NaCl.
[0123] In yet another case, the cryoprotectant solution comprises about 60% DMSO and about 10% human platelet lysate in 0.9% NaCl.
[0124] In a further case, the cryoprotectant solution comprises about 80% DMSO and about 10% human platelet lysate in 0.9% NaCl.
[0125] In a case, the cryoprotectant solution comprises about 100% DMSO in 0.9% NaCl.
Two-Step Chilling of Cadaver Bone
[0126] After the pressure in the closed container comprising the cadaver bone and cryoprotectant solution has been raised and held for a suitable amount of time to allow infiltration of the cryoprotectant solution into the cadaver bone, the cadaver bone, now infiltrated with cryoprotectant, is removed from the closed container.
[0127] The cadaver bone then undergoes an initial chilling period. For this, the cadaver bone is placed in a static minus 80 freezer set at a temperature of colder than about - 60 °C or colder than about -100 °C, e.g., from about - 70 °C to about -80 °C. There, the cadaver bone undergoes an initial chilling period. In some embodiments, the cadaver bone is initially chilled in a static minus 80 freezer set at a temperature of about - 86 °C. Data showing the dynamics of the initial chilling period is shown in PCT/US2021/042064 at FIG. 6A.
[0128] In some cases, the static freezer is set at a range of temperature from about -60 °C, about -65 °C, about -70 °C, about -75 °C, about -80 °C, about -82 °C, about -84 °C, about -86 °C, about -88 °C, about -90 °C, about -95 °C, or about -100 °C. In some cases, the freezer can be set at a range of temperature from at least about -60 °C, about -65 °C, about -70 °C, about - 75 °C, about -80 °C, about -82 °C, about -84 °C, about -86 °C, about -88 °C, about -90 °C, or about -95 °C. In some cases, the freezer can be set at a range of temperature from at most about -65 °C, about -70 °C, about -75 °C, about -80 °C, about -82 °C, about -84 °C, about -86 °C, about -88 °C, about -90 °C, about -95 °C, or about -100 °C.
[0129] The cadaver bone may be initially chilled at a rate of from about -0.3 °C/min to about -5 °C/min. In some embodiments, the cadaver bone is initially chilled at a rate of from about -0.4 °C/min to about -0.9 °C/min. As examples, the initial chilling rate may be about - 0.3 °C/min, -0.4 °C/min, -0.5 °C/min, -0.6 °C/min, -0.7 °C/min, -0.8 °C/min, -0.9 °C/min, to about -1 °C/min. In other examples, the initial chilling rate may be about -1 °C/min, -2 °C/min, -3 °C/min, -4 °C/min, or about -5 °C/min. In these rates, the minus sign
Figure imgf000024_0001
means that the temperature is dropping by the stated amount.
[0130] The duration of the initial chilling period may vary from a few hours to overnight. The time should be sufficient for the cadaver bone to reach a temperature of colder than about -50 °C, e.g., at -60 °C to -80 °C. In some embodiments, the bone reaches the desired temperature in about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or about 12 hours. Is some embodiments, the cadaver bone is initially chilled in the minus 80 freezer for at least 12 hours or at least overnight.
[0131] Without wishing to be bound by theory, it appears that the period of initial chilling in the presence of extracellular ice increases intracellular solute concentrations to an amount that allows intracellular vitrification in the subsequent chilling.
[0132] During the period of initial chilling, the cadaver bone is not held in a static freezer having its temperature set to from about - 5 °C to about - 15 °C and for a period of time between about 1 and about 30 minutes. The cadaver bone may temporarily acquire a temperature of between about - 5 °C and about - 15 ° C, but this occurs as the temperature of the cadaver bone is continuously dropping towards the desired temperature, e.g., colder than about -50 °C.
[0133] Once the cadaver bone has reached the desired temperature, the cadaver bone undergoes a subsequent chilling period. For this, the cadaver bone is placed in liquid nitrogen or in liquid nitrogen vapor, e.g., at a temperature of about -200 °C. Data showing the dynamics of the subsequent chilling period is shown in PCT/US2021/042064 at FIG. 6B. In some embodiments, the subsequent chilling period may occur in a suitable static freezer that is capable of maintaining temperatures equivalent to liquid nitrogen yet without use of liquid nitrogen, e.g., a cryogenic freezer.
[0134] During the subsequently chilling period, the cadaver bone is cooled at a rate of from about -2 °C/min to about -6 °C/min. In some embodiments, the cadaver bone is initially chilled at a rate of about -2 °C/min, -2,2 °C/min, -2,4 °C/min, -2,6 °C/min, -2,8 °C/min, -3 °C/min, -3,2 °C/min, -3,4 °C/min, -3,6 °C/min, -3,8 °C/min, -4 °C/min, -4,2 °C/min, -4,4 °C/min, -4,6 °C/min, -4,8 °C/min, -5 °C/min, -5,2 °C/min, -5,4 °C/min, -5,6 °C/min, -5,8 °C/min, or about -6 °C/min. In these rates, the minus sign (“-”) means that the temperature is dropping by the stated amount.
[0135] The cryopreserved cadaver bone may be held in liquid nitrogen, in liquid nitrogen vapor, or in a suitable static freezer indefinitely. As examples, the cryopreserved cadaver bone may be held for at least a day, at least a week, at least a month, at least a year, at least five years, or at least 20 years. The cryopreserved cadaver bone may be held in liquid nitrogen, in liquid nitrogen vapor, or suitable static freezer for hundreds or thousands of years.
[0136] Without wishing to be bound by theory, the two-step chilling of cadaver bone method, as disclosed herein, improves the viability of the extracted bone marrow cells (hematopoietic stem cells (HSCs; CD34+ cells) and/or mesenchymal stromal/stem cells (MSCs)) relative to methods that do not use the two-step chilling method. Therefore, using the methods of the present disclosure, a greater number of viable cells (HSCs and/or MSCs) are obtained relative to standard methods.
[0137] In some cases, the methods of the present disclosure provide from about 1% more viable cells to about 100% more viable cells, e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,
41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% more viable cells than from methods that do not use two-step chilling, as disclosed herein.
[0138] In some cases, the methods of the present disclosure provide from about 101% more viable cells to about 200% more viable cells, e.g., about 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 10%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%,
145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%,
158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%,
171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%,
184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%,
197%, 198%, 199%, or about 200% more viable cells than from methods that do not use two- step chilling, as disclosed herein.
[0139] In some cases, the methods of the present disclosure provide from about 2-fold more viable cells to about 10-fold more viable cells, e.g., about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, about 10-fold, or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein. As examples, the methods of the present disclosure provide 2-fold to 3-fold, 3-fold to 4-fold, 4-fold to 5-fold, 5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold, or 9-fold to 10-fold more viable cells than from methods that do not use two-step chilling, as disclosed herein.
[0140] In some cases, the methods of the present disclosure provide from about 10-fold more viable cells to about 100-fold more viable cells, e.g., about 10-fold, 20-fold, 30-fold, 40- fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, about 100-fold or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein. As examples, the methods of the present disclosure provide 10-fold to 20-fold, 20-fold to 30-fold, 30-fold to 40-fold, 40-fold to 50-fold, 50-fold to 60-fold, 60-fold to 70-fold, 70-fold to 80-fold, 80-fold to 90-fold, or 90-fold to 100-fold more viable cells than from methods that do not use two-step chilling, as disclosed herein.
[0141] In some cases, the methods of the present disclosure provide from about 100-fold more viable cells to about 1000-fold more viable cells, e.g., about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, about 1000-fold or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein. As examples, the methods of the present disclosure provide 100-fold to 200 fold, 200-fold to 300-fold, 300-fold to 400-fold, 400-fold to 500-fold, 500-fold to 600-fold, 600-fold to 700-fold, 700-fold to 800-fold, 800-fold to 900-fold, or 900-fold to 1000-fold more viable cells than from methods that do not use two-step chilling, as disclosed herein.
[0142] In some cases, the methods of the present disclosure provide from about 1000-fold more viable cells to about 10000-fold more viable cells, e.g., about 1000-fold, 2000-fold, 3000- fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, about 10000-fold or any fold therebetween more viable cells than from methods that do not use two-step chilling, as disclosed herein. As examples, the methods of the present disclosure provide 1000-fold to 200 fold, 2000-fold to 3000-fold, 3000-fold to 4000-fold, 4000-fold to 5000-fold, 5000-fold to 6000-fold, 6000-fold to 7000-fold, 7000-fold to 8000-fold, 8000-fold to 9000-fold, or 9000- fold to 10000-fold more viable cells than from methods that do not use two-step chilling, as disclosed herein.
Methods for Rapidly Warming a Cryopreserved Cadaver Bone
[0143] Another aspect of the present disclosure is a method for rapidly warming cadaver bone for providing bone marrow or a derivative thereof. PCT/US2021/042064 discloses methods for rapidly warming cryopreserved bone; the contents of which are incorporated by reference in its entirety. These disclosed methods may be useful in the methods of the present disclosure.
[0144] In some cases, the method for rapidly warming cadaver bone comprise steps of: obtaining a cryopreserved cadaver bone; dividing the cryopreserved cadaver bone to obtain fragments of the cryopreserved bone; transferring the fragments of the cryopreserved bone into a grinding medium having a temperature of from about 35 °C to 45 °C for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20 °C.
[0145] In some embodiments, the cryopreserved cadaver bone is cryopreserved by an above-described method, e.g., comprising vacuum-infiltration of a cryoprotectant and/or using a two-step chilling method.
[0146] In other embodiments, the cryopreserved cadaver bone is not cryopreserved by an above-described method, e.g., without vacuum-infiltration of a cryoprotectant and/or not using a two-step chilling method.
[0147] In some embodiments, a cryopreserved cadaver bone transferred into a grinding medium without having been divided into fragments. Preferably, the cryopreserved cadaver bone has a temperature of at least below 0 °C when transferred into a grinding medium.
[0148] In alternate embodiments, the method comprises dividing the cryopreserved cadaver bone to obtain fragments of the cryopreserved bone. Preferably, the cryopreserved cadaver bone has a temperature of below 0 °C when dividing into fragments.
[0149] In order to simplify the process and for increased safety to the processing personnel, a custom bone cutting tool as described in US 2019/0343112, which is hereby incorporated by reference in its entirety, is used to divide the cryopreserved cadaver bone into smaller pieces. Another bone cutting tool may be used in combination, or in lieu of the custom bone cutting tool as described in US 2019/0343112.
[0150] The elements of the bone cutting tool are formed of medical grade stainless steel. The steel is preferably hardened steel capable of withstanding the forces required to cut through frozen bone. In the cleaning process, the tool is subjected to steam sterilization, which can be deleterious to the steel. Thus, in one feature of the present disclosure, the surfaces of the stainless-steel elements are passivated to prevent oxidation of the steel elements during sterilization.
[0151] The manual bone-cutting device for dividing the cryopreserved cadaver bone is capable of generating up to 1000 Ibf when less than 50 Ibf is applied. Such a manual bonecutting device comprises: a force transmission mechanism, wherein the force transmission mechanism comprises an elongated force transducing member pivotally coupled to a gear mechanism; and a manually operable handle coupled to an end of the elongated force transducing member, wherein the end is opposite of the gear mechanism. The manual bonecutting device comprises an upper cutting element and/or a lower cutting element. Its upper cutting element and/or lower cutting element each comprises one or more cutting blades that radiate outwards from a central portion of the upper cutting element and/or the lower cutting element. When the one or more cutting blades divide the cryopreserved cadaver bone into fragments that are generally sector-shaped.
[0152] The manual bone-cutting device divides the cryopreserved cadaver bone into fragments of the cryopreserved bone. The fragments of the cryopreserved bone are transferred into a grinding medium having a temperature of from about 35 °C to about 45 °C for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20 °C. Alternately, whole cryopreserved bone, which has not been divided, is transferred into a grinding medium having a temperature of from about 35 °C to about 45 °C for a time sufficient to warm the cadaver bone fragments to a surface temperature of about 20 °C. In some embodiments, the surface temperature of the cadaver bone fragments is higher than 20 °C, e.g., 25 °C or higher.
[0153] A volume of grinding medium is warmed and held at a temperature of from about 35 °C to about 45 °C, for example, by placing a container holding the grinding medium on a hot plate or in a water bath. In some examples, 300ml, 500ml or one liter of grinding medium is used to warm the cadaver bone. Preferably, the grinding medium has a temperature of about 37 °C to about 40 °C when the fragments of the cryopreserved bone are transferred to the grinding medium.
[0154] The cadaver bone fragments are warmed to a surface temperature of about 20 °C at a rate of from about 100 °C/min to about 500 °C/min. Is some embodiments, the warming rate is greater than about 300 °C/min, e.g., about 300 °C/min, 310 °C/min, 320 °C/min, 330 °C/min, 340 °C/min, 350 °C/min, 360 °C/min, 370 °C/min, 380 °C/min, 390 °C/min, 400 °C/min, 410 °C/min, 420 °C/min, 430 °C/min, 440 °C/min, 450 °C/min, 460 °C/min, 470 °C/min, 480 °C/min, 490 °C/min, and about 500 °C/min. In some embodiments, the warming rate is from about 400 °C/min to about 500 °C/min. In some instances, the cadaver bone fragments are warmed to a surface temperature of about 20 °C in less than one minute. In some cases, the cadaver bone fragments are warmed to a surface temperature of about 20 °C in about one minute or more, e.g., about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or about 10 minutes. Data showing the dynamics of the fast warming is shown in PCT/US21/42064 at FIG. 15.
[0155] When whole cadaver bone is warmed in the grinding medium, the warming rate will be slower than when bone fragments are warmed. As examples, the cadaver bone is warmed to a surface temperature of about 20 °C at a rate of from about 100 °C/min to about 250 °C/min.
[0156] Without wishing to be bound by theory, the fast warming rate of the present disclosed methods prevents ice recrystalization during thawing of the bone fragments (or whole cadaver bone) and improves the viability of the extracted bone marrow cells (hematopoietic stem cells (HSCs; CD34+ cells) and/or mesenchymal stromal/stem cells (MSCs)) relative to methods that do not use the rapid warming method. Therefore, using the methods of the present disclosure, a greater number of viable cells (HSCs and/or MSCs) are obtained relative to standard methods.
Extracting the Bone Marrow
[0157] A fresh cadaver vertebral body (VB) or a wanned, previously cryopreserved VB is prepared for grinding.
[0158] In an approach, a saw and/or anvil shears are used to cut the VBs are cut into smaller pieces, such as 1.5 cm2 pieces, that are small enough for fragmenting with a bone grinder. To simplify the process and for increased safety to the processing personnel, a custom bone cutting tool as described in US 2019/0343112, which is hereby incorporated by reference in its entirety, is provided is used to cut the VBs into the smaller pieces. Another custom bone cutting tool can be used in combination, or in lieu of the custom bone cutting tool as described in US 2019/0343112. The additional bone cutting tool is described in US2020/0325451, which is hereby incorporated by reference in its entirety.
[0159] The elements of the bone cutting tool are formed of medical grade stainless steel. The steel is preferably hardened steel capable of withstanding the forces required to cut through bone. In the cleaning process, the tool is subjected to steam sterilization, which can be deleterious to the steel. Thus, in one feature of the present disclosure, the surfaces of the stainless-steel elements are passivated to prevent oxidation of the steel elements during sterilization.
[0160] The pieces produced by the bone cutting tool are immediately placed into a sterile pitcher and submerged in 300-500 ml of a grind media. In one aspect of the present system and method, the grind media uses PLASMA-LYTE™-A as a base with heparin, human serum albumin (HSA), and a nuclease (Merck KGAA Corporation). Heparin is used as an anticoagulant. Other anticoagulants at various quantities can also be used. HSA provides a protein source to prevent cell adherence and adsorption to surfaces, as well as reactive oxygen scavenging. It is noted that conventional grind media utilizes DNase, but for the present disclosure Benzonase® or Denarase® reagent is substituted for DNase™ reagent (Qiagen Sciences LLC). Whereas DNase works only on DNA, modern pharmaceutical biotechnology processing relies on enzymes that can cleave all forms of DNA and RNA, and can reduce the viscosity of the solution in which the cells are suspended. It is noted that IMDM (Iscove's Modified Dulbecco's Media) can substitute for the PLASMA-LYTE™-A, since IMDM is suitable for rapidly proliferating high-density cell cultures and ideal for supporting T- and B-lymphocytes. It is further noted that Denarase® reagent (C-Lecta GmbH) is equivalent to Benzonase reagent in the same quantity in the present process.
[0161] In some embodiments, the amount of heparin in the grind media is about 5 U/ml to about 15 U/ml. In some embodiments, the amount of heparin in the grind media is about 5 U/ml to about 6 U/ml, about 5 U/ml to about 7 U/ml, about 5 U/ml to about 8 U/ml, about 5 U/ml to about 9 U/ml, about 5 U/ml to about 10 U/ml, about 5 U/ml to about 11 U/ml, about 5 U/ml to about 12 U/ml, about 5 U/ml to about 13 U/ml, about 5 U/ml to about 14 U/ml, about 5 U/ml to about 15 U/ml, about 6 U/ml to about 7 U/ml, about 6 U/ml to about 8 U/ml, about 6 U/ml to about 9 U/ml, about 6 U/ml to about 10 U/ml, about 6 U/ml to about 11 U/ml, about 6 U/ml to about 12 U/ml, about 6 U/ml to about 13 U/ml, about 6 U/ml to about 14 U/ml, about 6 U/ml to about 15 U/ml, about 7 U/ml to about 8 U/ml, about 7 U/ml to about 9 U/ml, about 7 U/ml to about 10 U/ml, about 7 U/ml to about 11 U/ml, about 7 U/ml to about 12 U/ml, about 7 U/ml to about 13 U/ml, about 7 U/ml to about 14 U/ml, about 7 U/ml to about 15 U/ml, about 8 U/ml to about 9 U/ml, about 8 U/ml to about 10 U/ml, about 8 U/ml to about 11 U/ml, about 8 U/ml to about 12 U/ml, about 8 U/ml to about 13 U/ml, about 8 U/ml to about 14 U/ml, about 8 U/ml to about 15 U/ml, about 9 U/ml to about 10 U/ml, about 9 U/ml to about 11 U/ml, about 9 U/ml to about 12 U/ml, about 9 U/ml to about 13 U/ml, about 9 U/ml to about 14 U/ml, about 9 U/ml to about 15 U/ml, about 10 U/ml to about 11 U/ml, about 10 U/ml to about 12 U/ml, about 10 U/ml to about 13 U/ml, about 10 U/ml to about 14 U/ml, about 10 U/ml to about 15 U/ml, about 11 U/ml to about 12 U/ml, about 11 U/ml to about 13 U/ml, about 11 U/ml to about 14 U/ml, about 11 U/ml to about 15 U/ml, about 12 U/ml to about 13 U/ml, about 12 U/ml to about 14 U/ml, about 12 U/ml to about 15 U/ml, about 13 U/ml to about 14 U/ml, about 13 U/ml to about 15 U/ml, or about 14 U/ml to about 15 U/ml. In some embodiments, the amount of heparin in the grind media is about 5 U/ml, about 6 U/ml, about 7 U/ml, about 8 U/ml, about 9 U/ml, about 10 U/ml, about 11 U/ml, about 12 U/ml, about 13 U/ml, about 14 U/ml, or about 15 U/ml. In some embodiments, the amount of heparin in the grind media is at least about 5 U/ml, about 6 U/ml, about 7 U/ml, about 8 U/ml, about 9 U/ml, about 10 U/ml, about 11 U/ml, about 12 U/ml, about 13 U/ml, or about 14 U/ml. In some embodiments, the amount of heparin in the grind media is at most about 6 U/ml, about 7 U/ml, about 8 U/ml, about 9 U/ml, about 10 U/ml, about 11 U/ml, about 12 U/ml, about 13 U/ml, about 14 U/ml, or about 15 U/ml.
[0162] In various embodiments, heparin is omitted from a grind medium.
[0163] In some embodiments, the amount of Benzonase® in the grind media is about 1 U/ml to about 10 U/ml. In some embodiments, the amount of Benzonase in the grind media is about 1 U/ml to about 2 U/ml, about 1 U/ml to about 3 U/ml, about 1 U/ml to about 4 U/ml, about 1 U/ml to about 5 U/ml, about 1 U/ml to about 6 U/ml, about 1 U/ml to about 7 U/ml, about 1 U/ml to about 8 U/ml, about 1 U/ml to about 9 U/ml, about 1 U/ml to about 10 U/ml, about 2 U/ml to about 3 U/ml, about 2 U/ml to about 4 U/ml, about 2 U/ml to about 5 U/ml, about 2 U/ml to about 6 U/ml, about 2 U/ml to about 7 U/ml, about 2 U/ml to about 8 U/ml, about 2 U/ml to about 9 U/ml, about 2 U/ml to about 10 U/ml, about 3 U/ml to about 4 U/ml, about 3 U/ml to about 5 U/ml, about 3 U/ml to about 6 U/ml, about 3 U/ml to about 7 U/ml, about 3 U/ml to about 8 U/ml, about 3 U/ml to about 9 U/ml, about 3 U/ml to about 10 U/ml, about 4 U/ml to about 5 U/ml, about 4 U/ml to about 6 U/ml, about 4 U/ml to about 7 U/ml, about 4 U/ml to about 8 U/ml, about 4 U/ml to about 9 U/ml, about 4 U/ml to about 10 U/ml, about 5 U/ml to about 6 U/ml, about 5 U/ml to about 7 U/ml, about 5 U/ml to about 8 U/ml, about 5 U/ml to about 9 U/ml, about 5 U/ml to about 10 U/ml, about 6 U/ml to about 7 U/ml, about 6 U/ml to about 8 U/ml, about 6 U/ml to about 9 U/ml, about 6 U/ml to about 10 U/ml, about 7 U/ml to about 8 U/ml, about 7 U/ml to about 9 U/ml, about 7 U/ml to about 10 U/ml, about 8 U/ml to about 9 U/ml, about 8 U/ml to about 10 U/ml, or about 9 U/ml to about 10 U/ml. In some embodiments, the amount of Benzonase in the grind media is about 1 U/ml, about 2 U/ml, about 3 U/ml, about 4 U/ml, about 5 U/ml, about 6 U/ml, about 7 U/ml, about 8 U/ml, about 9 U/ml, or about 10 U/ml. In some embodiments, the amount of Benzonase in the grind media is at least about 1 U/ml, about 2 U/ml, about 3 U/ml, about 4 U/ml, about 5 U/ml, about 6 U/ml, about 7 U/ml, about 8 U/ml, or about 9 U/ml. In some embodiments, the amount of Benzonase in the grind media is at most about 2 U/ml, about 3 U/ml, about 4 U/ml, about 5 U/ml, about 6 U/ml, about 7 U/ml, about 8 U/ml, about 9 U/ml, or about 10 U/ml.
[0164] In some embodiments, the amount of Benzonase® or Denarase® in the grind media is about 11 U/ml to about 55 U/ml. In some embodiments, the amount of Benzonase in the grind media is about 11 U/ml to about 15 U/ml, about 11 U/ml to about 20 U/ml, about 11 U/ml to about 25 U/ml, about 11 U/ml to about 30 U/ml, about 11 U/ml to about 35 U/ml, about 11 U/ml to about 40 U/ml, about 11 U/ml to about 45 U/ml, about 11 U/ml to about 50 U/ml, about 11 U/ml to about 55 U/rnl, about 15 U/rnl to about 20 U/rnl, about 15 U/rnl to about 25 U/ml, about 15 U/ml to about 30 U/ml, about 15 U/ml to about 35 U/ml, about 15 U/ml to about 40 U/ml, about 15 U/ml to about 45 U/ml, about 15 U/ml to about 50 U/ml, about 15 U/ml to about 55 U/ml, about 20 U/ml to about 25 U/ml, about 20 U/ml to about 30 U/ml, about 20 U/ml to about 35 U/ml, about 20 U/ml to about 40 U/ml, about 20 U/ml to about 45 U/ml, about 20 U/ml to about 50 U/ml, about 20 U/ml to about 55 U/ml, about 25 U/ml to about 30 U/ml, about 25 U/ml to about 35 U/ml, about 25 U/ml to about 40 U/ml, about 25 U/ml to about 45 U/ml, about 25 U/ml to about 50 U/ml, about 25 U/ml to about 55 U/ml, about 30 U/ml to about 35 U/ml, about 30 U/ml to about 40 U/ml, about 30 U/ml to about 45 U/ml, about 30 U/ml to about 50 U/ml, about 30 U/ml to about 55 U/ml, about 35 U/ml to about 40 U/ml, about 35 U/ml to about 45 U/ml, about 35 U/ml to about 50 U/ml, about 35 U/ml to about 55 U/ml, about 40 U/ml to about 45 U/ml, about 40 U/ml to about 50 U/ml, about 40 U/ml to about 55 U/ml, about 45 U/ml to about 50 U/ml, about 45 U/ml to about 55 U/ml, or about 50 U/ml to about 55 U/ml. In some embodiments, the amount of Benzonase in the grind media is about 11 U/ml, about 15 U/ml, about 20 U/ml, about 25 U/ml, about 30 U/ml, about 35 U/ml, about 40 U/ml, about 45 U/ml, about 50 U/ml, or about 55 U/ml. In some embodiments, the amount of Benzonase in the grind media is at least about 11 U/ml, about 15 U/ml, about 20 U/ml, about 25 U/ml, about 30 U/ml, about 35 U/ml, about 40 U/ml, about 45 U/ml, or about 50 U/ml. In some embodiments, the amount of Benzonase in the grind media is at most about 15 U/ml, about 20 U/ml, about 25 U/ml, about 30 U/ml, about 35 U/ml, about 40 U/ml, about 45 U/ml, about 50 U/ml, or about 55 U/ml.
[0165] In some cases, the amount of Benzonase® in a grind medium is about 3 U/ml and the amount of heparin in the grind medium is about 10 U/ml.
[0166] It is noted that Denarase® reagent (C-Lecta GmbH) is equivalent to Benzonase® reagent in the same quantity in the present process.
[0167] Notably, it has been discovered that a relationship exists between the amount of Benzonase® in a grinding medium and the amount of heparin, such that progressively lower amounts of Benzonase can be used as the amounts of heparin is reduced. Without wishing to be bound by theory, it is likely that heparin, through calcium chelation, helps prevent clumping of cells; however, more importantly, heparin chelates magnesium. Magnesium is an important co-factor for Benzonase. Therefore, in the presence of heparin, the presence and/or relative amounts of magnesium in a solution is reduced and this reduction in magnesium amounts reduces Benzonase activity. Thus, in some embodiments, the amount of heparin is lowered and in some embodiments, heparin is omitted.
[0168] In some embodiments, HSA is present in the grind media at about 0.5 % to about 5 %. In some embodiments, HSA is present in the grind media at about 0.5 % to about 1 %, about 0.5 % to about 1.5 %, about 0.5 % to about 2 %, about 0.5 % to about 2.5 %, about 0.5 % to about 3 %, about 0.5 % to about 3.5 %, about 0.5 % to about 4 %, about 0.5 % to about 4.5 %, about 0.5 % to about 5 %, about 1 % to about 1.5 %, about 1 % to about 2 %, about 1 % to about 2.5 %, about 1 % to about 3 %, about 1 % to about 3.5 %, about 1 % to about 4 %, about 1 % to about 4.5 %, about 1 % to about 5 %, about 1.5 % to about 2 %, about 1.5 % to about 2.5 %, about 1.5 % to about 3 %, about 1.5 % to about 3.5 %, about 1.5 % to about 4 %, about 1.5 % to about 4.5 %, about 1.5 % to about 5 %, about 2 % to about 2.5 %, about 2 % to about 3 %, about
2 % to about 3.5 %, about 2 % to about 4 %, about 2 % to about 4.5 %, about 2 % to about 5 %, about 2.5 % to about 3 %, about 2.5 % to about 3.5 %, about 2.5 % to about 4 %, about 2.5 % to about 4.5 %, about 2.5 % to about 5 %, about 3 % to about 3.5 %, about 3 % to about 4 %, about
3 % to about 4.5 %, about 3 % to about 5 %, about 3.5 % to about 4 %, about 3.5 % to about 4.5 %, about 3.5 % to about 5 %, about 4 % to about 4.5 %, about 4 % to about 5 %, or about 4.5 % to about 5 %. In some embodiments, HSA is present in the grind media at about 0.5 %, about 1 %, about 1.5 %, about 2 %, about 2.5 %, about 3 %, about 3.5 %, about 4 %, about 4.5 %, or about 5 %. In some embodiments, HSA is present in the grind media at least about 0.5 %, about
1 %, about 1.5 %, about 2 %, about 2.5 %, about 3 %, about 3.5 %, about 4 %, or about 4.5 %. In some embodiments, HSA is present in the grind media at most about 1 %, about 1.5 %, about
2 %, about 2.5 %, about 3 %, about 3.5 %, about 4 %, about 4.5 %, or about 5 %.
[0169] Another pitcher of 300-500 ml of grind media is retained for collecting the bone fragments after grinding, and another supply of about 100 ml of the grind media is retained for rinsing through the grinder during the grinding process to prevent bone fragments from sticking to the surface of the pitcher of the grinding components. In some embodiments, the additional grind media may have different quantities of heparin, HSA, and Benzonase as compared to the initial grind media.
[0170] An electric bone grinder or a purpose-built bone grinder, such as the grinder of Biorep Technologies Inc, (Miami, FL) can be used in an ISO-5 environment within an ISO- 7 clean room. Bone types are kept separate if both VB and ilium from the same donor are being processed. The bone is kept submerged in grind media at all times during and after the grinding process. Once all of the donor bone pieces are ground, the chamber of the bone grinder is thoroughly rinsed with fresh processing media. The bone fragments are discharged from the grinder into the pitcher containing grind media.
[0171] In some cases, bone marrow and bone grindings are shaken for 10 minutes at 150 RPM.
[0172] The contents of the pitcher are transferred to sterile bags. Next, the contents of the sterile bags are filtered to extract the solid components. In one embodiment, the contents of each bag are passed through a series of stainless-steel sieves. In this embodiment, a No. 40 (425 pm) sieve is stacked on top of a No. 80 (177 pm) sieve, which is seated over a catch-pan to receive the liquid filter contents. The sterile bags containing the output from the grinder is swirled and then poured evenly over the sieve stack or filtration sets. The filtering process is observed to ensure that excessive clumping is not occurring, which can signal the presence of soft tissue or other contaminants. Bone fragments retained on the surface of the sieves are distributed evenly on the sieves and rinsed with 250 ml of fresh processing medium. In one embodiment, the processing medium used for rinsing is the grind media described above or PLASMA-LYTE™ with 2.5% HSA. The sieved bone marrow product, which can be approximately 1000 ml in a well-performed process, is transferred to sterile packs for subsequent processing and analysis. The contents of each bag are visually inspected to confirm that the contents do not include any visible bone fragments or soft tissue.
[0173] In some embodiments, the rinse media can contain the various amounts of HSA as described for the grind media. In some embodiments, the rinse media can contain, additionally, heparin and/or Benzonase.
[0174] In some cases, the amount of Benzonase® in a rinse medium is about 3 U/ml and the amount of heparin in the rinse medium is about 10 U/ml.
[0175] In another embodiment, the contents of each bag are passed through bone marrow filtration units. In this embodiment, the system includes a stand configured to support a sterile collection bag which contains the bone fragments and media from the grinding operation described above. The stand includes a container hanger configured to engage the cap of the sterile bag to suspend the container. The bottom of the bag includes a discharge assembly that includes a pre-filter projecting into the body of the collection bag. In one specific embodiment the pre-filter is an 850pm filter. In some embodiments, the bone marrow passes first through an 800 pm pre-filter. The filter is connected to an output tube that is connected by a container claim to the input line of a first in-line filter. In the specific embodiment, the first in-line filter is a 200pm or a 500pm filter. The output line of the first in-line filter is connected to the input line of a second in-line filter. The second in-line filter is a 200pm or a 500pm filter. The two in-line filters are initially both 500pm for a first pass through the filter system. A second rinse is then performed on the grindings with the two in-line filters being 200pm. This double-pass filtration results in a cleaner suspension and enhances removal of fat from the suspension. The second in-line filter has an output line that can be engaged to a sterile bag, such as bag for the second filtration pass. On the second pass through the system, the output line of the second in-line filter can be engaged to a container clamp of a transfer pack container. The transfer pack container can be a 600-2000 ml bag to accommodate the filtered bone marrow product, which can be approximately 1000 ml in a well -performed process.
Agitation of Bone Grindings and/or Bone Grinding Filtrate
[0176] Described herein, in some embodiments, is a method for processing bone marrow or derivative thereof, the method comprises mechanically agitating the bone grindings and/or bone grinding filtrate during the grinding and filtration portion of the processing of the bone marrow. In some instances, the bone marrow can be obtained from a deceased donor. In some cases, the bone marrow can be obtained from a sample (e.g. bone or VB) that was previously chilled. In some cases, the bone marrow can be obtained from a sample (e.g. bone or VB) that was previously chilled but not frozen. In some cases, the bone marrow can be obtained from a sample (e.g. bone or VB) that is thawed. In some cases, the bone marrow can be processed for obtaining bone marrow cells. In some embodiments, the bone marrow cells can be hematopoietic stem cells (HSCs). In some embodiments, the bone marrow cells can be mesenchymal stem cells (MSCs).
[0177] Aspect disclosed in the present disclosure comprises a method for processing bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone or bone fragment from a deceased donor, optionally, processing the bone into bone fragments; mechanically grinding the bone or bone fragment in the presence of a grinding solution to generate a plurality of bone grindings; placing the plurality of bone grindings on a shaker at about 100 to about 200 rounds per minute (“RPM”) for about 1 to about 20 minutes; and removing the solution from the shaker, wherein the solution comprises the bone marrow or the derivative thereof and wherein the bone marrow or the derivative thereof comprises at least about 1,500,000 CD34+ cells/ml of the bone marrow or the derivative thereof. In some embodiments, the method further comprises contacting the solution with a rinse media and repeating the placing of the bone grindings on the shaker and then removing the solution from the shaker. In some embodiments, the method further comprises repeating step placing the bone grinding on the shaker and then removing the solution from the shaker one or more times. In some embodiments, the at least about 1,500,000 CD34+ cells/ml of the bone marrow or the derivative thereof comprises at least 85% viable CD34+ cells. In some embodiments, the method further comprises the at least about 1,500,000 CD34+ cells/ml of the bone marrow or the derivative thereof comprises at least 90% viable CD34+ cells.
[0178] The mechanical agitation can comprise agitating the bone grindings in a linear fashion. In some embodiments, the mechanical agitation can comprise agitating the bone grindings in a three-dimensional fashion. In some cases, the mechanical agitation of the bone grindings can comprise orbital shaking (via an orbital shaker) such as placing the bone grinding on a shaker. In some cases, the bone grindings can be mechanically agitated by the shaker at a rate at least about 10 rounds per minute (RPM), 20 RPM, 30 RPM, 40 RPM, 50 RPM, 60 RPM, 70 RPM, 80 RPM, 90 RPM, 100 RPM, 110 RPM, 120 RPM, 130 RPM, 140 RPM, 150 RPM, 160 RPM, 170 RPM, 180 RPM, 190 RPM, 200 RPM, 210 RPM, 220 RPM, 230 RPM, 240 RPM, 250 RPM, or more. In some cases, the bone grindings can be mechanically agitated by centrifugation (e.g. spinning). In some embodiments, the bone grindings can be spun at least 10 RPM, 20 RPM, 30 RPM, 40 RPM, 50 RPM, 60 RPM, 70 RPM, 80 RPM, 90 RPM, 100 RPM, 110 RPM, 120 RPM, 130 RPM, 140 RPM, 150 RPM, 160 RPM, 170 RPM, 180 RPM, 190 RPM, 200 RPM, 210 RPM, 220 RPM, 230 RPM, 240 RPM, 250 RPM, or more. In some embodiments, the bone grindings can be spun at least 300 RPM, 400 RPM, 500 RPM, 600 RPM, or more. In some embodiments, the bone grindings can be mechanically agitated by both shaking and spinning. In some embodiments, the mechanical agitation of the bone grindings can be for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or longer.
[0179] In some embodiments, the mechanical agitation of the bone grindings increases the yield of the bone marrow cells obtained. In some instances, the yield of the bone marrow cells obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to yield of bone marrow cells obtained without the mechanical agitation.
[0180] In some embodiments, the mechanical agitation of the bone grindings increases the viability of the bone marrow cells obtained. In some instances, the viability of the bone marrow cells obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the viability of bone marrow cells obtained without the mechanical agitation.
[0181] In some embodiments, the mechanical agitation of the bone grindings increases the number of CD34 expressing bone marrow cells obtained. In some instances, the number of CD34 expressing bone marrow cells obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the number of CD34 expressing bone marrow cells obtained without the mechanical agitation.
[0182] In some embodiments, the mechanical agitation of the bone grindings increases the number of CD45 expressing bone marrow cells obtained by the methods described herein. In some instances, the number of CD45 expressing bone marrow cells obtained by mechanical agitation of the bone grindings is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the number of CD45 expressing bone marrow cells obtained without the mechanical agitation.
[0183] The agitation can occur before the filtration steps described previously.
[0184] In certain embodiments, the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least about 100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,050,000, 1,100,000, 1,150,000, 1,200,000, 1,250,000, 1,300,000, 1,350,000, 1,400,000, 1,450,000, 1,500,000, 1,550,000, 1,600,000, 1,650,000, 1,700,000, 1,750,000, 1,800,000, 1,850,000, 1,900,000, 1950,000, 2,000,000, or more than 2,000,000 CD34+ cells/ml. In some embodiments, the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least about 1,500,000 CD34+ cells/ml to about 2,000,000 CD34+ cells/ml. In some embodiments, the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least about 1,500,000 CD34+ cells/ml to about 1,750,000 CD34+ cells/ml, about 1,500,000 CD34+ cells/ml to about 2,000,000 CD34+ cells/ml, or about 1,750,000 CD34+ cells/ml to about 2,000,000 CD34+ cells/ml. In some embodiments, the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least about 1,500,000 CD34+ cells/ml, about 1,750,000 CD34+ cells/ml, or about 2,000,000 CD34+ cells/ml. In some embodiments, the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least at least about 1,500,000 CD34+ cells/ml, or about 1,750,000 CD34+ cells/ml. In some embodiments, the amount of CD34+ cells/ml of the bone marrow or the derivative thereof obtained is at least at most about 1,750,000 CD34+ cells/ml, or about 2,000,000 CD34+ cells/ml. [0185] In some embodiments, the viability of the CD34+ cells is at least about 70% to about 95%. In some embodiments, the viability of the CD34+ cells is at least about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95%. In some embodiments, the viability of the CD34+ cells is at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the viability of the CD34+ cells is at least at least about 70%, about 75%, about 80%, about 85%, or about 90%. In some embodiments, the viability of the CD34+ cells is at least at most about 75%, about 80%, about 85%, about 90%, or about 95%.
[0186] For quality control, a small quantity of bone marrow, such as 0.3 mL, is extracted from the sterile pack using a syringe at an injection site and conducting inversion mixing before pulling the sample. The sample can be tested by a hematology analyzer, such as a Sysmex Hematology Analyzer, to determine the total nucleated cell (TNC) content of the sample, as an indicator of the TNC content of the bone marrow being subsequently processed.
Fat Removal and Concentration
[0187] The bone marrow product collected from the filtering is essentially a fatty emulsion. The fat content of the suspension obtained from the sieve filtering approach disclosed above is greater than the fat content of the suspension obtained from the doublepass filtration system. However, in both cases, there is a need to remove the fat content from the suspension. The suspension obtained from the filtering is recovered into 250 ml bags which are hermetically sealed with tube welders. Pairs of sterile bags and taring sticks are mounted within a centrifuge with bag ports facing down, and balanced. Volume compensating plates are used to prevent creasing of the bags during centrifugation. In one embodiment, the bags are centrifuged at 500xg for 15 minutes at room temperature to concentrate the cells, preferably to 2-3xl08/ml. After centrifugation is complete, each bag is individually hung on a ring stand. The distinct layers within the bag are visible, with the fat layer clearly delineated on top of the supernatant with the bone marrow pellet at the bottom. A new sterile bag is welded to the bag removed from the centrifuge. A bag clamp or clip is placed on the bag just below the fat layer, to clamp off or squeeze the bag closed beneath the fat layer. The pellet is then drained from the centrifuge bag into the new sterile bag, with the bag clip preventing passage of the fat layer. The pellet is agitated as it is drained to resuspend all of the pellet. After about half of the pellet has drained into the new bag, the tubing is closed with a hemostat or tube sealer. The second centrifuge bag is then welded to the new bag containing the pellet, and the contents of this second centrifuge bag are drained into the new bag.
[0188] The result is new sterile bags containing the bone marrow centrifuged to remove the fat. These bags of de-fatted bone marrow are then centrifuged at 500xg for 15 minutes at room temperature, with volume compensating plates to prevent creasing of the bags. Each bag is removed and suspended on a ring stand and a waste bag is welded to the bag, and a plasma extractor is used to remove the supernatant into the waste bag. The tubing is clamped with a hemostat when the pellet rises or breaks. The tubing is then sealed and severed to remove the pellet — containing bag from the waste bag, which is discarded. A Luer connection is welded to the pellet-containing bag. The pellets from each bag are combined into a bulk bag using a large syringe. The pellet-containing bags are rinsed into the bulk bag using a rinse media. The bulk bag is inverted several times to ensure that all of the pellet is resuspended. A small quantity of the processed BM, such as 0.5 mL, can be removed for quality control testing for density and cell count. The test sample can also be evaluated for human leukocyte antigens, CCR5delta 32 mutation and apolipoprotein (APOE), among other things.
[0189] In some embodiments, the centrifuge settings at one or more steps can be increased. In some embodiments, the centrifuge is spun at about 400 g to about 650 g. In some embodiments, the centrifuge is spun at about 400 g to about 450 g, about 400 g to about 500 g, about 400 g to about 550 g, about 400 g to about 600 g, about 400 g to about 650 g, about 450 g to about 500 g, about 450 g to about 550 g, about 450 g to about 600 g, about 450 g to about 650 g, about 500 g to about 550 g, about 500 g to about 600 g, about 500 g to about 650 g, about 550 g to about 600 g, about 550 g to about 650 g, or about 600 g to about 650 g. In some embodiments, the centrifuge is spun at about 400 g, about 450 g, about 500 g, about 550 g, about 600 g, or about 650 g. In some embodiments, the centrifuge is spun at least about 400 g, about 450 g, about 500 g, about 550 g, or about 600 g. In some embodiments, the centrifuge is spun at most about 450 g, about 500 g, about 550 g, about 600 g, or about 650 g. In some embodiments, the centrifuge is spun for about 10 minutes to about 40 minutes. In some embodiments, the centrifuge is spun for about 10 minutes to about 15 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 25 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 35 minutes, about 10 minutes to about 40 minutes, about 15 minutes to about 20 minutes, about 15 minutes to about 25 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 35 minutes, about 15 minutes to about 40 minutes, about 20 minutes to about 25 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 35 minutes, about 20 minutes to about 40 minutes, about 25 minutes to about 30 minutes, about 25 minutes to about 35 minutes, about 25 minutes to about 40 minutes, about 30 minutes to about 35 minutes, about 30 minutes to about 40 minutes, or about 35 minutes to about 40 minutes. In some embodiments, the centrifuge is spun for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes. In some embodiments, the centrifuge is spun for at least about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 35 minutes. In some embodiments, the centrifuge is spun for at most about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes. In some embodiments, the centrifuge is stopped without the use of a brake. In some embodiments, the centrifuge is stopped with a brake. In some embodiments, the centrifuge brake is set at about 25% to about 100%. In some embodiments, the centrifuge brake is set at about 25% to about 50%, about 25% to about 75%, about 25% to about 100%, about 50% to about 75%, about 50% to about 100%, or about 75% to about 100%. In some embodiments, the centrifuge brake is set at about 25%, about 50%, about 75%, or about 100%. In some embodiments, the centrifuge brake is set at least about 25%, about 50%, or about 75%. In some embodiments, the centrifuge brake is set at most about 50%, about 75%, or about 100%.
[0190] In some cases, fat removal can be occur using a commercial cell processing device (e.g., COBE® 2991 cell processor, TerumoBCT). See the World Wide Web (al) terumobct.com/2991. Such commercial cell processing devices may also concentrate cell products.
Cryopreservation of the Bone Marrow
[0191] Methods for extracting and banking bone marrow for future clinical use according to the processing methods described herein are summarized in the flowchart of FIG. 6 in PCT/US2021/005081. This method can eliminate the failures of the current methods of matching bone marrow donors to groups that are tough to match, such as certain minorities. Once the bone marrow is cryopreserved and banked there is no uncertainty as to the source of the bone marrow, there is no wait for a future recipient and the bone marrow is available in large, repeatable volumes.
[0192] The freeze media is a solution of a rinse media and a cry opreservation composition. The cryoprotectant can be a cell-permeable media, such as dimethyl sulfoxide (DMSO); 1, 2 propane diol (also known as propylene glycol); ethylene glycol; glycerol; foramamide; ethanediol or butane 2, 3 diol; and/or a non-permeable media, such as hydroxyethyl starch (HES), dextran, sucrose, trehalose, lactose, raffinose, ribotol, Mannitol or polyvinylpyrrolidone (PVP). Each bone donor can also provide at least three surrogate vials, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more surrogate vial. The greater number of cry opreservation bags obtained from a donor, the greater number of surrogate vials that can be prepared, so that each cryopreservation bag has at least one vial, preferably, two, three, four, or more vials per cryopreservation bag.
[0193] HSA also provides cryoprotection through oncotic pressure, cell surface protein stabilization and reactive oxygen scavenging. In a preferred embodiment, the cryoprotectant is DMSO. The rinse media can be an electrolyte medium, such as PlasmaLyte, Isolyte, IMDM or other electrolyte solutions suitable for infusion. The freeze media can also include concentrations of oxyrase to reduce oxygen content to less than atmospheric, such as to less than 3% of atmospheric concentrations. The addition of oxyrase produces a hypobaric composition that can facilitate cry opreservation.
[0194] In some embodiments, for a method provided herein, a bone marrow product is cryopreserved in a freeze media, wherein said freeze media comprises an electrolyte formulation, human serum albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof.
[0195] In some embodiments, said freeze media and/or rinse media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, about 5% to about 6%, about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, about 6% to about 7%, about 7% to about 10%, about 7% to about 9%, about 7% to about 8%, about 8% to about 10%, about 8% to about 9%, or about 9% to about 10% HSA. In some embodiments, said freeze media and/or rinse media comprises about 1% to about 5% HSA. In some embodiments, said freeze media and/or rinse media comprises about 2.5% HSA. [0196] In some embodiments, said freeze media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about
1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about
2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about
2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about
3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, about 5% to about 6%, about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, about 6% to about 7%, about 7% to about 10%, about 7% to about 9%, about 7% to about 8%, about 8% to about 10%, about 8% to about 9%, or about 9% to about 10% DMSO. In some embodiments, said freeze media comprises about 1% to about 10% DMSO. In some embodiments, said freeze media comprises about 2.5% DMSO, about 5% DMSO, or about 10% DMSO.
[0197] In some embodiments, said electrolyte formulation is Plasmalyte A.
[0198] In various embodiments, a rinse medium and/or freeze medium lacks heparin.
[0199] In some embodiments, the rinse media is fresh.
[0200] In some cases, the freeze media is <25°C before adding it to the bone marrow bulk bag. [0201] The freeze media may be added to the bone marrow bulk bag at a predetermined rate (10% of the Freeze Media volume per minute) based on the following formula:
Volume of Freeze Media to add per minute = Total Volume of Freeze Media [ml * 0.1
[0202] Preferably, the elapsed time for adding the cryoprotectant to the bone marrow bulk bag does not exceed 9-11 minutes.
[0203] Additional methods for cryopreserving bone marrow cell is further disclosed in PCT/US2021/005081, the entire contents of which is incorporated by reference in its entirety.
Isolation of CD34+ cells
[0204] Described herein, in some aspects, is a method for processing (e.g. isolating) CD34+ cells obtained from bone marrow or bone marrow derivative. In some cases, the bone marrow or bone marrow derivative can be fresh (e.g. never frozen) or thawed from being previously frozen. In some embodiments, the bone marrow or bone marrow derivative can be ground by the methods and systems described herein. In some embodiments, ground bone marrow or bone marrow cells can be contacted with the stabilization buffer described herein. In some embodiments, the stabilization prevents formation of aggregates of the bone marrow cells. In some instances, the bone marrow cells contacted and suspended in the stabilization buffer can be isolated by attaching to antibody such as a conjugated antibody. For example, bone marrow cells expressing CD34+ can be isolated and enriched by contacting the bone marrow cells with the CD34 antibody conjugated with iron, where the bone marrow cells expressing CD34 are then trapped a magnetic separation column (e.g. “CliniMACS®”). The bone marrow cells not expressing CD34 are can be washed away. The trapped CD34+ bone marrow cells can be harvested by removing the magnetic field and eluting the targeted CD34+ bone marrow cells. Such approach does not require isolating the bone marrow cells with a Ficoll gradient.
[0205] Aspect described in the present disclosure comprises a method for processing a population of CD34+ cells obtained from bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone or bone fragment from a deceased donor, optionally, processing the bone into bone fragments; extracting the bone marrow or derivative thereof from the bone or bone fragment; and contacting the bone marrow or derivative thereof with a stabilization buffer, wherein the stabilization buffer comprises more than about 3 U/ml of a nuclease; performing a CD34+ cell isolation assay to generate a cellular composition comprising the population of CD34+ cells, wherein the composition comprising the population of CD34+ cells comprises at least about 80,000 CD34+ cells/750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer. In some embodiments, the at least about 80,000 CD34+ cells/750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 70% viable CD34+ cells. In some embodiments, the at least about 80,000 CD34+ cells/750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 80% viable CD34+ cells. In some embodiments, the at least about 80,000 CD34+ cells/750 pl of the bone marrow or the derivative thereof contacted with the stabilization buffer comprise at least 90% viable CD34+ cells.
[0206] Another aspect of the present disclosure comprises a stabilization buffer comprising: at least 5 U/ml of an anticoagulant; and more than 3 U/ml of a nuclease. In some embodiments, stabilization buffer comprises more than about 5 U/ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 10 U/ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 15 U/ml of a nuclease. In some embodiments, the stabilization buffer comprises more than about 20 U/ml of a nuclease. In some embodiments, the stabilization buffer comprises about 20 U/ml of a nuclease. In some embodiments, the nuclease is Benzonase® or Denarase®. In some embodiments, the stabilization buffer further comprises more than about 10 U/ml of an anticoagulant. In some embodiments, the stabilization buffer further comprises about 10 U/ml of an anticoagulant. In some embodiments, the anticoagulant is heparin. In some embodiments, the stabilization buffer further comprises human serum albumin (HSA). In some embodiments, the stabilization buffer comprises 0.5% HSA.
[0207] In some embodiments, the stabilization buffer comprises nuclease. In some embodiments, the nuclease is Benzonase® or Denarase®. In some embodiments, the stabilization buffer comprises nuclease at about 3 U/ml, 4 U/ml, 5 U/ml, 6 U/ml, 7 U/ml, 8 U/ml, 9 U/ml, 10 U/ml, 11 U/ml, 12 U/ml, 13 U/ml, 14 U/ml, 15 U/ml, 16 U/ml, 17 U/ml, 18 U/ml, 19 U/ml, 20 U/ml, 21 U/ml, 22 U/ml, 23 U/ml, 24 U/ml, 25 U/ml, 26 U/ml, 27 U/ml, 28 U/ml, 29 U/ml, 30 U/ml, 50 U/ml, 100 U/ml, 200 U/ml, or more U/ml. In some embodiments, the stabilization buffer comprises an anticoagulant. In some cases, the anticoagulant is Heparin. In some instances, the stabilization buffer comprises anticoagulant at about 0.1 U/ml, 0.2 U/ml, 0.3 U/ml, 0.4 U/ml, 0.5 U/ml, 0.6 U/ml, 0.7 U/ml, 0.8 U/ml, 0.9 U/ml, 1.0 U/ml, 2.0 U/ml, 3.0 U/ml, 4.0 U/ml, 5.0 U/ml, 6.0 U/ml, 7.0 U/ml, 8.0 U/ml, 9.0 U/ml, 10 U/ml, 11 U/ml, 12 U/ml, 13 U/ml, 14 U/ml, 15 U/ml, 16 U/ml, 17 U/ml, 18 U/ml, 19 U/ml, 20 U/ml, 21 U/ml, 22 U/ml, 23 U/ml, 24 U/ml, 25 U/ml, 26 U/ml, 27 U/ml, 28 U/ml, 29 U/ml, 30 U/ml, 50 U/ml, 100 U/ml, 200 U/ml, or more U/ml.
[0208] In various embodiments, a stabilization buffer lacks heparin.
[0209] In some embodiments, the stabilization buffer comprises about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05% HSA, 0.1% HSA, 0.2% HSA, 0.3% HSA, 0.4% HSA, 0.5% HSA, 0.6% HSA, 0.7% HSA, 0.8% HSA, 0.9% HSA, 1.0% HSA, 1.5 % HSA, 2% HSA, 2.5% HSA, 5% HSA, 10% HSA, 20% HSA, or more HSA.
[0210] Described herein, in some embodiments, is a method of processing bone marrow to obtain bone marrow cells. In some embodiments, the method comprises contacting the bone marrow or the bone marrow cells with the stabilization buffer described herein.
[0211] An aspect of the present disclosure comprises a method for processing a population of CD34+ cells comprised in bone marrow or a derivative thereof, wherein the bone marrow or the derivative thereof is derived from a deceased donor, the method comprising: obtaining a bone or bone fragment from a deceased donor, optionally, processing the bone into bone fragments; extracting the bone marrow or derivative thereof from the bone or bone fragment; and contacting the bone marrow or derivative thereof with a stabilization buffer, wherein the stabilization buffer comprises more than about 3 U/ml of a nuclease; performing a CD34+ cell isolation assay to generate a cellular composition comprising the population of CD34+ cells, wherein the composition comprising the population of CD34+ cells comprises at least about 80,000 CD34+ cells/750 ul of the bone marrow or the derivative thereof contacted with the stabilization buffer.
[0212] In some embodiments, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the yield of the bone marrow cells obtained from the methods described herein compared to the yield of the bone marrow cells processed in the absence of the stabilization buffer. In some instances, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the yield of the bone marrow cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to yield of bone marrow cells processed in the absence of the stabilization buffer. In some embodiments, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the viability of the bone marrow cells obtained from the methods described herein compared to the viability of the bone marrow cells processed in the absence of the stabilization buffer. In some instances, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the viability of the bone marrow cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to viability of bone marrow cells processed in the absence of the stabilization buffer.
[0213] In some embodiments, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the number of CD34+ bone marrow cells compared to the number of CD34+ bone marrow cells processed in the absence of the stabilization buffer. In some cases, the number of CD34+ bone marrow obtained from processing with the stabilization buffer is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the number of CD34+ bone marrow obtained from processing in the absence of stabilization buffer. In some embodiments, processing or contacting the bone marrow or bone marrow cells described herein with the stabilization buffer increases the number of CD45+ bone marrow cells compare to the number of CD45+ bone marrow cells processed in the absence of the stabilization buffer. In some cases, the number of CD45+ bone marrow obtained from processing with the stabilization buffer is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, or more compared to the number of CD45+ bone marrow obtained from processing in the absence of stabilization buffer.
[0214] In some embodiments, cellular compositions comprising CD34+ cells derived from bone marrow samples processed with the stabilization buffers described herein have an increased amount of CD34+ cells, as compared to cellular compositions generated from known CD34+ isolation methods. In some embodiments. The amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 CD34+ cells/750 ul of bone marrow or a derivative thereof contacted with the stabilization buffers described herein. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 cells/750 ul to about 100,000 cells/750 ul. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 cells/750 ul to about 75,000 cells/750 ul, about 70,000 cells/750 ul to about 80,000 cells/750 ul, about 70,000 cells/750 ul to about 85,000 cells/750 ul, about 70,000 cells/750 ul to about 90,000 cells/750 ul, about 70,000 cells/750 ul to about 95,000 cells/750 ul, about 70,000 cells/750 ul to about 100,000 cells/750 ul, about 75,000 cells/750 ul to about 80,000 cells/750 ul, about 75,000 cells/750 ul to about 85,000 cells/750 ul, about 75,000 cells/750 ul to about 90,000 cells/750 ul, about 75,000 cells/750 ul to about 95,000 cells/750 ul, about 75,000 cells/750 ul to about 100,000 cells/750 ul, about 80,000 cells/750 ul to about 85,000 cells/750 ul, about 80,000 cells/750 ul to about 90,000 cells/750 ul, about 80,000 cells/750 ul to about 95,000 cells/750 ul, about 80,000 cells/750 ul to about 100,000 cells/750 ul, about 85,000 cells/750 ul to about 90,000 cells/750 ul, about 85,000 cells/750 ul to about 95,000 cells/750 ul, about 85,000 cells/750 ul to about 100,000 cells/750 ul, about 90,000 cells/750 ul to about 95,000 cells/750 ul, about 90,000 cells/750 ul to about 100,000 cells/750 ul, or about 95,000 cells/750 ul to about 100,000 cells/750 ul. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least about 70,000 cells/750 ul, about 75,000 cells/750 ul, about 80,000 cells/750 ul, about 85,000 cells/750 ul, about 90,000 cells/750 ul, about 95,000 cells/750 ul, or about 100,000 cells/750 ul. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least at least about 70,000 cells/750 ul, about 75,000 cells/750 ul, about 80,000 cells/750 ul, about 85,000 cells/750 ul, about 90,000 cells/750 ul, or about 95,000 cells/750 ul. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein is at least at most about 75,000 cells/750 ul, about 80,000 cells/750 ul, about 85,000 cells/750 ul, about 90,000 cells/750 ul, about 95,000 cells/750 ul, or about 100,000 cells/750 ul.
[0215] In some embodiments, the CD34+ cells derived from bone marrow samples processed with the stabilization buffers described herein also exhibit higher viability as compared to cellular compositions generated from known CD34+ isolation methods.
[0216] In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70% to about 95%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70% to about 95%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least at least about 70%, about 75%, about 80%, about 85%, or about 90%. In some embodiments, the amount of CD34+ cells isolated from the bone marrow samples contacted with the stabilization buffers described herein comprise a percent viability of at least at most about 75%, about 80%, about 85%, about 90%, or about 95%. Viability may relate to either or both of functional viability which measures the cells’ ability to proliferate and routine viability which relates to the numbers or percentages of live cells, e.g., as measured by Trypan Blue.
[0217] In an aspect of the present disclosure, a method is provided for selecting CD34 expressing (CD34+) cells from deceased donor bone marrow using density reduced Ficoll and an immunomagnetic CD34+ cell isolation kit. Surprisingly, it has been found that cell isolation using density reduced Ficoll prior to CD34 selection is beneficial to obtain high purity and viability CD45/CD34+ cells from freshly prepared deceased donor bone marrow. On the other hand, Ficoll at conventional density has been found to be optimal for CD45/CD34+ cell selection from thawed cryopreserved deceased donor bone marrow.
[0218] Vertebral sections obtained from a recently deceased donor were processed as described above. Thus, in one embodiment, the bone is cleaned of all soft tissue and then cut into small pieces that were immediately submerged into 500 ml of grinding media. The grinding media can be PLASMA-LYTE™ A injection pH 7.4, multiple electrolytes, injection type 1 USP (PLASMA-LYTE™) containing 2.5% human serum albumin (HSA), 3 U/ml denarase, and 10 U/ml heparin. The sectioned VB are ground using a bone grinder, filtered and rinsed with rinse media (such as PLASMA-LYTE™ with 2.5% HSA). The entire cell suspension is centrifuged to concentrate cells to 2-3xl08/ml and the cell concentration is extracted. A portion or all of the resulting BM preparation can be used immediately for CD34 selection, while the remainder can be prepared for cry opreservation. The cryopreserved portion involves adding a final concentration of 10% DMSO and 5% HSA to the BM cells and bringing the preparation to - 86°C, either by passive cooling or by controlled cooling at a rate of approximately -l°C/min, after which the cryopreserved portion is plunged into liquid nitrogen. [0219] For selection of CD34+ cells, either the newly processed BM preparation is used or a previously cryopreserved portion is thawed for use. Ficoll-Paque PLUS is added to the BM preparation to separate the desired CD34+ cell component of the bone marrow. It has been found for cell selection from cryopreserved bone marrow that the conventional density for the Ficoll of 1.077 g/ml produces acceptable results. However, in one aspect of the present disclosure, for cell selection from freshly prepared deceased donor bone marrow the Ficoll density is reduced from the conventional density. In particular, the density is reduced by mixing Ficoll-Paque PLUS (density 1.077 g/mL, GE Company) with Plasma Lyte-A Injection pH 7.4 (Baxter Healthcare 2B2544X) in specific proportions to obtain an overall density of less than 1.077 g/ml, particularly 1.063 — 1.052 g/ml. In one specific embodiment, the density of 1.063 g/ml was found to be optimal for isolation of CD34+ cells, taking into account quantity, viability and purity of the CD34+ cells.
[0220] In one embodiment, 5 ml of the 1.063 g/ml density Ficoll solutions is pipetted into 15-ml centrifuge tubes, and the BM solution generated from VBs of deceased donors is carefully layered over the Ficoll gradient. The tubes are centrifuged for 30 min at 400 g without break at room temperature. After centrifugation, buffy coat cells are harvested carefully, and the cells are washed in phosphate-buffered saline (PBS) containing 0.5% HSA and 2mM Ethylenediaminetetraacetic acid (EDTA) (MACS buffer, Miltenyi). In one specific embodiment, centrifugation is performed for 5 min at 400 g, and the resulting cell pellets are resuspended in 10 ml PBS, followed by a second centrifugation for 5 min at 400 g.
[0221] Nucleated cells in the isolated buffy coat can be counted using a Sysmex XP-300. A Cellometer Vision (Nexcellom) or flow cytometer can be used to determine cell counts of purified CD34 cells. 20 microliters of AOPI can be added to 20 microliters of cells and after mixing total viable cells can be determined. The CD34+ cells can be selected by a positive immune separation method using a CliniMAX system (Miltenyi, Bergisch Gladbach, Germany) or an EasySep CD34 kit (Stemcell Technologies, Vancouver, BC, Canada) in accordance with the protocol of the manufacturer. From testing at various Ficoll densities it has been surprisingly determined that the lower Ficoll density contemplated in the present disclosure (i.e., 1.063 — 1.052 gm/ml vs. the conventional 1.077 gm/ml density) leads to more optimum cell recovery. Optimization is based on purity, viability and yield of selected CD34 cells. A target of >90% purity and >90% viable CD34+ cells is preferred. While lower Ficoll densities resulted in greater purity and fewer dead cells, it was surprisingly found that a greater portion of the CD34+ cells present in the deceased donor whole bone marrow before selection are lost using the lower Ficoll densities to prepare buffy coat. Thus, the high viability and purity of CD45/CD34+ cells achieved at the conventional Ficoll density gradient also leads to a large loss in yield (approximately 60% loss of input CD34+ cells).
[0222] Thus, in accordance with one aspect of the present disclosure, for freshly prepared the optimal density of Ficoll for selection of CD45/CD34+ cells at >90% purity and viability is less than 1.077 and particularly 1.063- 1.052. This Ficoll density provides a higher yield of CD45/CD34+ cells with similar purity and cell viability to the conventional Ficoll density approach.
[0223] In another aspect of the present disclosure, the CD34+ cells can be initially acquired from a freshly prepared deceased donor bone marrow using the reduced density Ficoll-Paque described above. The BM can be cryogenically frozen and then the CD34+ cells can be acquired later using conventional density Ficoll-Paque. This approach essentially allows selective recovery of cells from deceased donor bone marrow — either before freezing using the modified Ficoll density or after freezing and thawing using conventional Ficoll density.
[0224] Once CD34+ have been isolated from bone marrow product, it could be said that the remaining cell product is enriched for MSCs, and especially vBM-MSCs. Recovery of MSC from processed bone marrow
[0225] Bone marrow is a well-known source for mesenchymal stromal/stem cells (MSCs) which can be harvested from bone marrow obtained using the methods described above. MSCs are self-renewing, multipotent progenitor cells with multilineage potential to differentiate into cell types of mesodermal origin, such as adipocytes, osteocytes, and chondrocytes. In addition, MSCs can migrate to sites of inflammation and exert potent immunosuppressive and antiinflammatory effects through interactions between lymphocytes associated with both the innate and adaptive immune system. MSCs can be used in treating osteogenesis imperfect, cartilage defects, myocardial infarction, Crohn's disease, multiple sclerosis, autoimmune disease such as Lupus, liver cirrhosis, osteo arthritis, and rheumatoid arthritis. Matched HSC/MSC units which can be used in co-transplant for treatment of graft vs. host disease (GVHD), and for hematopoietic stem cell transplant support.
[0226] In another feature of the systems and methods disclosed herein, a method is provided for preparing a composition of human MSCs from bone. In some embodiments, the preparation may include providing a bone derived from a deceased donor, grinding the bone into one or more ground bone segments, filtering the one or more ground bone segments and extracting the human MSCs from the one or more ground bone segments. In some embodiments, the MSCs may be recovered from thawed or cryopreserved VB bone fragments. In some embodiments, the MSC cells are readily released from the VB; these MSCs are referred to herein as bone marrow vertebral body mesenchymal stem cells (vBM-MSCs). In some embodiments, the extracted human MSCs may be vertebral bone marrow MSCs (vBM-MSCs), adherent vertebral body mesenchymal stem cells (vBA-MSCs), or both. In some embodiments, the extracted human MSCs are derived from a bone or fragments thereof that has already been processed to remove bone marrow or derivates thereof associated with the bone or fragment thereof (e.g. bone marrow derived cells, hematopoietic stem cells). In some embodiments, the extracted human MSCs are derived from a bone or fragments thereof that has been processed for bone marrow and/or bone marrow-derived cells (e.g. hematopoietic stem cells) as described herein. In some embodiments, the extracted human MSCs are derived from the bone grindings and/or segments described herein following filtration and/or extraction and/or isolation of bone marrow and/or bone marrow-derived cells as described herein. The processing and extraction of viable vBA-MSCs from the bone and/or derivates thereof (e.g. bone grindings described herein, bone segments described herein) results in significant improvements in cell yield, especially with respect to total cell yield (vBA-MSCs and hematopoietic stem cells) per weight of bone derived from a donor, and viability of cells with respect to the state of the art. In some embodiments, the vBA-MSCs described herein can be combined with bone marrow-derived MSCs (vBM-MSCs) isolated from bone marrow isolated and processed as described herein.
[0227] Some methods recover MSCs from enzymatically digested vertebral body (VB) bone fragments that are the byproduct of the VB grinding and elution of the methods described herein. In this method, a mixture of both collagenase and neutral protease is used to obtain the highest possible yields of vertebral bone adherent MSC (vBA-MSC). The MSCs can be recovered from cryopreserved VB bone fragments that are later processed according to the present disclosure. In one specific aspect, recombinant Clostridium histolyticum collagenase, comprised of the two active isoforms, is used in effective amounts in the MSC extraction process. The mixture of cells liberated by digesting VB bone fragment is cultured on tissue-coated plastic in the presence of Mesencult medium to select proliferative vBA-MSC. Freshly digested preparations as well as different passages of vBA-MSC can be characterized by flow cytometry, colony forming unitfibroblast (CFU-F) potential, population doubling time (PDT) and trilineage (adipogenic, chondrogenic and osteogenic) differentiation in vitro. In some embodiments, the mesenchymal stem cells can be recovered or cultured in Alpha-MEM supplemented with human platelet lysate and epidermal growth factor and/or fibroblast growth factor.
[0228] In some embodiments, the extraction of human MSCs may include contacting the bone or derivatives thereof with a digestion solution. In some embodiments, the digestion solution may include one or more distinct enzymes. In some embodiments, the one or more distinct enzymes may include one or more collagenases and neutral proteases. In some embodiments, the combination of one or more collagenases and neutral proteases is used to obtain the highest possible yields of vBA-MSC.
[0229] The present disclosure thus contemplates a method for optimizing digestion and MSC recovery from vertebral bone fragments using a combination of purified collagenase and neutral protease. In one specific embodiment, the collagenase is DE collagenase (Vitacyte), which is comprised of purified Clostridium histolyticum collagenase and Paneibacillus polymyxa neutral protease. In accordance with one aspect of the disclosure, optimal neutral protease concentration and collagenase concentrations (Cl and C2 collagenase) and optimal ratio of solution volume (mis) to bone fragment weight (mgs) are determined.
[0230] In some embodiments, a collagenase may include Clostridium histolyticum further comprising two active isoforms, Cl and C2. In some embodiments, one or more collagenases comprising isoforms Cl and C2 may be present in the digestion solution at a ratio comprising more collagenase isoform Cl than collagenase isoform C2. In some embodiments, the ratio of collagenase isoform Cl to collagenase isoform C2 may be about 30 to about 70: about 10 to about 29. In some embodiments, the ratio of collagenase isoform Cl to collagenase C2 may be 35: 15. In some embodiments, the mass ratio of Cl and C2 for each concentration may be 70:30, 54:46, 37:63, 82: 18, 54:46, and 90: 10.
[0231] In some embodiments, the neutral protease may be Paneibacillus polymyxa neutral protease. In some embodiments, the neutral protease concentration may be about 2 U/ml to about 21 U/ml. In some embodiments, the neutral protease concentration may be about 2 U/ml to about 7 U/ml, about 2 U/ml to about 12 U/ml, about 2 U/ml to about 17 U/ml, about 2 U/ml to about 21 U/ml, about 7 U/ml to about 12 U/ml, about 7 U/ml to about 17 U/ml, about 7 U/ml to about 21 U/ml, about 12 U/ml to about 17 U/ml, about 12 U/ml to about 21 U/ml, or about 17 U/ml to about 21 U/ml. In some embodiments, the neutral protease concentration may be about 2 U/ml, about 7 U/ml, about 12 U/ml, about 17 U/ml, or about 21 U/ml. In some embodiments, the neutral protease concentration may be at least about 2 U/ml, about 7 U/ml, about 12 U/ml, or about 17 U/ml. In some embodiments, the neutral protease concentration may be at most about 7 U/ml, about 12 U/ml, about 17 U/ml, or about 21 U/ml. In some embodiments, the digestion solution may comprise the neutral protease at an activity of about 19.6 U/ml.
[0232] In some embodiments, the collagenase concentration is about 0.05 U/ml to about 1.6 U/ml. In some embodiments, the collagenase concentration is about 0.05 U/ml to about 0.1 U/ml, about 0.05 U/ml to about 0.15 U/ml, about 0.05 U/ml to about 0.2 U/ml, about 0.05 U/ml to about 0.25 U/ml, about 0.05 U/ml to about 0.3 U/ml, about 0.05 U/ml to about 0.35 U/ml, about 0.05 U/ml to about 0.4 U/ml, about 0.05 U/ml to about 0.8 U/ml, about 0.05 U/ml to about 1.2 U/ml, about 0.05 U/ml to about 1.6 U/ml, about 0.1 U/ml to about 0.15 U/ml, about 0.1 U/ml to about 0.2 U/ml, about 0.1 U/ml to about 0.25 U/ml, about 0.1 U/ml to about 0.3 U/ml, about 0.1 U/ml to about 0.35 U/ml, about 0.1 U/ml to about 0.4 U/ml, about 0.1 U/ml to about 0.8 U/ml, about 0.1 U/ml to about 1.2 U/ml, about 0.1 U/ml to about 1.6 U/ml, about 0.15 U/ml to about 0.2 U/ml, about 0.15 U/ml to about 0.25 U/ml, about 0.15 U/ml to about 0.3 U/ml, about 0.15 U/ml to about 0.35 U/ml, about 0.15 U/ml to about 0.4 U/ml, about 0.15 U/ml to about 0.8 U/ml, about 0.15 U/ml to about 1.2 U/ml, about 0.15 U/ml to about 1.6 U/ml, about 0.2 U/ml to about 0.25 U/ml, about 0.2 U/ml to about 0.3 U/ml, about 0.2 U/ml to about 0.35 U/ml, about 0.2 U/ml to about 0.4 U/ml, about 0.2 U/ml to about 0.8 U/ml, about 0.2 U/ml to about 1.2 U/ml, about 0.2 U/ml to about 1.6 U/ml, about 0.25 U/ml to about 0.3 U/ml, about 0.25 U/ml to about 0.35 U/ml, about 0.25 U/ml to about 0.4 U/ml, about 0.25 U/ml to about 0.8 U/ml, about 0.25 U/ml to about 1.2 U/rnl, about 0.25 U/rnl to about 1.6 U/rnl, about 0.3 U/rnl to about 0.35 U/ml, about 0.3 U/ml to about 0.4 U/ml, about 0.3 U/ml to about 0.8 U/ml, about 0.3 U/ml to about 1.2 U/ml, about 0.3 U/ml to about 1.6 U/ml, about 0.35 U/ml to about 0.4 U/ml, about 0.35 U/ml to about 0.8 U/ml, about 0.35 U/ml to about 1.2 U/ml, about 0.35 U/ml to about 1.6 U/ml, about 0.4 U/ml to about 0.8 U/ml, about 0.4 U/ml to about 1.2 U/ml, about 0.4 U/ml to about 1.6 U/ml, about 0.8 U/ml to about 1.2 U/ml, about 0.8 U/ml to about 1.6 U/ml, or about 1.2 U/ml to about 1.6 U/ml. In some embodiments, the collagenase concentration is about 0.05 U/ml, about 0.1 U/ml, about 0.15 U/ml, about 0.2 U/ml, about 0.25 U/ml, about 0.3 U/ml, about 0.35 U/ml, about 0.4 U/ml, about 0.8 U/ml, about 1.2 U/ml, or about 1.6 U/ml. In some embodiments, the collagenase concentration is at least about 0.05 U/ml, about 0.1 U/ml, about 0.15 U/ml, about 0.2 U/ml, about 0.25 U/ml, about 0.3 U/ml, about 0.35 U/ml, about 0.4 U/ml, about 0.8 U/ml, or about 1.2 U/ml. In some embodiments, the collagenase concentration is at most about 0.1 U/ml, about 0.15 U/ml, about 0.2 U/ml, about 0.25 U/ml, about 0.3 U/ml, about 0.35 U/ml, about 0.4 U/ml, about 0.8 U/ml, about 1.2 U/ml, or about 1.6 U/ml.
[0233] In accordance with one aspect of the disclosure, neutral protease concentration and collagenase concentrations (Cl and C2 collagenase) and ratio of solution volume (mis) to bone fragment weight (mgs) are determined.
[0234] In some embodiments, the total collagenase concentrations (Cl and C2 collagenase) are about 25 pg/ml to about 100 pg/ml. In some embodiments, the total collagenase concentrations are about 25 pg/ml to about 32.5 pg/ml, about 25 pg/ml to about 47.5 pg/ml, about 25 pg/ml to about 42.5 pg/ml, about 25 pg/ml to about 50 pg/ml, about 25 pg/ml to about 65 pg/ml, about 25 pg/ml to about 77.5 pg/ml, about 25 pg/ml to about 85 pg/ml, about 25 pg/ml to about 100 pg/ml, about 32.5 pg/ml to about 47.5 pg/ml, about 32.5 pg/ml to about
42.5 pg/ml, about 32.5 pg/ml to about 50 pg/ml, about 32.5 pg/ml to about 65 pg/ml, about
32.5 pg/ml to about 77.5 pg/ml, about 32.5 pg/ml to about 85 pg/ml, about 32.5 pg/ml to about 100 pg/ml, about 47.5 pg/ml to about 42.5 pg/ml, about 47.5 pg/ml to about 50 pg/ml, about
47.5 pg/ml to about 65 pg/ml, about 47.5 pg/ml to about 77.5 pg/ml, about 47.5 pg/ml to about 85 pg/ml, about 47.5 pg/ml to about 100 pg/ml, about 42.5 pg/ml to about 50 pg/ml, about
42.5 pg/ml to about 65 pg/ml, about 42.5 pg/ml to about 77.5 pg/ml, about 42.5 pg/ml to about 85 pg/ml, about 42.5 pg/ml to about 100 pg/ml, about 50 pg/ml to about 65 pg/ml, about 50 pg/ml to about 77.5 pg/ml, about 50 pg/ml to about 85 pg/ml, about 50 pg/ml to about 100 pg/ml, about 65 pg/ml to about 77.5 pg/ml, about 65 pg/ml to about 85 pg/ml, about 65 pg/ml to about 100 gg/ml, about 77.5 gg/ml to about 85 gg/ml, about 77.5 gg/ml to about 100 gg/ml, or about 85 gg/ml to about 100 gg/ml. In some embodiments, the total collagenase concentrations are about 25 gg/ml, about 32.5 gg/ml, about 47.5 gg/ml, about 42.5 gg/ml, about 50 gg/ml, about 65 gg/ml, about 77.5 gg/ml, about 85 gg/ml, or about 100 gg/ml. In some embodiments, the total collagenase concentrations are at least about 25 gg/ml, about 32.5 gg/ml, about 47.5 gg/ml, about 42.5 gg/ml, about 50 gg/ml, about 65 gg/ml, about 77.5 gg/ml, or about 85 gg/ml. In some embodiments, the total collagenase concentrations are at most about
32.5 gg/ml, about 47.5 gg/ml, about 42.5 gg/ml, about 50 gg/ml, about 65 gg/ml, about 77.5 gg/ml, about 85 gg/ml, or about 100 gg/ml.
[0235] In some embodiments, the mass ratio of Cl and C2 for each concentration are 70:30, 54:46, 37:63, 82: 18 and 90: 10, respectively.
[0236] The volume to weight ratio of digestion solution to captured ground bone is about 1 : 1 to about 15: 1, e.g., about 5: 1. In some embodiments, the ratio may be 1 : 1, 2.5:1, 5:1, 7.5: 1, 10: 1 and 15: 1 (volume:weight). In some embodiments, the incubation period is about 1 hour to about 4 hours. In some embodiments, the incubation period is about 1 hour to about 1.5 hours, about 1 hour to about 2 hours, about 1 hour to about 2.5 hours, about 1 hour to about 3 hours, about 1.5 hours to about 2 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 3 hours, about 2 hours to about 2.5 hours, about 2 hours to about 3 hours, or about 2.5 hours to about 3 hours. In some embodiments, the incubation period is about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. In some embodiments, the incubation period is at least about 1 hour, about 1.5 hours, about 2 hours, or about 2.5 hours. In some embodiments, the incubation period is at most about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, or about 4 hours. In some cases, the digestion solution is contacted with the captured ground bone for up to about 4 hours.
[0237] In some cases, the optimal volume-to- weight ratio has been found to be 5:1 at an optimal incubation time of 2.5 hours. The optimal protease produced neutral protease activity of
19.6 U/ml. On the other hand, it was found that total viable MSC cell count is generally insensitive to collagenase concentration. It was also found that the yields produced by recombinant collagenase isoforms Cl and C2 are similar to the yields with purified collagenase, regardless of the C1/C2 ratio. Further details of the MSC recovery process of the present disclosure are found in the technical article in Johnstone et al., “Identification and characterization of a large source of primary mesenchymal stem cells tightly adhered to bone surfaces of human vertebral body marrow cavities ” bioRxiv 2020.05.04.076950; doi.org/10.1101/2020.05.04.076950, the entire disclosure of which is incorporated herein by reference.
[0238] According to the process, fragments of VB bone are placed in cryoprotectant solution comprised of PLASMA-LYTE™, 2.5% human serum albumin and 10% dimethyl sulfoxide (DMSO) and incubated for 1 hour at 4°C. In some embodiments, the incubation period is about 1 hour to about 3 hours. In some embodiments, the incubation period is about 1 hour to about 1.5 hours, about 1 hour to about 2 hours, about 1 hour to about 2.5 hours, about 1 hour to about 3 hours, about 1.5 hours to about 2 hours, about 1.5 hours to about 2.5 hours, about 1.5 hours to about 3 hours, about 2 hours to about 2.5 hours, about 2 hours to about 3 hours, or about 2.5 hours to about 3 hours. In some embodiments, the incubation period is about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. In some embodiments, the incubation period is at least about 1 hour, about 1.5 hours, about 2 hours, or about 2.5 hours. In some embodiments, the incubation period is at most about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. The solution is removed and the bone fragments cooled at a rate of ~l°/min to -86°C and then plunged into liquid nitrogen. After 24-48 hours in liquid nitrogen, the bone fragments are thawed rapidly in a water bath set at 37°C and then washed in saline and digested using the collagenase/protease solution described above.
[0239] In some embodiments, the volume-to-weight ratio was 5: 1 at an incubation time of 2.5 hours. In some embodiments, the protease produced neutral protease activity of 19.6 U/ml. [0240] The mixture of cells liberated by digesting VB bone fragment is cultured on tissue- coated plastic in the presence of Mesencult medium to select proliferative vBA-MSC. Freshly digested preparations as well as different passages of VB-MSC can be characterized by flow cytometry, colony forming unit-fibroblast (CFU-F) potential, population doubling time (PDT) and trilineage (adipogenic, chondrogenic, and osteogenic) differentiation in vitro.
[0241] In some embodiments, the method of human MSC extraction disclosed herein may be capable of extracting quantities of about 10 million to about 10 billion. In some embodiments, human MSCs may be administered in quantities of about 10 million to about 100 million, about 10 million to about 1 billion, about 10 million to about 10 billion, about 100 million to about 1 billion, about 100 million to about 10 billion, or about 1 billion to about 10 billion. In some embodiments, human MSCs may be administered in quantities of about 10 million, about 100 million, about 1 billion, or about 10 billion. In some embodiments, human MSCs may be administered in quantities of at least about 10 million, about 100 million, or about 1 billion. In some embodiments, human MSCs may be administered in quantities of at most about 100 million, about 1 billion, or about 10 billion. Culturing of MS Cs
[0242] In one aspect of the present disclosure, extracted MSCs may be (e.g., vBA-MSCs and/or vBM-MSCs) cultured and passaged to realize clinical scale MSC preparation having a desired number of MSCs with the antigen profiles taught herein. In some embodiments, a clinical scale preparation may be obtained by serial passage expansion where each passage includes a step of splitting the previous culture into a plurality of cultures at a given ratio. Each passaging step increases the number of concurrent cultures in the preparation. In some embodiments, clinical scale preparations having the instant preparation profiles, e.g. antigen profile, TNFRI profile, cryopreservation profile, differentiation profile, and/or sterility (with respect to pathogens) are successfully produced.
[0243] MSCs for clinical use are derived from the continuous manufacturing process of obtained bone marrow from deceased-donor vertebral bodies to selection of MSC for further cell culture. MSCs are cultured out of the bone marrow units collected. The overall manufacturing process to derive the final MSC product is illustrated in FIG 1. As shown in FIG. 1, MSCs are cultured out of primary bone marrow units using a tiered culture system.
Passage 0 to Passage 1: Primary Cell Culture and Master Cell Bank (MCB) Preparation [0244] As shown in FIG. 1, a unit of bone marrow cells (as obtained from a herein disclosed method and either freshly collected or previously cryopreserved) is utilized for primary cell culture. A unit is thawed if previously frozen. The bone marrow cells are plated in MSC Culture Media and cultured for about 14 days (e.g., from about 10 days to about 16 days). These are termed Passage 0 or P0 cells. MSC Culture Media may comprise Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM) supplemented with human platelet lysate (hPL) and basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). In some embodiments, no heparin is included in the MSC Culture Media. In some embodiments, the MSC Culture Media comprises no bovine or porcine components. In various embodiments, no antibiotics and/or antimycotics are used in the MSC Culture Media. During culturing of the P0 cells, fresh media changes occur about every three to about four days.
[0245] When the P0 cells have reached greater than about 75% confluence, cells are detached and immediately replated in MSC Culture Media. These are termed Passage 1 or Pl cells. The Pl cells are cultured for about five to about six days and until they have reached greater than about 75% confluence.
[0246] When the Pl cells are to be cryofrozen, the cells are detached, resuspended in PlasmaLyte-A which may be supplemented with human serum albumin (HSA) and dimethyl sulfoxide (DMSO). About 13 million cells/mL of Pl cells can be packaged in 2-mL CellSeal® closed-system cryovials at 2 mL per cryovial using an automatic filler. The cryovials can be cryopreserved and placed into the vapor phase above LN2 in alarm monitored cryogenic tanks for storage at <-140°C. These cells constitute the Master Cell Bank (MCB).
[0247] In other embodiments, the Pl cells are detached and further cultured into passage 2 cells.
Passage 1 to Passage 3: Working Cell Bank (WCB) Preparation
[0248] One vial of Pl cells, if cryofrozen, is thawed and plated in MSC Culture Media for about five to about six days. These are now termed Passage 2 or P2 cells. When the Pl cells reach greater than 75% confluence, the P2 cells are detached and immediately replated in MSC Culture Media. These cells are considered Passage 3 or P3 cells.
[0249] Alternately, unfrozen Pl cells are detached from the Pl culture and further cultured into passage 2 cells and into P3 cells (as described above).
[0250] The P3 cells are cultured for about five to about 6 days. Once the culturing container have reached greater than 75% confluence, cells are detached, resuspended in PlasmaLyte-A, optionally supplemented with HSA and DMSO. About 6 million cells/mL of P3 cells can be packaged in cryobags at about 410 million P3 cells in 65 mL per bag. The bags are then cryopreserved and placed into the vapor phase above LN2 in alarm monitored cryogenic tanks for storage at <-140°C. These cells now constitute the Working Cell Bank (WCB).
[0251] In other embodiments, the P3 cells are detached and further cultured into passage 4 cells.
Passage 4: End of Production (EOP) Cell Preparation
[0252] One bag from the WCB, if cryofrozen, is thawed and plated in MSC Culture Media. These cells are now termed Passage 4 or P4 cells. The P4 cells are cultured for about four days to about five days. Once the culturing container has reached greater than 75% confluence, P4 cells are detached and resuspended in PlasmaLyte-A, optionally supplemented with HSA and DMSO. About 20 million cells/mL of P4 cells can packaged in 5-mL CellSeal closed-system cryovials at 5 mL per cryovial using an automatic filler. Cells are then cryopreserved and placed into the vapor phase above LN2 in alarm monitored cryogenic tanks for storage at <-140°C. These cells constitute the End of Production Bank (EOP).
[0253] In other embodiments, the P4 cells are detached and then further cultured to become a cell composition for administration to a subject in need.
Cryo-recovered (CR) Cell Preparation
[0254] For one clinical dose, one vial from the EOP bank is thawed, if cryofrozen, and plated in MSC Culture Media at a density from about 32,000 to about 40,000 cells/cm2. These cells are cultured briefly (from about 20 hours to about 28 hours) to allow cell recovery only and prior to entering logarithmic phase. After this brief culturing, cells are detached, resuspended in PlasmaLyte-A, optionally supplemented with HSA. About 10 million cells/mL can be packaged in 5 mL vials (e.g., CellSeal closed-system cryovials). A clinical dose equates to two (2) 5 mL vials. Cells can be used at the site where this final culturing occurred. Alternately, cells can be shipped to a treatment site under cold, non-freezing temperatures, e.g., from about 2 to about 8°C. Up to 10 clinical doses may be prepared at one time, depending on the treatment schedule.
[0255] Alternately, P4 cells are detached and plated in MSC Culture Media at a density from about 32,000 to about 40,000 cells/cm2. These cells are cultured briefly (from about 20 hours to about 28 hours) to allow cell recovery only (e.g., to allow metabolic activity to resume post-thaw) and prior to entering logarithmic phase. After this brief culturing, cells are detached, resuspended in PlasmaLyte-A, optionally supplemented with HSA. About 10 million cells/mL can be packaged in 5 mL vials (e.g., CellSeal closed-system cryovials). A clinical dose equates to two (2) 5 mL vials, e.g., about 100 million cells. Cells can be used at the site where this final culturing occurred. Alternately, cells can be shipped to a treatment site under cold, non-freezing temperatures, e.g., from about 2 to about 8°C. Up to 10 clinical doses may be prepared at one time, depending on the treatment schedule.
[0256] In some embodiments, the primary MSCs (obtained from bone marrow) may be further passaged to non-primary cells (e.g. removed from the culture surface and expanded into additional area) by seeding at a density of about 1,000 to about one million nucleated cells/cm2 of culture dish (e.g. about 5,900 cells/cm2 plus and minus about 1,200), and then culturing for additional days, e.g. about 14±about 2 days. In suitable embodiments, the primary cells may be grown to confluence, and in some instances may be passaged to a second culture of nonprimary cells by seeding the primary cells from a confluent primary cell culture in the second culture surface in an amount below confluence and growing the non-primary culture to confluence. This method can be repeated for additional passages.
[0257] In some embodiments, the MSCs in the treatment composition may originate from sequential generation number (i.e., they are within about 1 or about 2 or about 3 or about 4 cell doublings of each other). Optionally, the average number of cell doublings in the present composition treatment composition may be about 20 to about 25 doublings. Optionally, the average number of cell doublings in the present treatment composition may be about 9 to about 13 (e.g., about 11 or about 11.2) doublings arising from the primary culture, plus about 1, about 2, about 3, or about 4 doublings per passage (for example, about 2.5 doublings per passage). Exemplary average cell doublings in present preparations may be of about 13.5, about 16, about 18.5, about 21, about 23.5, about 26, about 28.5, about 31, about 33.5, or about 36 when produced by about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passages, respectively.
[0258] In some embodiments, notwithstanding one or more population doublings, the MSCs in the treatment composition (e.g., vB A-MSCs and/or vBM-MSCs) may originate from MSCs that were cultured through about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 passages.
[0259] Confluence refers to the percentage of the surface of a culture dish that is covered by adherent cells. For example, 50 percent confluence means roughly half of the surface is covered, while 100 percent confluence means the surface is completely covered by the cells, and no more room is left for the cells to grow as a monolayer. By “75% confluence or greater” is meant about 75% confluence, about 76% confluence, about 77% confluence, about 78% confluence, about 79% confluence, about 80% confluence, about 81% confluence, about 82% confluence, about 83% confluence, about 84% confluence, about 85% confluence, about 86% confluence, about 87% confluence, about 88% confluence, about 89% confluence, about 90% confluence, about 91% confluence, about 92% confluence, about 93% confluence, about 94% confluence, about 95% confluence, about 96% confluence, about 97% confluence, about 98% confluence, about 99% confluence, or about 100% confluence. Confluence can be determined by any standard method used in the field. See, e.g., Haenel, Frauke, and Norbert Garbow. "Cell counting and confluency analysis as quality controls in cell-based assays." Multimode Detection (2014): 1-5.
[0260] Additional details regarding the method of manufacturing cultured MSCs for administering to a subject in need, e.g., with a perianal fistula, is disclosed below.
[0261] In some embodiments, no heparin is included in the MSC Culture Media. In some embodiments, the MSC Culture Media comprises no bovine or porcine components. In various embodiments, no antibiotics and/or antimycotics are used in the MSC Culture Media.
[0262] In some embodiments, extracted MSCs are cultured in a MSC Culture Medium wherein the medium is configured to generate MSCs having the instant preparation profiles, e.g. antigen profile, TNFRI profile, cryopreservation profile, differentiation profile, and/or sterility (with respect to pathogens). In some embodiments, the medium comprises minimal essential medium (MEM). In some embodiments, the medium comprises alpha MEM. In some embodiments, the medium comprises human platelet lysate (hPL), e.g., Stemulate™. In some embodiments, the medium comprises fibroblast growth factor (FGF) e.g., carrier free FGF and/or FGF-2). In some embodiments, the medium comprises epidermal growth factor (EGF, e.g., carrier free EGF). In some embodiments, the medium comprises alpha MEM, hPL, FGF, EGF, or any combination thereof. In some embodiments, the medium comprises alpha MEM, hPL, FGF, and EGF. In some embodiments, the medium does not further require heparin.
[0263] In some embodiments, hPL is present in the MSC Culture Medium at about 1 % to about 21 %. In some embodiments, hPL is present in the medium at about 1 % to about 3 %, about 1 % to about 5 %, about 1 % to about 7 %, about 1 % to about 9 %, about 1 % to about
10 %, about 1 % to about 11 %, about 1 % to about 13 %, about 1 % to about 15 %, about 1 % to about 17 %, about 1 % to about 19 %, about 1 % to about 21 %, about 3 % to about 5 %, about 3 % to about 7 %, about 3 % to about 9 %, about 3 % to about 10 %, about 3 % to about
11 %, about 3 % to about 13 %, about 3 % to about 15 %, about 3 % to about 17 %, about 3 % to about 19 %, about 3 % to about 21 %, about 5 % to about 7 %, about 5 % to about 9 %, about 5 % to about 10 %, about 5 % to about 11 %, about 5 % to about 13 %, about 5 % to about 15 %, about 5 % to about 17 %, about 5 % to about 19 %, about 5 % to about 21 %, about 7 % to about 9 %, about 7 % to about 10 %, about 7 % to about 11 %, about 7 % to about 13 %, about 7 % to about 15 %, about 7 % to about 17 %, about 7 % to about 19 %, about 7 % to about 21 %, about 9 % to about 10 %, about 9 % to about 11 %, about 9 % to about 13 %, about 9 % to about 15 %, about 9 % to about 17 %, about 9 % to about 19 %, about 9 % to about 21 %, about 10 % to about 11 %, about 10 % to about 13 %, about 10 % to about 15 %, about 10 % to about 17 %, about 10 % to about 19 %, about 10 % to about 21 %, about 11 % to about 13 %, about 11 % to about 15 %, about 11 % to about 17 %, about 11 % to about 19 %, about 11 % to about 21 %, about 13 % to about 15 %, about 13 % to about 17 %, about 13 % to about 19 %, about 13 % to about 21 %, about 15 % to about 17 %, about 15 % to about 19 %, about 15 % to about 21 %, about 17 % to about 19 %, about 17 % to about 21 %, or about 19 % to about 21 %. In some embodiments, hPL is present in the medium at about 1 %, about 3 %, about 5 %, about 7 %, about 9 %, about 10 %, about 11 %, about 13 %, about 15 %, about 17 %, about 19 %, or about 21 %. In some embodiments, hPL is present in the medium at least about 1 %, about 3 %, about 5 %, about 7 %, about 9 %, about 10 %, about 11 %, about 13 %, about 15 %, about 17 %, or about 19 %. In some embodiments, hPL is present in the medium at most about 3 %, about 5 %, about 7 %, about 9 %, about 10 %, about 11 %, about 13 %, about 15 %, about 17 %, about 19 %, or about 21 %.In some embodiments, FGF is present in the medium at about 0.5 ng/mL to about 5 ng/mL.
[0264] In some embodiments, FGF is present in the MSC Culture Medium at about 0.5 ng/mL to about 1 ng/mL, about 0.5 ng/mL to about 1.5 ng/mL, about 0.5 ng/mL to about 2 ng/mL, about 0.5 ng/mL to about 2.5 ng/mL, about 0.5 ng/mL to about 3 ng/mL, about 0.5 ng/mL to about 3.5 ng/mL, about 0.5 ng/mL to about 4 ng/mL, about 0.5 ng/mL to about 4.5 ng/mL, about 0.5 ng/mL to about 5 ng/mL, about 1 ng/mL to about 1.5 ng/mL, about 1 ng/mL to about 2 ng/mL, about 1 ng/mL to about 2.5 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 3.5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 4.5 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1.5 ng/mL to about 2 ng/mL, about 1.5 ng/mL to about 2.5 ng/mL, about
1.5 ng/mL to about 3 ng/mL, about 1.5 ng/mL to about 3.5 ng/mL, about 1.5 ng/mL to about 4 ng/mL, about 1.5 ng/mL to about 4.5 ng/mL, about 1.5 ng/mL to about 5 ng/mL, about 2 ng/mL to about 2.5 ng/mL, about 2 ng/mL to about 3 ng/mL, about 2 ng/mL to about 3.5 ng/mL, about
2 ng/mL to about 4 ng/mL, about 2 ng/mL to about 4.5 ng/mL, about 2 ng/mL to about 5 ng/mL, about 2.5 ng/mL to about 3 ng/mL, about 2.5 ng/mL to about 3.5 ng/mL, about 2.5 ng/mL to about 4 ng/mL, about 2.5 ng/mL to about 4.5 ng/mL, about 2.5 ng/mL to about 5 ng/mL, about
3 ng/mL to about 3.5 ng/mL, about 3 ng/mL to about 4 ng/mL, about 3 ng/mL to about 4.5 ng/mL, about 3 ng/mL to about 5 ng/mL, about 3.5 ng/mL to about 4 ng/mL, about 3.5 ng/mL to about
4.5 ng/mL, about 3.5 ng/mL to about 5 ng/mL, about 4 ng/mL to about 4.5 ng/mL, about 4 ng/mL to about 5 ng/mL, or about 4.5 ng/mL to about 5 ng/mL. In some embodiments, FGF is present in the medium at about 0.5 ng/mL, about 1 ng/mL, about 1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, about 4.5 ng/mL, or about 5 ng/mL. In some embodiments, FGF is present in the medium at least about 0.5 ng/mL, about 1 ng/mL, about
1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, or about 4.5 ng/mL. In some embodiments, FGF is present in the medium at most about 1 ng/mL, about 1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, about 4.5 ng/mL, or about 5 ng/mL.
[0265] In some embodiments, EGF is present in the MSC Culture Medium at about 0.5 ng/mL to about 5 ng/mL. In some embodiments, EGF is present in the medium at about 0.5 ng/mL to about 1 ng/mL, about 0.5 ng/mL to about 1.5 ng/mL, about 0.5 ng/mL to about 2 ng/mL, about 0.5 ng/mL to about 2.5 ng/mL, about 0.5 ng/mL to about 3 ng/mL, about 0.5 ng/mL to about 3.5 ng/mL, about 0.5 ng/mL to about 4 ng/mL, about 0.5 ng/mL to about 4.5 ng/mL, about 0.5 ng/mL to about 5 ng/mL, about 1 ng/mL to about 1.5 ng/mL, about 1 ng/mL to about 2 ng/mL, about 1 ng/mL to about 2.5 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 3.5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 4.5 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1.5 ng/mL to about 2 ng/mL, about 1.5 ng/mL to about 2.5 ng/mL, about
1.5 ng/mL to about 3 ng/mL, about 1.5 ng/mL to about 3.5 ng/mL, about 1.5 ng/mL to about 4 ng/mL, about 1.5 ng/mL to about 4.5 ng/mL, about 1.5 ng/mL to about 5 ng/mL, about 2 ng/mL to about 2.5 ng/mL, about 2 ng/mL to about 3 ng/mL, about 2 ng/mL to about 3.5 ng/mL, about
2 ng/mL to about 4 ng/mL, about 2 ng/mL to about 4.5 ng/mL, about 2 ng/mL to about 5 ng/mL, about 2.5 ng/mL to about 3 ng/mL, about 2.5 ng/mL to about 3.5 ng/mL, about 2.5 ng/mL to about 4 ng/mL, about 2.5 ng/mL to about 4.5 ng/mL, about 2.5 ng/mL to about 5 ng/mL, about
3 ng/mL to about 3.5 ng/mL, about 3 ng/mL to about 4 ng/mL, about 3 ng/mL to about 4.5 ng/mL, about 3 ng/mL to about 5 ng/mL, about 3.5 ng/mL to about 4 ng/mL, about 3.5 ng/mL to about
4.5 ng/mL, about 3.5 ng/mL to about 5 ng/mL, about 4 ng/mL to about 4.5 ng/mL, about 4 ng/mL to about 5 ng/mL, or about 4.5 ng/mL to about 5 ng/mL. In some embodiments, EGF is present in the medium at about 0.5 ng/mL, about 1 ng/mL, about 1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, about 4.5 ng/mL, or about 5 ng/mL. In some embodiments, EGF is present in the medium at least about 0.5 ng/mL, about 1 ng/mL, about 1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, or about 4.5 ng/mL. In some embodiments, EGF is present in the medium at most about 1 ng/mL, about 1.5 ng/mL, about 2 ng/mL, about 2.5 ng/mL, about 3 ng/mL, about 3.5 ng/mL, about 4 ng/mL, about 4.5 ng/mL, or about 5 ng/mL.
[0266] In some embodiments, the MSC Culture Medium comprises a modified alpha MEM. In some embodiments, the modified alpha MEM comprises one or more inorganic salts, one or more amino acids, one or more vitamins, glucose, lipoic acid, sodium bicarbonate, sodium pyruvate, or any combination thereof.
[0267] In some embodiments, the one or more inorganic salts comprise calcium chloride (dihydrate), magnesium sulfate (heptahydrate), potassium chloride, sodium chloride, sodium phosphate monobasic (dehydrate), or any combination thereof. In some embodiments, each inorganic salt present in the MSC Culture Medium is present at about 100 mg/Liter to about 800 mg/Liter. In some embodiments, each inorganic salt present in the medium is present at about 100 mg/Liter to about 200 mg/Liter, about 100 mg/Liter to about 300 mg/Liter, about 100 mg/Liter to about 400 mg/Liter, about 100 mg/Liter to about 500 mg/Liter, about 100 mg/Liter to about 600 mg/Liter, about 100 mg/Liter to about 700 mg/Liter, about 100 mg/Liter to about 800 mg/Liter, about 200 mg/Liter to about 300 mg/Liter, about 200 mg/Liter to about 400 mg/Liter, about 200 mg/Liter to about 500 mg/Liter, about 200 mg/Liter to about 600 mg/Liter, about 200 mg/Liter to about 700 mg/Liter, about 200 mg/Liter to about 800 mg/Liter, about 300 mg/Liter to about 400 mg/Liter, about 300 mg/Liter to about 500 mg/Liter, about 300 mg/Liter to about 600 mg/Liter, about 300 mg/Liter to about 700 mg/Liter, about 300 mg/Liter to about 800 mg/Liter, about 400 mg/Liter to about 500 mg/Liter, about 400 mg/Liter to about 600 mg/Liter, about 400 mg/Liter to about 700 mg/Liter, about 400 mg/Liter to about 800 mg/Liter, about 500 mg/Liter to about 600 mg/Liter, about 500 mg/Liter to about 700 mg/Liter, about 500 mg/Liter to about 800 mg/Liter, about 600 mg/Liter to about 700 mg/Liter, about 600 mg/Liter to about 800 mg/Liter, or about 700 mg/Liter to about 800 mg/Liter. In some embodiments, each inorganic salt present in the medium is present at about 100 mg/Liter, about 200 mg/Liter, about 300 mg/Liter, about 400 mg/Liter, about 500 mg/Liter, about 600 mg/Liter, about 700 mg/Liter, or about 800 mg/Liter. In some embodiments, each inorganic salt present in the medium is present at least about 100 mg/Liter, about 200 mg/Liter, about 300 mg/Liter, about 400 mg/Liter, about 500 mg/Liter, about 600 mg/Liter, or about 700 mg/Liter. In some embodiments, each inorganic salt present in the medium is present at most about 200 mg/Liter, about 300 mg/Liter, about 400 mg/Liter, about 500 mg/Liter, about 600 mg/Liter, about 700 mg/Liter, or about 800 mg/Liter.
[0268] In some embodiments, the one or more amino acids comprise glycine, alanine, alanyl-glutamine, arginine (HC1), asparagine (monohydrate), aspartic acid, cysteine (HC1) (monohydrate), cystine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or any combination thereof. In some embodiments, the one or more amino acids are present in the L isoform. In some embodiments, the one or more amino acids are present in the D isoform. In some embodiments, the one or more amino acids are present in both isoforms. In some embodiments, each amino acid present in the medium is present at about 10 mg/Liter to about 100 mg/Liter. In some embodiments, each amino acid present in the MSC Culture Medium is present at about 10 mg/Liter to about 20 mg/Liter, about 10 mg/Liter to about 30 mg/Liter, about 10 mg/Liter to about 40 mg/Liter, about 10 mg/Liter to about 50 mg/Liter, about 10 mg/Liter to about 60 mg/Liter, about 10 mg/Liter to about 70 mg/Liter, about 10 mg/Liter to about 80 mg/Liter, about 10 mg/Liter to about 90 mg/Liter, about 10 mg/Liter to about 100 mg/Liter, about 20 mg/Liter to about 30 mg/Liter, about 20 mg/Liter to about 40 mg/Liter, about 20 mg/Liter to about 50 mg/Liter, about 20 mg/Liter to about 60 mg/Liter, about 20 mg/Liter to about 70 mg/Liter, about 20 mg/Liter to about 80 mg/Liter, about 20 mg/Liter to about 90 mg/Liter, about 20 mg/Liter to about 100 mg/Liter, about 30 mg/Liter to about 40 mg/Liter, about 30 mg/Liter to about 50 mg/Liter, about 30 mg/Liter to about 60 mg/Liter, about 30 mg/Liter to about 70 mg/Liter, about 30 mg/Liter to about 80 mg/Liter, about 30 mg/Liter to about 90 mg/Liter, about 30 mg/Liter to about 100 mg/Liter, about 40 mg/Liter to about 50 mg/Liter, about 40 mg/Liter to about 60 mg/Liter, about 40 mg/Liter to about 70 mg/Liter, about 40 mg/Liter to about 80 mg/Liter, about 40 mg/Liter to about 90 mg/Liter, about 40 mg/Liter to about 100 mg/Liter, about 50 mg/Liter to about 60 mg/Liter, about 50 mg/Liter to about 70 mg/Liter, about 50 mg/Liter to about 80 mg/Liter, about 50 mg/Liter to about 90 mg/Liter, about 50 mg/Liter to about 100 mg/Liter, about 60 mg/Liter to about 70 mg/Liter, about 60 mg/Liter to about 80 mg/Liter, about 60 mg/Liter to about 90 mg/Liter, about 60 mg/Liter to about 100 mg/Liter, about 70 mg/Liter to about 80 mg/Liter, about 70 mg/Liter to about 90 mg/Liter, about 70 mg/Liter to about 100 mg/Liter, about 80 mg/Liter to about 90 mg/Liter, about 80 mg/Liter to about 100 mg/Liter, or about 90 mg/Liter to about 100 mg/Liter. In some embodiments, each amino acid present in the medium is present at about 10 mg/Liter, about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, or about 100 mg/Liter. In some embodiments, each amino acid present in the medium is present at least about 10 mg/Liter, about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, or about 90 mg/Liter. In some embodiments, each amino acid present in the medium is present at most about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, or about 100 mg/Liter. In some embodiments, each amino acid present in the medium is present at about 100 mg/Liter to about 500 mg/Liter. In some embodiments, each amino acid present in the medium is present at about 100 mg/Liter to about 200 mg/Liter, about 100 mg/Liter to about 300 mg/Liter, about 100 mg/Liter to about 400 mg/Liter, about 100 mg/Liter to about 500 mg/Liter, about 200 mg/Liter to about 300 mg/Liter, about 200 mg/Liter to about 400 mg/Liter, about 200 mg/Liter to about 500 mg/Liter, about 300 mg/Liter to about 400 mg/Liter, about 300 mg/Liter to about 500 mg/Liter, or about 400 mg/Liter to about 500 mg/Liter. In some embodiments, each amino acid present in the medium is present at about 100 mg/Liter, about 200 mg/Liter, about 300 mg/Liter, about 400 mg/Liter, or about 500 mg/Liter. In some embodiments, each amino acid present in the medium is present at least about 100 mg/Liter, about 200 mg/Liter, about 300 mg/Liter, or about 400 mg/Liter. In some embodiments, each amino acid present in the medium is present at most about 200 mg/Liter, about 300 mg/Liter, about 400 mg/Liter, or about 500 mg/Liter.
[0269] In some embodiments, the one or more vitamins comprise ascorbic acid, biotin, choline chloride, calcium pantothenate, folic acid, myo-inositol, niacinamide, pyridoxal (HC1), pyruvic acid (sodium salt), riboflavin, thiamine (HC1), vitamin B12, or any combination thereof. In some embodiments, the one or more vitamins are present in the L isoform. In some embodiments, the one or more vitamins are present in the D isoform. In some embodiments, the one or more vitamins are present in both isoforms. In some embodiments, each vitamin present in the MSC Culture Medium is present at about 0.1 mg/Liter to about 2 mg/Liter. In some embodiments, each vitamin present in the medium is present at about 0.1 mg/Liter to about 0.3 mg/Liter, about 0.1 mg/Liter to about 0.5 mg/Liter, about 0.1 mg/Liter to about 0.7 mg/Liter, about 0.1 mg/Liter to about 0.9 mg/Liter, about 0.1 mg/Liter to about 1.1 mg/Liter, about 0.1 mg/Liter to about 1.3 mg/Liter, about 0.1 mg/Liter to about 1.5 mg/Liter, about 0.1 mg/Liter to about 1.7 mg/Liter, about 0.1 mg/Liter to about 1.9 mg/Liter, about 0.1 mg/Liter to about 2 mg/Liter, about 0.3 mg/Liter to about 0.5 mg/Liter, about 0.3 mg/Liter to about 0.7 mg/Liter, about 0.3 mg/Liter to about 0.9 mg/Liter, about 0.3 mg/Liter to about 1.1 mg/Liter, about 0.3 mg/Liter to about 1.3 mg/Liter, about 0.3 mg/Liter to about 1.5 mg/Liter, about 0.3 mg/Liter to about 1.7 mg/Liter, about 0.3 mg/Liter to about 1.9 mg/Liter, about 0.3 mg/Liter to about 2 mg/Liter, about 0.5 mg/Liter to about 0.7 mg/Liter, about 0.5 mg/Liter to about 0.9 mg/Liter, about 0.5 mg/Liter to about 1.1 mg/Liter, about 0.5 mg/Liter to about 1.3 mg/Liter, about 0.5 mg/Liter to about 1.5 mg/Liter, about 0.5 mg/Liter to about 1.7 mg/Liter, about 0.5 mg/Liter to about 1.9 mg/Liter, about 0.5 mg/Liter to about 2 mg/Liter, about 0.7 mg/Liter to about 0.9 mg/Liter, about 0.7 mg/Liter to about 1.1 mg/Liter, about 0.7 mg/Liter to about 1.3 mg/Liter, about 0.7 mg/Liter to about 1.5 mg/Liter, about 0.7 mg/Liter to about 1.7 mg/Liter, about 0.7 mg/Liter to about 1.9 mg/Liter, about 0.7 mg/Liter to about 2 mg/Liter, about 0.9 mg/Liter to about 1.1 mg/Liter, about 0.9 mg/Liter to about 1.3 mg/Liter, about 0.9 mg/Liter to about 1.5 mg/Liter, about 0.9 mg/Liter to about 1.7 mg/Liter, about 0.9 mg/Liter to about 1.9 mg/Liter, about 0.9 mg/Liter to about 2 mg/Liter, about 1.1 mg/Liter to about 1.3 mg/Liter, about 1.1 mg/Liter to about 1.5 mg/Liter, about 1.1 mg/Liter to about 1.7 mg/Liter, about 1.1 mg/Liter to about 1.9 mg/Liter, about 1.1 mg/Liter to about 2 mg/Liter, about 1.3 mg/Liter to about 1.5 mg/Liter, about 1.3 mg/Liter to about 1.7 mg/Liter, about 1.3 mg/Liter to about 1.9 mg/Liter, about 1.3 mg/Liter to about 2 mg/Liter, about 1.5 mg/Liter to about 1.7 mg/Liter, about 1.5 mg/Liter to about 1.9 mg/Liter, about 1.5 mg/Liter to about 2 mg/Liter, about 1.7 mg/Liter to about 1.9 mg/Liter, about 1.7 mg/Liter to about 2 mg/Liter, or about 1.9 mg/Liter to about 2 mg/Liter. In some embodiments, each vitamin present in the medium is present at about 0.1 mg/Liter, about 0.3 mg/Liter, about 0.5 mg/Liter, about 0.7 mg/Liter, about 0.9 mg/Liter, about 1.1 mg/Liter, about 1.3 mg/Liter, about 1.5 mg/Liter, about 1.7 mg/Liter, about 1.9 mg/Liter, or about 2 mg/Liter. In some embodiments, each vitamin present in the medium is present at least about 0.1 mg/Liter, about 0.3 mg/Liter, about 0.5 mg/Liter, about 0.7 mg/Liter, about 0.9 mg/Liter, about 1.1 mg/Liter, about 1.3 mg/Liter, about 1.5 mg/Liter, about 1.7 mg/Liter, or about 1.9 mg/Liter. In some embodiments, each vitamin present in the medium is present at most about 0.3 mg/Liter, about 0.5 mg/Liter, about 0.7 mg/Liter, about 0.9 mg/Liter, about 1.1 mg/Liter, about 1.3 mg/Liter, about 1.5 mg/Liter, about 1.7 mg/Liter, about 1.9 mg/Liter, or about 2 mg/Liter. In some embodiments, each vitamin present in the medium is present at about 10 mg/Liter to about 120 mg/Liter. In some embodiments, each vitamin present in the medium is present at about 10 mg/Liter to about 20 mg/Liter, about 10 mg/Liter to about 30 mg/Liter, about 10 mg/Liter to about 40 mg/Liter, about 10 mg/Liter to about 50 mg/Liter, about 10 mg/Liter to about 60 mg/Liter, about 10 mg/Liter to about 70 mg/Liter, about 10 mg/Liter to about 80 mg/Liter, about 10 mg/Liter to about 90 mg/Liter, about 10 mg/Liter to about 100 mg/Liter, about 10 mg/Liter to about 110 mg/Liter, about 10 mg/Liter to about 120 mg/Liter, about 20 mg/Liter to about 30 mg/Liter, about 20 mg/Liter to about 40 mg/Liter, about 20 mg/Liter to about 50 mg/Liter, about 20 mg/Liter to about 60 mg/Liter, about 20 mg/Liter to about 70 mg/Liter, about 20 mg/Liter to about 80 mg/Liter, about 20 mg/Liter to about 90 mg/Liter, about 20 mg/Liter to about 100 mg/Liter, about 20 mg/Liter to about 110 mg/Liter, about 20 mg/Liter to about 120 mg/Liter, about 30 mg/Liter to about 40 mg/Liter, about 30 mg/Liter to about 50 mg/Liter, about 30 mg/Liter to about 60 mg/Liter, about 30 mg/Liter to about 70 mg/Liter, about 30 mg/Liter to about 80 mg/Liter, about 30 mg/Liter to about 90 mg/Liter, about 30 mg/Liter to about 100 mg/Liter, about 30 mg/Liter to about 110 mg/Liter, about 30 mg/Liter to about 120 mg/Liter, about 40 mg/Liter to about 50 mg/Liter, about 40 mg/Liter to about 60 mg/Liter, about 40 mg/Liter to about 70 mg/Liter, about 40 mg/Liter to about 80 mg/Liter, about 40 mg/Liter to about 90 mg/Liter, about 40 mg/Liter to about 100 mg/Liter, about 40 mg/Liter to about 110 mg/Liter, about 40 mg/Liter to about 120 mg/Liter, about 50 mg/Liter to about 60 mg/Liter, about 50 mg/Liter to about 70 mg/Liter, about 50 mg/Liter to about 80 mg/Liter, about 50 mg/Liter to about 90 mg/Liter, about 50 mg/Liter to about 100 mg/Liter, about 50 mg/Liter to about 110 mg/Liter, about 50 mg/Liter to about 120 mg/Liter, about 60 mg/Liter to about 70 mg/Liter, about 60 mg/Liter to about 80 mg/Liter, about 60 mg/Liter to about 90 mg/Liter, about 60 mg/Liter to about 100 mg/Liter, about 60 mg/Liter to about 110 mg/Liter, about 60 mg/Liter to about 120 mg/Liter, about 70 mg/Liter to about 80 mg/Liter, about 70 mg/Liter to about 90 mg/Liter, about 70 mg/Liter to about 100 mg/Liter, about 70 mg/Liter to about 110 mg/Liter, about 70 mg/Liter to about 120 mg/Liter, about 80 mg/Liter to about 90 mg/Liter, about 80 mg/Liter to about 100 mg/Liter, about 80 mg/Liter to about 110 mg/Liter, about 80 mg/Liter to about 120 mg/Liter, about 90 mg/Liter to about 100 mg/Liter, about 90 mg/Liter to about 110 mg/Liter, about 90 mg/Liter to about 120 mg/Liter, about 100 mg/Liter to about 110 mg/Liter, about 100 mg/Liter to about 120 mg/Liter, or about 110 mg/Liter to about 120 mg/Liter. In some embodiments, each vitamin present in the medium is present at about 10 mg/Liter, about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, about 110 mg/Liter, or about 120 mg/Liter. In some embodiments, each vitamin present in the medium is present at least about 10 mg/Liter, about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, or about 110 mg/Liter. In some embodiments, each vitamin present in the medium is present at most about 20 mg/Liter, about 30 mg/Liter, about 40 mg/Liter, about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, about 110 mg/Liter, or about 120 mg/Liter.
[0270] In some embodiments, the glucose comprised in the medium is anhydrous. In some embodiments, the glucose is present in the L isoform. In some embodiments, the glucose is present in the D isoform. In some embodiments, the glucose is present in both isoforms. In some embodiments, glucose present in the MSC Culture Medium is present at about 500 mg/Liter to about 1,600 mg/Liter. In some embodiments, glucose present in the medium is present at about 500 mg/Liter to about 600 mg/Liter, about 500 mg/Liter to about 700 mg/Liter, about 500 mg/Liter to about 800 mg/Liter, about 500 mg/Liter to about 900 mg/Liter, about 500 mg/Liter to about 1,000 mg/Liter, about 500 mg/Liter to about 1,100 mg/Liter, about 500 mg/Liter to about 1,200 mg/Liter, about 500 mg/Liter to about 1,300 mg/Liter, about 500 mg/Liter to about 1,400 mg/Liter, about 500 mg/Liter to about 1,500 mg/Liter, about 500 mg/Liter to about 1,600 mg/Liter, about 600 mg/Liter to about 700 mg/Liter, about 600 mg/Liter to about 800 mg/Liter, about 600 mg/Liter to about 900 mg/Liter, about 600 mg/Liter to about 1,000 mg/Liter, about 600 mg/Liter to about 1,100 mg/Liter, about 600 mg/Liter to about 1,200 mg/Liter, about 600 mg/Liter to about 1,300 mg/Liter, about 600 mg/Liter to about 1,400 mg/Liter, about 600 mg/Liter to about 1,500 mg/Liter, about 600 mg/Liter to about 1,600 mg/Liter, about 700 mg/Liter to about 800 mg/Liter, about 700 mg/Liter to about 900 mg/Liter, about 700 mg/Liter to about 1,000 mg/Liter, about 700 mg/Liter to about 1,100 mg/Liter, about
700 mg/Liter to about 1,200 mg/Liter, about 700 mg/Liter to about 1,300 mg/Liter, about 700 mg/Liter to about 1,400 mg/Liter, about 700 mg/Liter to about 1,500 mg/Liter, about 700 mg/Liter to about 1,600 mg/Liter, about 800 mg/Liter to about 900 mg/Liter, about 800 mg/Liter to about 1,000 mg/Liter, about 800 mg/Liter to about 1,100 mg/Liter, about 800 mg/Liter to about 1,200 mg/Liter, about 800 mg/Liter to about 1,300 mg/Liter, about 800 mg/Liter to about 1,400 mg/Liter, about 800 mg/Liter to about 1,500 mg/Liter, about 800 mg/Liter to about 1,600 mg/Liter, about 900 mg/Liter to about 1,000 mg/Liter, about 900 mg/Liter to about 1,100 mg/Liter, about 900 mg/Liter to about 1,200 mg/Liter, about 900 mg/Liter to about 1,300 mg/Liter, about 900 mg/Liter to about 1,400 mg/Liter, about 900 mg/Liter to about 1,500 mg/Liter, about 900 mg/Liter to about 1,600 mg/Liter, about 1,000 mg/Liter to about 1,100 mg/Liter, about 1,000 mg/Liter to about 1,200 mg/Liter, about 1,000 mg/Liter to about 1,300 mg/Liter, about 1,000 mg/Liter to about 1,400 mg/Liter, about 1,000 mg/Liter to about 1,500 mg/Liter, about 1,000 mg/Liter to about 1,600 mg/Liter, about 1,100 mg/Liter to about 1,200 mg/Liter, about 1,100 mg/Liter to about 1,300 mg/Liter, about 1,100 mg/Liter to about 1,400 mg/Liter, about 1,100 mg/Liter to about 1,500 mg/Liter, about 1,100 mg/Liter to about 1,600 mg/Liter, about 1,200 mg/Liter to about 1,300 mg/Liter, about 1,200 mg/Liter to about 1,400 mg/Liter, about 1,200 mg/Liter to about 1,500 mg/Liter, about 1,200 mg/Liter to about 1,600 mg/Liter, about 1,300 mg/Liter to about 1,400 mg/Liter, about 1,300 mg/Liter to about 1,500 mg/Liter, about 1,300 mg/Liter to about 1,600 mg/Liter, about 1,400 mg/Liter to about 1,500 mg/Liter, about 1,400 mg/Liter to about 1,600 mg/Liter, or about 1,500 mg/Liter to about 1,600 mg/Liter. In some embodiments, glucose present in the medium is present at about 500 mg/Liter, about 600 mg/Liter, about 700 mg/Liter, about 800 mg/Liter, about 900 mg/Liter, about 1,000 mg/Liter, about 1,100 mg/Liter, about 1,200 mg/Liter, about 1,300 mg/Liter, about 1,400 mg/Liter, about 1,500 mg/Liter, or about 1,600 mg/Liter. In some embodiments, glucose present in the medium is present at least about 500 mg/Liter, about 600 mg/Liter, about 700 mg/Liter, about 800 mg/Liter, about 900 mg/Liter, about 1,000 mg/Liter, about 1,100 mg/Liter, about 1,200 mg/Liter, about 1,300 mg/Liter, about 1,400 mg/Liter, or about 1,500 mg/Liter. In some embodiments, glucose present in the medium is present at most about 600 mg/Liter, about 700 mg/Liter, about 800 mg/Liter, about 900 mg/Liter, about 1,000 mg/Liter, about 1,100 mg/Liter, about 1,200 mg/Liter, about 1,300 mg/Liter, about 1,400 mg/Liter, about 1,500 mg/Liter, or about 1,600 mg/Liter.
[0271] In some embodiments, lipoic acid present in the MSC Culture Medium is present at about 0.05 mg/Liter to about 0.5 mg/Liter. In some embodiments, the lipoic acid is present in the medium in the form of DL-thiotic acid. In some embodiments, lipoic acid present in the medium is present at about 0.05 mg/Liter to about 0.1 mg/Liter, about 0.05 mg/Liter to about 0.15 mg/Liter, about 0.05 mg/Liter to about 0.2 mg/Liter, about 0.05 mg/Liter to about 0.25 mg/Liter, about 0.05 mg/Liter to about 0.3 mg/Liter, about 0.05 mg/Liter to about 0.35 mg/Liter, about 0.05 mg/Liter to about 0.4 mg/Liter, about 0.05 mg/Liter to about 0.45 mg/Liter, about 0.05 mg/Liter to about 0.5 mg/Liter, about 0.1 mg/Liter to about 0.15 mg/Liter, about 0.1 mg/Liter to about 0.2 mg/Liter, about 0.1 mg/Liter to about 0.25 mg/Liter, about 0.1 mg/Liter to about 0.3 mg/Liter, about 0.1 mg/Liter to about 0.35 mg/Liter, about 0.1 mg/Liter to about 0.4 mg/Liter, about 0.1 mg/Liter to about 0.45 mg/Liter, about 0.1 mg/Liter to about 0.5 mg/Liter, about 0.15 mg/Liter to about 0.2 mg/Liter, about 0.15 mg/Liter to about 0.25 mg/Liter, about 0.15 mg/Liter to about 0.3 mg/Liter, about 0.15 mg/Liter to about 0.35 mg/Liter, about 0.15 mg/Liter to about 0.4 mg/Liter, about 0.15 mg/Liter to about 0.45 mg/Liter, about 0.15 mg/Liter to about 0.5 mg/Liter, about 0.2 mg/Liter to about 0.25 mg/Liter, about 0.2 mg/Liter to about 0.3 mg/Liter, about 0.2 mg/Liter to about 0.35 mg/Liter, about 0.2 mg/Liter to about 0.4 mg/Liter, about 0.2 mg/Liter to about 0.45 mg/Liter, about 0.2 mg/Liter to about 0.5 mg/Liter, about 0.25 mg/Liter to about 0.3 mg/Liter, about 0.25 mg/Liter to about 0.35 mg/Liter, about 0.25 mg/Liter to about 0.4 mg/Liter, about 0.25 mg/Liter to about 0.45 mg/Liter, about 0.25 mg/Liter to about 0.5 mg/Liter, about 0.3 mg/Liter to about 0.35 mg/Liter, about 0.3 mg/Liter to about 0.4 mg/Liter, about 0.3 mg/Liter to about 0.45 mg/Liter, about 0.3 mg/Liter to about 0.5 mg/Liter, about 0.35 mg/Liter to about 0.4 mg/Liter, about 0.35 mg/Liter to about 0.45 mg/Liter, about 0.35 mg/Liter to about 0.5 mg/Liter, about 0.4 mg/Liter to about 0.45 mg/Liter, about 0.4 mg/Liter to about 0.5 mg/Liter, or about 0.45 mg/Liter to about 0.5 mg/Liter. In some embodiments, lipoic acid present in the medium is present at about 0.05 mg/Liter, about 0.1 mg/Liter, about 0.15 mg/Liter, about 0.2 mg/Liter, about 0.25 mg/Liter, about 0.3 mg/Liter, about 0.35 mg/Liter, about 0.4 mg/Liter, about 0.45 mg/Liter, or about 0.5 mg/Liter. In some embodiments, lipoic acid present in the medium is present at least about 0.05 mg/Liter, about 0.1 mg/Liter, about 0.15 mg/Liter, about 0.2 mg/Liter, about 0.25 mg/Liter, about 0.3 mg/Liter, about 0.35 mg/Liter, about 0.4 mg/Liter, or about 0.45 mg/Liter. In some embodiments, lipoic acid present in the medium is present at most about 0.1 mg/Liter, about 0.15 mg/Liter, about 0.2 mg/Liter, about 0.25 mg/Liter, about 0.3 mg/Liter, about 0.35 mg/Liter, about 0.4 mg/Liter, about 0.45 mg/Liter, or about 0.5 mg/Liter.
[0272] In some embodiments, sodium bicarbonate present in the MSC Culture Medium is present at about 250 mg/Liter to about 2,000 mg/Liter. In some embodiments, sodium bicarbonate present in the medium is present at about 250 mg/Liter to about 500 mg/Liter, about 250 mg/Liter to about 750 mg/Liter, about 250 mg/Liter to about 1,000 mg/Liter, about
250 mg/Liter to about 1,250 mg/Liter, about 250 mg/Liter to about 1,500 mg/Liter, about 250 mg/Liter to about 1,750 mg/Liter, about 250 mg/Liter to about 2,000 mg/Liter, about 500 mg/Liter to about 750 mg/Liter, about 500 mg/Liter to about 1,000 mg/Liter, about 500 mg/Liter to about 1,250 mg/Liter, about 500 mg/Liter to about 1,500 mg/Liter, about 500 mg/Liter to about 1,750 mg/Liter, about 500 mg/Liter to about 2,000 mg/Liter, about 750 mg/Liter to about 1,000 mg/Liter, about 750 mg/Liter to about 1,250 mg/Liter, about 750 mg/Liter to about 1,500 mg/Liter, about 750 mg/Liter to about 1,750 mg/Liter, about 750 mg/Liter to about 2,000 mg/Liter, about 1,000 mg/Liter to about 1,250 mg/Liter, about 1,000 mg/Liter to about 1,500 mg/Liter, about 1,000 mg/Liter to about 1,750 mg/Liter, about 1,000 mg/Liter to about 2,000 mg/Liter, about 1,250 mg/Liter to about 1,500 mg/Liter, about 1,250 mg/Liter to about 1,750 mg/Liter, about 1,250 mg/Liter to about 2,000 mg/Liter, about 1,500 mg/Liter to about 1,750 mg/Liter, about 1,500 mg/Liter to about 2,000 mg/Liter, or about 1,750 mg/Liter to about 2,000 mg/Liter. In some embodiments, sodium bicarbonate present in the medium is present at about 250 mg/Liter, about 500 mg/Liter, about 750 mg/Liter, about 1,000 mg/Liter, about 1,250 mg/Liter, about 1,500 mg/Liter, about 1,750 mg/Liter, or about 2,000 mg/Liter. In some embodiments, sodium bicarbonate present in the medium is present at least about 250 mg/Liter, about 500 mg/Liter, about 750 mg/Liter, about 1,000 mg/Liter, about 1,250 mg/Liter, about 1,500 mg/Liter, or about 1,750 mg/Liter. In some embodiments, sodium bicarbonate present in the medium is present at most about 500 mg/Liter, about 750 mg/Liter, about 1,000 mg/Liter, about 1,250 mg/Liter, about 1,500 mg/Liter, about 1,750 mg/Liter, or about 2,000 mg/Liter.
[0273] In some embodiments, sodium pyruvate present in the MSC Culture Medium is present at about 50 mg/Liter to about 160 mg/Liter. In some embodiments, sodium pyruvate present in the medium is present at about 50 mg/Liter to about 60 mg/Liter, about 50 mg/Liter to about 70 mg/Liter, about 50 mg/Liter to about 80 mg/Liter, about 50 mg/Liter to about 90 mg/Liter, about 50 mg/Liter to about 100 mg/Liter, about 50 mg/Liter to about 110 mg/Liter, about 50 mg/Liter to about 120 mg/Liter, about 50 mg/Liter to about 130 mg/Liter, about 50 mg/Liter to about 140 mg/Liter, about 50 mg/Liter to about 150 mg/Liter, about 50 mg/Liter to about 160 mg/Liter, about 60 mg/Liter to about 70 mg/Liter, about 60 mg/Liter to about 80 mg/Liter, about 60 mg/Liter to about 90 mg/Liter, about 60 mg/Liter to about 100 mg/Liter, about 60 mg/Liter to about 110 mg/Liter, about 60 mg/Liter to about 120 mg/Liter, about 60 mg/Liter to about 130 mg/Liter, about 60 mg/Liter to about 140 mg/Liter, about 60 mg/Liter to about 150 mg/Liter, about 60 mg/Liter to about 160 mg/Liter, about 70 mg/Liter to about 80 mg/Liter, about 70 mg/Liter to about 90 mg/Liter, about 70 mg/Liter to about 100 mg/Liter, about 70 mg/Liter to about 110 mg/Liter, about 70 mg/Liter to about 120 mg/Liter, about 70 mg/Liter to about 130 mg/Liter, about 70 mg/Liter to about 140 mg/Liter, about 70 mg/Liter to about 150 mg/Liter, about 70 mg/Liter to about 160 mg/Liter, about 80 mg/Liter to about 90 mg/Liter, about 80 mg/Liter to about 100 mg/Liter, about 80 mg/Liter to about 110 mg/Liter, about 80 mg/Liter to about 120 mg/Liter, about 80 mg/Liter to about 130 mg/Liter, about 80 mg/Liter to about 140 mg/Liter, about 80 mg/Liter to about 150 mg/Liter, about 80 mg/Liter to about 160 mg/Liter, about 90 mg/Liter to about 100 mg/Liter, about 90 mg/Liter to about 110 mg/Liter, about 90 mg/Liter to about 120 mg/Liter, about 90 mg/Liter to about 130 mg/Liter, about 90 mg/Liter to about 140 mg/Liter, about 90 mg/Liter to about 150 mg/Liter, about 90 mg/Liter to about 160 mg/Liter, about 100 mg/Liter to about 110 mg/Liter, about 100 mg/Liter to about 120 mg/Liter, about 100 mg/Liter to about 130 mg/Liter, about 100 mg/Liter to about 140 mg/Liter, about 100 mg/Liter to about 150 mg/Liter, about 100 mg/Liter to about 160 mg/Liter, about 110 mg/Liter to about 120 mg/Liter, about 110 mg/Liter to about 130 mg/Liter, about 110 mg/Liter to about 140 mg/Liter, about 110 mg/Liter to about 150 mg/Liter, about 110 mg/Liter to about 160 mg/Liter, about 120 mg/Liter to about 130 mg/Liter, about 120 mg/Liter to about 140 mg/Liter, about 120 mg/Liter to about 150 mg/Liter, about 120 mg/Liter to about 160 mg/Liter, about 130 mg/Liter to about 140 mg/Liter, about 130 mg/Liter to about 150 mg/Liter, about 130 mg/Liter to about 160 mg/Liter, about 140 mg/Liter to about 150 mg/Liter, about 140 mg/Liter to about 160 mg/Liter, or about 150 mg/Liter to about 160 mg/Liter. In some embodiments, sodium pyruvate present in the medium is present at about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, about 110 mg/Liter, about 120 mg/Liter, about 130 mg/Liter, about 140 mg/Liter, about 150 mg/Liter, or about 160 mg/Liter. In some embodiments, sodium pyruvate present in the medium is present at least about 50 mg/Liter, about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, about 110 mg/Liter, about 120 mg/Liter, about 130 mg/Liter, about 140 mg/Liter, or about 150 mg/Liter. In some embodiments, sodium pyruvate present in the medium is present at most about 60 mg/Liter, about 70 mg/Liter, about 80 mg/Liter, about 90 mg/Liter, about 100 mg/Liter, about 110 mg/Liter, about 120 mg/Liter, about 130 mg/Liter, about 140 mg/Liter, about 150 mg/Liter, or about 160 mg/Liter.
[0274] In some embodiments, the pH of the alpha MEM is from about 7.0 to about 7.4.
[0275] In some embodiments, the alpha MEM comprises the ingredients are presented in
Table 5
Figure imgf000072_0001
[0278] In some embodiments, the vBA-MSCs and/or vBM-MSCs are cultured in a MSC Culture Medium comprising alpha MEM as described in Table 5, 10% hPL (e.g., Stemulate™, 2 ng/mL recombinant, FGF (e.g., basic fibroblast growth factor (FGF-2) and/or carrier free FGF), and 2 ng/mL recombinant, epidermal growth factor (EGF, e.g., carrier free EGF). [0279] In some embodiments, the following reagents may be used during the manufacture of an MSC composition, as described above, and at the described concentrations: Phosphate Buffered Saline (PBS) at 100% Dimethyl Sulfoxide (DMSO) at 5%; Plasma Lyte-A Injection pH 7.4 at 100%; Human Serum Albumin, 25% at 2.5%; MSC Growth Media (Alpha MEM) at 100%; FGF2 at 2ng/mL; EGF at 2ng/mL; TrypLE™ Select at 100%; and Human Platelet Lysate at 10%.
Cryopreservation and Rinse Media
[0280] In some embodiments, for a method provided herein, said cell culture was cryopreserved in a cry opreservation media, wherein said cry opreservation media comprises an electrolyte formulation, human serum albumin (HSA), dimethyl sulfoxide (DMSO), or any combination thereof.
[0281] In some embodiments, cryovials are cryopreserved in a method comprising a precooling step or equilibration at 4 °C followed by passive cry opreservation at a rate of -1 °C per minute to about - 80 °C or less; then the cryovials are placed into the vapor phase above liquid nitrogen in cryogenic tanks for storage at less than or equal to -140 °C.
[0282] In some embodiments, said cryopreservation media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about
8%, about 5% to about 7%, about 5% to about 6%, about 6% to about 10%, about 6% to about
9%, about 6% to about 8%, about 6% to about 7%, about 7% to about 10%, about 7% to about
9%, about 7% to about 8%, about 8% to about 10%, about 8% to about 9%, or about 9% to about
10% HSA. In some embodiments, said cry opreservation media comprises about 1% to about 5% HSA. In some embodiments, said cryopreservation media comprises about 2.5% HSA.
[0283] In some embodiments, said cryopreservation media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, about 5% to about 6%, about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, about 6% to about 7%, about 7% to about 10%, about 7% to about 9%, about 7% to about 8%, about 8% to about 10%, about 8% to about 9%, or about 9% to about 10% DMSO. In some embodiments, said cry opreservation media comprises about 1% to about 10% DMSO. In some embodiments, said cryopreservation media comprises about 5% DMSO. [0284] In some embodiments, said electrolyte formulation is Plasmalyte A.
[0285] In some embodiments, for a method provided herein, the method further comprises, prior to (c), resuspending said cell culture in a rinse media, wherein said rinse media comprises an electrolyte formulation, human serum albumin (HSA), or both. In some embodiments, for a method provided herein, the method further comprises, prior to (c), resuspending said cell culture in a rinse media, wherein said rinse media comprises an electrolyte formulation, human serum albumin (HSA), or both.
[0286] In some embodiments, the rinse media is fresh.
[0287] In some embodiments, said rinse media comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, about 5% to about 6%, about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, about 6% to about 7%, about 7% to about 10%, about 7% to about 9%, about 7% to about 8%, about 8% to about 10%, about 8% to about 9%, or about 9% to about 10% HSA. In some embodiments, said rinse media comprises about 1% to about 5% HSA. In some embodiments, said rinse media comprises about 2.5% HSA. [0288] In some embodiments, said electrolyte formulation is Plasmalyte A.
[0289] In some embodiments, the systems and methods described herein have the ability to generate about 220 cryovials (26 million cells/vial) of passage 1 (Pl) master cell bank (MCB), which can generate approximately 193,600 vials (100 million cells/vial) of passage 4 (P4) marrow-derived mesenchymal stem cell final product with the potential to treat about 193,600 subjects at a dose of 100 million cells/dose. In some embodiments, the systems and methods described herein generate between about 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, or more than 300 cryovials of passage 1 master cell bank. In some embodiments, each cryovial comprises about 10 million, about 11 million, about 12 million, about 13 million, about 14 million, about 15 million, about 16 million, about 17 million, about 18 million, about 19 million, about 20 million, about 21 million, about 22 million, about 23 million, about24 million, about 25 million, about 26 million, about 27 million, about 28 million, about 29 million, about 30 million, about 32 million, about 32 million, about 33 million, about 34 million, about 35 million, about 36 million, about 37 million, about 38 million, about 48 million, or 40 million cells per vial of passage 1 master bank cells. In some embodiments, this allows the generation of about 100,000, about 110,000, about 120,000, about 130,000, about
140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about
200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about
260,000, about 270,000, about 280,000, about 290,000, about or 300,000 vials of passage 4
(P4) cells. In some embodiments, this allows the generation of at least about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about
170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about
230,000, about 240,000, about 250,000, about 260,000, about 270,000, about 280,000, about
290,000, about or 300,000 vials of passage 4 (P4) cells. In some embodiments, each vial of P4 cells comprises about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140, 150 million, about 160 million, about 170 million, about 180 million, about 290 million, about 200 million or more than 200 million cells. In some embodiments, each vial of P4 cells comprises at least about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140, 150 million, about 160 million, about 170 million, about 180 million, about 290 million, about 200 million or more than 200 million cells. In some embodiments, this allows the treatment of about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about 260,000, about 270,000, about 280,000, about 290,000, about or 300,000 patients. In some embodiments, this allows the treatment of at least about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about 260,000, about 270,000, about 280,000, about 290,000, about or 300,000 patients.
Cryorecovery
[0290] In some embodiments, MSCs and compositions comprising the same are provided to an end user (e.g., treatment facility) in a condition where they can be immediately be used (i.e., injected into a subject) and with minimal processing. In practice, facilities where the use of MSCs would take place likely do not have personnel trained in MSC sample prep. Therefore, there is a need for methods and systems of MSC sample preparation for immediate injection by the end user and where the MSCs do not require further processing steps upon arrival to the treatment facility.
[0291] The methods and systems disclosed herein comprise sample preparation of MSCs to including thawing cryopreserved MSCs and maintaining the MSCs under specific conditions (e.g. specific temperature(s)) for a period of time. The methods and systems disclosed herein also comprise sample preparation of MSCs including thawing cryopreserved MSCs, maintaining the MSCs for a period of time under specific conditions (e.g. a specific/first temperature), and then maintaining the MSCs under different conditions (e.g. at a different temperature than the temperature under which the MSCs were maintained immediately post-thaw). In some embodiments, this change in temperature is a cooling. In some embodiments, the MSCs are maintained in hypothermic conditions post-thaw until direct infusion into a subject.
[0292] Provided herein, in some embodiments, is a method of warming a cryopreserved population of stem cells to a first temperature and storing said stem cells at a second temperature less than about 40 °C. In some embodiments, the first temperature that the cryopreserved population of stem cells is warmed to is greater than about 0 °C. In some embodiments, the first temperature is greater than about 20 °C. In some embodiments, the second temperature that the stem cells are stored at is a hypothermic temperature. [0293] In some embodiments, a frozen cryovial comprising cryopreserved cells (e.g., P3 cells) are placed into a 37 °C water bath. The vial will be kept in the water bath until approximately 80% of ice has melted (which takes about two to three minutes). A cryovial may then be sprayed with sterile 70% ethanol (EtOH), wiped with sterile wipes and transferred into a biosafety cabinet. Cells in the thawed vial can then transferred to a sterile conical tube, the cells diluted with PlasmaLyte-A + 0.5% HSA, centrifuged (e.g., at room temperature) to form a cell pellet, and the supernatant is removed and the pellet is resuspended in a culturing medium, e.g., MSC Culture Media, for further plating and culturing, e.g., as Passage 4 (P4).
[0294] Provided herein, in one aspect, is a method for preparing stem cells for infusion, the method comprising: (a) providing a cryopreserved population of cells comprising said stem cells; (b) warming said stem cells to a first temperature and holding said stem cells at said first temperature for a first period of time; and (c) changing said first temperature to a second temperature and maintaining said stem cells at said second temperature for a second period of time. [0295] In some embodiments, said first temperature is greater than about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C. In some embodiments, said first temperature is about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15
°C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22
°C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29
°C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36
°C, about 37 °C, about 38 °C, about 39 °C, about 40 °C. In some embodiments, said first temperature is greater than 0 °C.
[0296] In some embodiments, said second temperature is less than about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C. In some embodiments, said second temperature is about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22
°C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29
°C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36
°C, about 37 °C, about 38 °C, about 39 °C, about 40 °C. In some embodiments, said second temperature is hypothermic. In some embodiments, said second temperature is less than 40 °C. [0297] In some embodiments, said first time period is less than about one week. In some embodiments, said time period is less than about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In some embodiments, said time period is less than about 5 days. In some embodiments, said time period is less than about 2 days. In some embodiments, said time period is less than about 1 day. In some embodiments, said time period is less than about 24 hours, about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour. In some embodiments, said time period is less than about 12 hours. In some embodiments, said time period is less than about 6 hours. In some embodiments, said time period is less than about 2 hours. In some embodiments, said time period is less than about 60 minutes, about 59 minutes, about 58 minutes, about 57 minutes, about 56 minutes, about 55 minutes, about 54 minutes, about 53 minutes, about 52 minutes, about 51 minutes, about 50 minutes, about 49 minutes, about 48 minutes, about 47 minutes, about 46 minutes, about 45 minutes, about 44 minutes, about 43 minutes, about 42 minutes, about 41 minutes, about 40 minutes, about 39 minutes, about 38 minutes, about 37 minutes, about 36 minutes, about 35 minutes, about 34 minutes, about 33 minutes, about 32 minutes, about 31 minutes, about 30 minutes, about 29 minutes, about 28 minutes, about 27 minutes, about 26 minutes, about 25 minutes, about 24 minutes, about 23 minutes, about 22 minutes, about 21 minutes, about 20 minutes, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute.
[0298] In some embodiments, the stem cells are maintained after they are warmed/thawed. In some embodiments, this maintenance is not accounted for in the first period of time described herein. In some embodiments, this maintenance is accounted for in the first period of time. In some embodiments, this maintenance follows similar methods as the MSC culturing methods described herein. In some embodiments, the post-thaw maintenance methods comprise not allowing the MSCs to double in population. In some embodiments, the MSCs are recovered and/or packaged prior to doubling. In some embodiments, the post-thaw culturing maintenance methods comprise culturing plating said stem cells at about 500 cells/cm2 to about 4,000 cells/cm2. In some embodiments, the post-thaw culturing maintenance methods comprise culturing plating said stem cells at about 500 cells/cm2 to about 1,000 cells/cm2, about 500 cells/cm2 to about 1,500 cells/cm2, about 500 cells/cm2 to about 2,000 cells/cm2, about 500 cells/cm2 to about 2,500 cells/cm2, about 500 cells/cm2 to about 3,000 cells/cm2, about 500 cells/cm2 to about 3,500 cells/cm2, about 500 cells/cm2 to about 4,000 cells/cm2, about 1,000 cells/cm2 to about 1,500 cells/cm2, about 1,000 cells/cm2 to about 2,000 cells/cm2, about 1,000 cells/cm2 to about 2,500 cells/cm2, about 1,000 cells/cm2 to about 3,000 cells/cm2, about 1,000 cells/cm2 to about 3,500 cells/cm2, about 1,000 cells/cm2 to about 4,000 cells/cm2, about 1,500 cells/cm2 to about 2,000 cells/cm2, about 1,500 cells/cm2 to about 2,500 cells/cm2, about 1,500 cells/cm2 to about 3,000 cells/cm2, about 1,500 cells/cm2 to about 3,500 cells/cm2, about 1,500 cells/cm2 to about 4,000 cells/cm2, about 2,000 cells/cm2 to about 2,500 cells/cm2, about 2,000 cells/cm2 to about 3,000 cells/cm2, about 2,000 cells/cm2 to about 3,500 cells/cm2, about 2,000 cells/cm2 to about 4,000 cells/cm2, about 2,500 cells/cm2 to about 3,000 cells/cm2, about 2,500 cells/cm2 to about 3,500 cells/cm2, about 2,500 cells/cm2 to about 4,000 cells/cm2, about 3,000 cells/cm2 to about 3,500 cells/cm2, about 3,000 cells/cm2 to about 4,000 cells/cm2, or about
3.500 cells/cm2 to about 4,000 cells/cm2. In some embodiments, the post-thaw culturing maintenance methods comprise culturing plating said stem cells at about 500 cells/cm2, about 1,000 cells/cm2, about 1,500 cells/cm2, about 2,000 cells/cm2, about 2,500 cells/cm2, about 3,000 cells/cm2, about 3,500 cells/cm2, or about 4,000 cells/cm2. In some embodiments, the post-thaw culturing maintenance methods comprise culturing plating said stem cells at least about 500 cells/cm2, about 1,000 cells/cm2, about 1,500 cells/cm2, about 2,000 cells/cm2, about
2.500 cells/cm2, about 3,000 cells/cm2, or about 3,500 cells/cm2. In some embodiments, the post-thaw culturing maintenance methods comprise culturing plating said stem cells at most about 1,000 cells/cm2, about 1,500 cells/cm2, about 2,000 cells/cm2, about 2,500 cells/cm2, about 3,000 cells/cm2, about 3,500 cells/cm2, or about 4,000 cells/cm2. In some embodiments, the post-thaw maintenance methods comprise plating said stem cells at about 3,000 to about 10,000, about 3,000 to about 9,000, about 3,000 to about 8,000 about, about 3,000 to about 7,000, about 3,000 to about 6,000, about 3,000 to about 5,000, about 3,000 to about 4,000, about 10,000 to about 50,000, about 10,000 to about 40,000, about 10,000 to about 30,000, about 10,000 to about 20,000, about 20,000 to about 50,000, about 20,000 to about 40,000, about 20,000 to about 30,000, about 30,000 to about 50,000, about 30,000 to about 40,000, or about 40,000 to about 50,000 cells/cm2 at said first temperature. In some embodiments, for a method provided herein, the method further comprises, prior to (c), maintaining said stem cells at about 3,000 cells/cm2 to about 50,000 cells/cm2 at said first temperature. In some embodiments, the post-thaw culturing maintenance methods comprise culturing plating said stem cells at about 10,000 cells/cm2 to about 50,000 cells/cm2. In some embodiments, the postthaw culturing maintenance methods comprise culturing plating said stem cells at about 10,000 cells/cm2 to about 15,000 cells/cm2, about 10,000 cells/cm2 to about 20,000 cells/cm2, about 10,000 cells/cm2 to about 25,000 cells/cm2, about 10,000 cells/cm2 to about 30,000 cells/cm2, about 10,000 cells/cm2 to about 35,000 cells/cm2, about 10,000 cells/cm2 to about 40,000 cells/cm2, about 10,000 cells/cm2 to about 45,000 cells/cm2, about 10,000 cells/cm2 to about 50,000 cells/cm2, about 15,000 cells/cm2 to about 20,000 cells/cm2, about 15,000 cells/cm2 to about 25,000 cells/cm2, about 15,000 cells/cm2 to about 30,000 cells/cm2, about 15,000 cells/cm2 to about 35,000 cells/cm2, about 15,000 cells/cm2 to about 40,000 cells/cm2, about 15,000 cells/cm2 to about 45,000 cells/cm2, about 15,000 cells/cm2 to about 50,000 cells/cm2, about 20,000 cells/cm2 to about 25,000 cells/cm2, about 20,000 cells/cm2 to about 30,000 cells/cm2, about 20,000 cells/cm2 to about 35,000 cells/cm2, about 20,000 cells/cm2 to about 40,000 cells/cm2, about 20,000 cells/cm2 to about 45,000 cells/cm2, about 20,000 cells/cm2 to about 50,000 cells/cm2, about 25,000 cells/cm2 to about 30,000 cells/cm2, about 25,000 cells/cm2 to about 35,000 cells/cm2, about 25,000 cells/cm2 to about 40,000 cells/cm2, about 25,000 cells/cm2 to about 45,000 cells/cm2, about 25,000 cells/cm2 to about 50,000 cells/cm2, about 30,000 cells/cm2 to about 35,000 cells/cm2, about 30,000 cells/cm2 to about 40,000 cells/cm2, about 30,000 cells/cm2 to about 45,000 cells/cm2, about 30,000 cells/cm2 to about 50,000 cells/cm2, about 35,000 cells/cm2 to about 40,000 cells/cm2, about 35,000 cells/cm2 to about 45,000 cells/cm2, about 35,000 cells/cm2 to about 50,000 cells/cm2, about 40,000 cells/cm2 to about 45,000 cells/cm2, about 40,000 cells/cm2 to about 50,000 cells/cm2, or about 45,000 cells/cm2 to about 50,000 cells/cm2. In some embodiments, the post-thaw culturing maintenance methods comprise culturing plating said stem cells at about 10,000 cells/cm2, about 15,000 cells/cm2, about 20,000 cells/cm2, about 25,000 cells/cm2, about 30,000 cells/cm2, about 35,000 cells/cm2, about 40,000 cells/cm2, about 45,000 cells/cm2, or about 50,000 cells/cm2. In some embodiments, the post-thaw culturing maintenance methods comprise culturing plating said stem cells at least about 10,000 cells/cm2, about 15,000 cells/cm2, about 20,000 cells/cm2, about 25,000 cells/cm2, about 30,000 cells/cm2, about 35,000 cells/cm2, about 40,000 cells/cm2, or about 45,000 cells/cm2. In some embodiments, the post-thaw culturing maintenance methods comprise culturing plating said stem cells at most about 15,000 cells/cm2, about 20,000 cells/cm2, about 25,000 cells/cm2, about 30,000 cells/cm2, about 35,000 cells/cm2, about 40,000 cells/cm2, about 45,000 cells/cm2, or about 50,000 cells/cm2. In some embodiments, for a method provided herein, the method further comprises, prior to (c), maintaining said stem cells at about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, 10,000, about 20,000, about 30,000, about 40,000, or about 50,000 cells/cm2 at said first temperature. In some embodiments, for a method provided herein, the method further comprises, prior to (c), maintaining said stem cells at about 32,000 cells/cm2 at said first temperature. In some embodiments, the post-thaw maintaining takes place in a T25 flask. In some embodiments, the post-thaw maintaining takes place in a T75 flask. In some embodiments, the post-thaw maintaining takes place in another sized flasks. [0299] In some embodiments, the post-thaw culturing maintenance methods increases the number of cells in a culture vessel from about 5% to about 500%. As examples, the post-thaw culturing maintenance methods may increase the number of cells in a culture vessel by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200%, about 220%, about 240%, about 260%, about 280%, about 300%, about 320%, about 340%, about 360%, about 380%, about 400%, about 420%, about 440%, about 460%, about 480%, or about 500%. The post-thaw culturing maintenance methods may increase the number of cells in a culture vessel by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 200%, about 200% to about 300%, about 300% to about 400%, or about 400% to about 500%.
[0300] In some embodiments, the stem cells are packaged in 5 mL volumes of 10 * 106 live cells/mL in Plasma-Lyte A + 2.5% HSA (Rinse Media). In some embodiments, the stem cells are packaged in volumes of about 1 x 106 live cells/mL to about 12 x 106 live cells/mL. In some embodiments, the stem cells are packaged in volumes of about 1 x 10A6 live cells/mL to about 2 x 106 live cells/mL, about 1 x 106 live cells/mL to about 3 x 106 live cells/mL, about
1 x 106 live cells/mL to about 4 x 106 live cells/mL, about 1 x 106 live cells/mL to about 5 x 106 live cells/mL, about 1 x 106 live cells/mL to about 6 x 106 live cells/mL, about 1 x 106 live cells/mL to about 7 x 106 live cells/mL, about 1 x 106 live cells/mL to about 8 x 106 live cells/mL, about 1 x 106 live cells/mL to about 9 x 106 live cells/mL, about 1 x 106 live cells/mL to about 10 x 106 live cells/mL, about 1 x 106 live cells/mL to about 11 x 106 live cells/mL, about 1 x 106 live cells/mL to about 12 x 106 live cells/mL, about 2 x 106 live cells/mL to about 3 x 106 live cells/mL, about 2 x 106 live cells/mL to about 4 x 106 live cells/mL, about 2 x 106 live cells/mL to about 5 x 106 live cells/mL, about 2 x 106 live cells/mL to about 6 x 106 live cells/mL, about 2 x 106 live cells/mL to about 7 x 106 live cells/mL, about 2 x 106 live cells/mL to about 8 x 106 live cells/mL, about 2 x 106 live cells/mL to about 9 x 106 live cells/mL, about
2 x 106 live cells/mL to about 10 x 106 live cells/mL, about 2 x 106 live cells/mL to about 11 x 106 live cells/mL, about 2 x 106 live cells/mL to about 12 x 106 live cells/mL, about 3 x 106 live cells/mL to about 4 x 106 live cells/mL, about 3 x 106 live cells/mL to about 5 x 106 live cells/mL, about 3 x 106 live cells/mL to about 6 x 106 live cells/mL, about 3 x 106 live cells/mL to about 7 x 106 live cells/mL, about 3 x 106 live cells/mL to about 8 x 106 live cells/mL, about
3 x 106 live cells/mL to about 9 x 106 live cells/mL, about 3 x 106 live cells/mL to about 10 x 106 live cells/mL, about 3 x 106 live cells/mL to about 11 x 106 live cells/mL, about 3 x 106 live cells/mL to about 12 x 106 live cells/mL, about 4 x 106 live cells/mL to about 5 x 106 live cells/mL, about 4 x 106 live cells/mL to about 6 x 106 live cells/mL, about 4 x 106 live cells/mL to about 7 x 106 live cells/mL, about 4 x 106 live cells/mL to about 8 x 106 live cells/mL, about
4 x 106 live cells/mL to about 9 x 106 live cells/mL, about 4 x 106 live cells/mL to about 10 x 106 live cells/mL, about 4 x 106 live cells/mL to about 11 x 106 live cells/mL, about 4 x 106 live cells/mL to about 12 x 10A6 live cells/mL, about 5 x 10A6 live cells/mL to about 6 x 10A6 live cells/mL, about 5 x 106 live cells/mL to about 7 x 106 live cells/mL, about 5 x 106 live cells/mL to about 8 x 106 live cells/mL, about 5 x 106 live cells/mL to about 9 x 106 live cells/mL, about 5 x 106 live cells/mL to about 10 x 106live cells/mL, about 5 x 106 live cells/mL to about 11 x 106 live cells/mL, about 5 x 106 live cells/mL to about 12 x 106 live cells/mL, about 6 x 106 live cells/mL to about 7 x 106 live cells/mL, about 6 x 106 live cells/mL to about 8 x 106 live cells/mL, about 6 x 106 live cells/mL to about 9 x 106 live cells/mL, about 6 x 106 live cells/mL to about 10 x 106 live cells/mL, about 6 x 106 live cells/mL to about 11 x 106 live cells/mL, about 6 x 106 live cells/mL to about 12 x 106 live cells/mL, about 7 x 106 live cells/mL to about 8 x 106 live cells/mL, about 7 x 106 live cells/mL to about 9 x 106 live cells/mL, about 7 x 106 live cells/mL to about 10 x 106 live cells/mL, about 7 x 106 live cells/mL to about 11 x 106 live cells/mL, about 7 x 106 live cells/mL to about 12 x 106 live cells/mL, about 8 x 106 live cells/mL to about 9 x 106 live cells/mL, about 8 x 106 live cells/mL to about 10 x 106 live cells/mL, about 8 x 106 live cells/mL to about 11 x 106 live cells/mL, about 8 x 106 live cells/mL to about 12 x 106 live cells/mL, about 9 x 106 live cells/mL to about 10 x 106 live cells/mL, about 9 x 106 live cells/mL to about 11 x 106 live cells/mL, about 9 x 106 live cells/mL to about 12 x 106 live cells/mL, about 10 x 106 live cells/mL to about 11 x 106 live cells/mL, about 10 x 106 live cells/mL to about 12 x 106 live cells/mL, or about 11 x 106 live cells/mL to about 12 x 106 live cells/mL. In some embodiments, the stem cells are packaged in volumes of about 1 x 106 live cells/mL, about 2 x 106 live cells/mL, about 3 x 106 live cells/mL, about 4 x 106 live cells/mL, about 5 x 106 live cells/mL, about 6 x 106 live cells/mL, about 7 x 106 live cells/mL, about 8 x 106 live cells/mL, about 9 x 106 live cells/mL, about 10 x 106 live cells/mL, about 11 x 106 live cells/mL, or about 12 x 106 live cells/mL. In some embodiments, the stem cells are packaged in volumes of at least about 1 x 106 live cells/mL, about 2 x 106 live cells/mL, about 3 x 106 live cells/mL, about 4 x 106 live cells/mL, about 5 x 106 live cells/mL, about 6 x 106 live cells/mL, about 7 x 106 live cells/mL, about 8 x 106 live cells/mL, about 9 x 106 live cells/mL, about 10 x 106 live cells/mL, or about 11 x 106 live cells/mL. In some embodiments, the stem cells are packaged in volumes of at most about 2 x 106 live cells/mL, about 3 x 106 live cells/mL, about 4 x 106 live cells/mL, about 5 x 106 live cells/mL, about 6 x 106 live cells/mL, about 7 x 106 live cells/mL, about 8 x 106 live cells/mL, about 9 x 106 live cells/mL, about 10 x 106 live cells/mL, about 11 x 106 live cells/mL, or about 12 x 106 live cells/mL.
[0301] Once the stem cells are packaged, the stem cells are put under the second temperature for the second time period. In some embodiments, the second temperature is less than about 40 °C, about 39 °C, about 38 °C, about 37 °C, about 36 °C, about 35 °C, about 34 °C, about 33 °C, about 32 °C, about 31 °C, about 30 °C, about 29 °C, about 28 °C, about 27 °C, about 26 °C, about 25 °C, about 24 °C, about 23 °C, about 22 °C, about 21 °C, about 20 °C, about 19 °C, about 18 °C, about 17 °C, about 16 °C, about 15 °C, about 14 °C, about 13 °C, about 12 °C, about 11 °C, about 10 °C, about 9 °C, about 8 °C, about 7 °C, about 6 °C, about 5 °C, about 4 °C, about 3 °C, about 2 °C, or about 1 °C. In some embodiments, said cell culture is maintained at about 40 °C, about 39 °C, about 38 °C, about 37 °C, about 36 °C, about 35 °C, about 34 °C, about 33 °C, about 32 °C, about 31 °C, about 30 °C, about 29 °C, about 28 °C, about 27 °C, about 26 °C, about 25 °C, about 24 °C, about 23 °C, about 22 °C, about 21 °C, about 20 °C, about 19 °C, about 18 °C, about 17 °C, about 16 °C, about 15 °C, about 14 °C, about 13 °C, about 12 °C, about 11 °C, about 10 °C, about 9 °C, about 8 °C, about 7 °C, about 6 °C, about 5 °C, about 4 °C, about 3 °C, about 2 °C, or about 1 °C. In some embodiments, the second temperature is less than 37 °C. In some embodiments, the second temperature is less than 35 °C. In some embodiments, the second temperature is less than 30 °C. In some embodiments, the second temperature is less than 25 °C. In some embodiments, said the second temperature is less than 20 °C. In some embodiments, the second temperature is about 2 °C to about 8 °C.
[0302] In some embodiments, said second time period is less than about one week. In some embodiments, said time period is less than about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In some embodiments, said time period is less than about 5 days. In some embodiments, said time period is less than about 2 days. In some embodiments, said time period is less than about 1 day. In some embodiments, said time period is less than about 24 hours, about 23 hours, about 22 hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour. In some embodiments, said time period is less than about 12 hours. In some embodiments, said time period is less than about 6 hours. In some embodiments, said time period is less than about 2 hours. In some embodiments, said time period is less than about 60 minutes, about 59 minutes, about 58 minutes, about 57 minutes, about 56 minutes, about 55 minutes, about 54 minutes, about 53 minutes, about 52 minutes, about 51 minutes, about 50 minutes, about 49 minutes, about 48 minutes, about 47 minutes, about 46 minutes, about 45 minutes, about 44 minutes, about 43 minutes, about 42 minutes, about 41 minutes, about 40 minutes, about 39 minutes, about 38 minutes, about 37 minutes, about 36 minutes, about 35 minutes, about 34 minutes, about 33 minutes, about 32 minutes, about 31 minutes, about 30 minutes, about 29 minutes, about 28 minutes, about 27 minutes, about 26 minutes, about 25 minutes, about 24 minutes, about 23 minutes, about 22 minutes, about 21 minutes, about 20 minutes, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute. MSC preparations and compositions for administration to a subject in need, e.g., with a perianal fistula
[0303] Cultured P4 cells or the equivalent may be briefly cultured for about 20 to about 28 hours to allow metabolic activity to resume post-thaw. P4 or the equivalent cells are detached from the culturing vessel, rinsed, and washed using PBS via centrifugation, e.g., at 500xg. After centrifugation, the supernatant is aspirated, and cells are resuspended in PBS. An aliquot of this preparation is taken for a viable cell count via trypan blue or an equivalent assay using acridine orange/propidium iodide. This count dictates final concentration requirements for final formulation and packaging.
[0304] Once the cell count is known, the cells are centrifuged, supernatant is aspirated, and the pellet is resuspended in PlasmaLyte-A + 2.5% HSA at about 10 million cells/mL. The cells are then packaged in 5 mL vials, e.g., CellSeal closed-system cryovials. Two 5 ml vials at 10 million cells/mL equate to one dose (, e.g., of about 100 million cells) for a patient in need, e.g., having a perianal fistula.
[0305] In some embodiments, the preparations and compositions of the present disclosure may comprise at least about 100 million vBA-MSCs and/or vBM-MSCs having an antigen profile of more than about 1.75% CD45+ cells, at least about 95% CD105+ cells, and at least about 95% CD 166+ cells and the cells may be expanded ex vivo from passage 2 until passage 4 while maintaining population uniformity based upon the antigen profile (i.e. more than about 1.75% CD45+ cells, at least about 95% CD105+ cells, and at least about 95% CD166+ cells). [0306] In some embodiments, the preparations and compositions of the present disclosure may comprise vBA-MSCs and/or vBM-MSCs having an antigen profile of reduced expression of one or more senescent cell markers, as compared to bone marrow-derived MSCs prepared according to known MSC culturing techniques. In some embodiments, the one or more senescent cell markers comprise MIC-A, MIC-B, ULBP2, or any combination thereof. NK cell-mediated immune responses are stimulated by MIC-A, MIC-B, and/or ULBP2.
[0307] In various embodiments, an MSC preparation or compositions for administration to a subject in need, e.g., with a perianal fistula, comprises MSC cells suspended in PlasmaLyte- A with 2.5% Human Serum Albumin (HSA).
[0308] In some embodiments, the vBA-MSCs and/or vBM-MSCs preparations and compositions described herein comprise an amount of cells that express one or more senescent cell markers of about 1 % less than bone marrow-derived MSCs to about 100 % less than bone marrow-derived MSCs. In some embodiments, the vBA-MSCs and/or vBM-MSCs preparations and compositions described herein comprise an amount of cells that express one or more senescent cell markers of about 100 % less than bone marrow-derived MSCs to about 90 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 80 % less than bone marrow-derived MSCs, about 100 % less than bone marrow- derived MSCs to about 70 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 60 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 50 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 40 % less than bone marrow- derived MSCs, about 100 % less than bone marrow-derived MSCs to about 30 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 20 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 10 % less than bone marrow-derived MSCs, about 100 % less than bone marrow- derived MSCs to about 5 % less than bone marrow-derived MSCs, about 100 % less than bone marrow-derived MSCs to about 1 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs to about 80 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs to about 70 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs to about 60 % less than bone marrow- derived MSCs, about 90 % less than bone marrow-derived MSCs to about 50 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs to about 40 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs to about 30 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs to about 20 % less than bone marrow-derived MSCs, about 90 % less than bone marrow- derived MSCs to about 10 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs to about 5 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs to about 1 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs to about 70 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs to about 60 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs to about 50 % less than bone marrow- derived MSCs, about 80 % less than bone marrow-derived MSCs to about 40 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs to about 30 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs to about 20 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs to about 10 % less than bone marrow-derived MSCs, about 80 % less than bone marrow- derived MSCs to about 5 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs to about 1 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs to about 60 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs to about 50 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs to about 40 % less than bone marrow- derived MSCs, about 70 % less than bone marrow-derived MSCs to about 30 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs to about 20 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs to about 10 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs to about 5 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs to about 1 % less than bone marrow-derived MSCs, about 60 % less than bone marrow- derived MSCs to about 50 % less than bone marrow-derived MSCs, about 60 % less than bone marrow-derived MSCs to about 40 % less than bone marrow-derived MSCs, about 60 % less than bone marrow-derived MSCs to about 30 % less than bone marrow-derived MSCs, about 60 % less than bone marrow-derived MSCs to about 20 % less than bone marrow-derived MSCs, about 60 % less than bone marrow-derived MSCs to about 10 % less than bone marrow- derived MSCs, about 60 % less than bone marrow-derived MSCs to about 5 % less than bone marrow-derived MSCs, about 60 % less than bone marrow-derived MSCs to about 1 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs to about 40 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs to about 30 % less than bone marrow-derived MSCs, about 50 % less than bone marrow- derived MSCs to about 20 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs to about 10 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs to about 5 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs to about 1 % less than bone marrow-derived MSCs, about 40 % less than bone marrow-derived MSCs to about 30 % less than bone marrow-derived MSCs, about 40 % less than bone marrow-derived MSCs to about 20 % less than bone marrow- derived MSCs, about 40 % less than bone marrow-derived MSCs to about 10 % less than bone marrow-derived MSCs, about 40 % less than bone marrow-derived MSCs to about 5 % less than bone marrow-derived MSCs, about 40 % less than bone marrow-derived MSCs to about 1 % less than bone marrow-derived MSCs, about 30 % less than bone marrow-derived MSCs to about 20 % less than bone marrow-derived MSCs, about 30 % less than bone marrow- derived MSCs to about 10 % less than bone marrow-derived MSCs, about 30 % less than bone marrow-derived MSCs to about 5 % less than bone marrow-derived MSCs, about 30 % less than bone marrow-derived MSCs to about 1 % less than bone marrow-derived MSCs, about 20 % less than bone marrow-derived MSCs to about 10 % less than bone marrow-derived MSCs, about 20 % less than bone marrow-derived MSCs to about 5 % less than bone marrow-derived MSCs, about 20 % less than bone marrow-derived MSCs to about 1 % less than bone marrow- derived MSCs, about 10 % less than bone marrow-derived MSCs to about 5 % less than bone marrow-derived MSCs, about 10 % less than bone marrow-derived MSCs to about 1 % less than bone marrow-derived MSCs, or about 5 % less than bone marrow-derived MSCs to about 1 % less than bone marrow-derived MSCs. In some embodiments, the vBA-MSC preparations and compositions described herein comprise an amount of cells that express one or more senescent cell markers of about 100 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs, about 60 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs, about 40 % less than bone marrow-derived MSCs, about 30 % less than bone marrow-derived MSCs, about 20 % less than bone marrow- derived MSCs, about 10 % less than bone marrow-derived MSCs, about 5 % less than bone marrow-derived MSCs, or about 1 % less than bone marrow-derived MSCs. In some embodiments, the vBA-MSCs and/or vBM-MSCs preparations and compositions described herein comprise an amount of cells that express one or more senescent cell markers of at least about 100 % less than bone marrow-derived MSCs, about 90 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs, about 70 % less than bone marrow- derived MSCs, about 60 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs, about 40 % less than bone marrow-derived MSCs, about 30 % less than bone marrow-derived MSCs, about 20 % less than bone marrow-derived MSCs, about 10 % less than bone marrow-derived MSCs, or about 5 % less than bone marrow-derived MSCs. In some embodiments, the vBA-MSCs and/or vBM-MSCs preparations and compositions described herein comprise an amount of cells that express one or more senescent cell markers of at most about 90 % less than bone marrow-derived MSCs, about 80 % less than bone marrow-derived MSCs, about 70 % less than bone marrow-derived MSCs, about 60 % less than bone marrow-derived MSCs, about 50 % less than bone marrow-derived MSCs, about 40 % less than bone marrow-derived MSCs, about 30 % less than bone marrow-derived MSCs, about 20 % less than bone marrow-derived MSCs, about 10 % less than bone marrow-derived MSCs, about 5 % less than bone marrow-derived MSCs, or about 1 % less than bone marrow- derived MSCs.
[0309] In some embodiments, the preparations and compositions of the present disclosure generate a lessened NK cell-mediated immune response upon administration to a subject comprising mis-matched MHC molecules (e.g. mis-matched human leukocyte antigens when the subject is a human), as compared to administration of a composition comprising bone marrow-derived MSCs. In some embodiments, the preparations and compositions of the present disclosure do not generate a NK cell-mediated immune response upon administration to a subject comprising mis-matched MHC molecules (e.g. mis-matched human leukocyte antigens when the subject is a human).
Preparation of Mesenchymal Stem Cells for Administration to a Subject in Need
[0310] In certain aspects, described herein is a method of administering a composition comprising MSCs to a subject with a perianal fistula. In some embodiments, described here is a method of using GMP grade MSC product in patients with refractory fistulizing perianal CD. In some embodiments, the method of using allogeneic bone marrow derived mesenchymal stem cells (MSCs) produces regenerative signals.
[0311] In some embodiments, the compositions described herein are comprised of culture- expanded mesenchymal stromal cells (MSCs) isolated from the bone marrow of consented and disease-screened deceased organ and tissue donors. Treating a subject with an autoimmune disorder such as Crohn’s disease with allogenic cells can provide improved treatment outcomes by providing a balance of healthy cells within the subject. In some embodiments, the final product is composed of MSCs formulated at 20M cells/mL in Plasma Lyte A® with 2.5% human serum albumin (HSA) and 5% DMSO, and manufactured in 5mL cryopreserved units. [0312] MSCs for administration to subject having a perianal fistula are derived from the continuous manufacturing process of obtained bone marrow from deceased-donor vertebral bodies to selection of MSC for further cell culture. MSCs are cultured out of the bone marrow units collected. The overall manufacturing process to derive the final MSC product is illustrated in FIG 1. As shown in FIG. 1, MSCs are cultured out of primary bone marrow units using a tiered culture system.
[0313] In certain embodiments, the compositions and methods of the present disclosure include intentional HLA mis-matching between a donor and a recipient. In some embodiments, transplantation of HLA mis-matching bone marrow products of the present invention following NMA conditioning improves a patient’s outcome yet avoids GVHD. In some embodiments, the improved outcome and avoided GVHD occurs in the absence of an immunosuppressive prophylaxis. As used herein a mis-matching occurs when a donor and recipient differ by at least one HLA marker, e.g., at least two, three, four, five, six, seven, eight, nine, ten, or eleven HLA markers. In some embodiments, a mis-matching occurs when a donor and a recipient have fewer than six HLA matches out of eight HLA markers, fewer than four HLA matches out of six HLA markers, or fewer than five HLA matches out of ten HLA markers. In some embodiments, a mis-matching occurs when a donor and a recipient have fewer than half of the characterized HLA markers matching. In some embodiments, there is HLA mis-matching between the HSCs and the patient. In some embodiments, there is HLA mis-matching between the human cadaver and the patient.
[0314] In some embodiments, MSCs may administered prophylactically, perioperatively or postoperatively with SOT or other VCA procedures. In some embodiments, MSCs administered may comprise human MSCs derived from bone marrow, adherent vertebral body MSCs (vBA-MSCs), or both. In some embodiments, the MSCs are administered to a subject with a perianal fistula. In some embodiments, the MSCs are injected into the perianal fistula.
[0315] In some embodiments, the composition of human MSCs provided for inhibiting an immune response may be administered in quantities of about 10 million to about 10 billion. In some embodiments, human MSCs may be administered in quantities of about 10 million to about 100 million, about 10 million to about 1 billion, about 10 million to about 10 billion, about 100 million to about 1 billion, about 100 million to about 10 billion, or about 1 billion to about 10 billion. In some embodiments, human MSCs may be administered in quantities of about 10 million, about 100 million, about 1 billion, or about 10 billion. In some embodiments, human MSCs may be administered in quantities of at least about 10 million, about 100 million, or about 1 billion. In some embodiments, human MSCs may be administered in quantities of at most about 100 million, about 1 billion, or about 10 billion. In some embodiments, the MSCs are administered to a subject with a perianal fistula. In some embodiments, the MSCs are injected into the perianal fistula.
[0316] In some embodiments, the composition of MSCs may be comprised of less than 5% CD45+. In some embodiments, the composition of MSCs may be comprised of less than about 0.5 % CD45+ to about 10 % CD45+. In some embodiments, the composition of MSCs may be comprised of less than about 10 % CD45+ to about 9 % CD45+, about 10 % CD45+ to about 8 % CD45+, about 10 % CD45+ to about 7 % CD45+, about 10 % CD45+ to about 6 % CD45+, about 10 % CD45+ to about 5 % CD45+, about 10 % CD45+ to about 4 % CD45+, about 10 % CD45+ to about 3 % CD45+, about 10 % CD45+ to about 2 % CD45+, about 10 % CD45+ to about 1 % CD45+, about 10 % CD45+ to about 0.5 % CD45+, about 9 % CD45+ to about 8 % CD45+, about 9 % CD45+ to about 7 % CD45+, about 9 % CD45+ to about 6 % CD45+, about 9 % CD45+ to about 5 % CD45+, about 9 % CD45+ to about 4 % CD45+, about 9 % CD45+ to about 3 % CD45+, about 9 % CD45+ to about 2 % CD45+, about 9 % CD45+ to about 1 % CD45+, about 9 % CD45+ to about 0.5 % CD45+, about 8 % CD45+ to about 7 % CD45+, about 8 % CD45+ to about 6 % CD45+, about 8 % CD45+ to about 5 % CD45+, about 8 % CD45+ to about 4 % CD45+, about 8 % CD45+ to about 3 % CD45+, about 8 % CD45+ to about 2 % CD45+, about 8 % CD45+ to about 1 % CD45+, about 8 % CD45+ to about 0.5 % CD45+, about 7 % CD45+ to about 6 % CD45+, about 7 % CD45+ to about 5 % CD45+, about 7 % CD45+ to about 4 % CD45+, about 7 % CD45+ to about 3 % CD45+, about 7 % CD45+ to about 2 % CD45+, about 7 % CD45+ to about 1 % CD45+, about 7 % CD45+ to about 0.5 % CD45+, about 6 % CD45+ to about 5 % CD45+, about 6 % CD45+ to about 4 % CD45+, about 6 % CD45+ to about 3 % CD45+, about 6 % CD45+ to about 2 % CD45+, about
6 % CD45+ to about 1 % CD45+, about 6 % CD45+ to about 0.5 % CD45+, about 5 % CD45+ to about 4 % CD45+, about 5 % CD45+ to about 3 % CD45+, about 5 % CD45+ to about 2 % CD45+, about 5 % CD45+ to about 1 % CD45+, about 5 % CD45+ to about 0.5 % CD45+, about 4 % CD45+ to about 3 % CD45+, about 4 % CD45+ to about 2 % CD45+, about 4 % CD45+ to about 1 % CD45+, about 4 % CD45+ to about 0.5 % CD45+, about 3 % CD45+ to about 2 % CD45+, about 3 % CD45+ to about 1 % CD45+, about 3 % CD45+ to about 0.5 % CD45+, about 2 % CD45+ to about 1 % CD45+, about 2 % CD45+ to about 0.5 % CD45+, or about 1 % CD45+ to about 0.5 % CD45+. In some embodiments, the composition of MSCs may be comprised of less than about 10 % CD45+, about 9 % CD45+, about 8 % CD45+, about
7 % CD45+, about 6 % CD45+, about 5 % CD45+, about 4 % CD45+, about 3 % CD45+, about 2 % CD45+, about 1 % CD45+, or about 0.5 % CD45+. In some embodiments, the composition of MSCs may be comprised of less than at least about 10 % CD45+, about 9 % CD45+, about 8 % CD45+, about 7 % CD45+, about 6 % CD45+, about 5 % CD45+, about 4 % CD45+, about 3 % CD45+, about 2 % CD45+, or about 1 % CD45+. In some embodiments, the composition of MSCs may be comprised of less than at most about 9 % CD45+, about 8 % CD45+, about 7 % CD45+, about 6 % CD45+, about 5 % CD45+, about 4 % CD45+, about 3 % CD45+, about 2 % CD45+, about 1 % CD45+, or about 0.5 % CD45+.
[0317] In some embodiments, the composition of MSCs may comprise more than 1% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.1% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.2% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.3% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.4% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.5% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.6% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.7% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.8% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 1.9% CD45+ cells. In some embodiments, the composition of MSCs may comprise more than 2% CD45+ cells. [0318] In some embodiments, the composition of MSCs may be comprised of at least 90% CD105+ cells. In some embodiments, the composition of MSCs may be comprised of at least about 70 % CD 105+ cells to about 100 % CD 105+ cells. In some embodiments, the composition of MSCs may be comprised of at least about 100 % CD 105+ cells to about 95 % CD 105+ cells, about 100 % CD 105+ cells to about 94 % CD 105+ cells, about 100 % CD 105+ cells to about 93 % CD 105+ cells, about 100 % CD 105+ cells to about 92 % CD 105+ cells, about 100 % CD 105+ cells to about 91 % CD 105+ cells, about 100 % CD 105+ cells to about 90 % CD 105+ cells, about 100 % CD 105+ cells to about 85 % CD 105+ cells, about 100 % CD105+ cells to about 80 % CD105+ cells, about 100 % CD105+ cells to about 75 % CD105+ cells, about 100 % CD 105+ cells to about 70 % CD 105+ cells, about 95 % CD 105+ cells to about 94 % CD105+ cells, about 95 % CD105+ cells to about 93 % CD105+ cells, about 95 % CD 105+ cells to about 92 % CD 105+ cells, about 95 % CD 105+ cells to about 91 % CD 105+ cells, about 95 % CD 105+ cells to about 90 % CD 105+ cells, about 95 % CD 105+ cells to about 85 % CD105+ cells, about 95 % CD105+ cells to about 80 % CD105+ cells, about 95 % CD105+ cells to about 75 % CD105+ cells, about 95 % CD105+ cells to about 70 % CD105+ cells, about 94 % CD 105+ cells to about 93 % CD 105+ cells, about 94 % CD 105+ cells to about 92 % CD105+ cells, about 94 % CD105+ cells to about 91 % CD105+ cells, about 94 % CD 105+ cells to about 90 % CD 105+ cells, about 94 % CD 105+ cells to about 85 % CD 105+ cells, about 94 % CD 105+ cells to about 80 % CD 105+ cells, about 94 % CD 105+ cells to about 75 % CD105+ cells, about 94 % CD105+ cells to about 70 % CD105+ cells, about 93 % CD 105+ cells to about 92 % CD 105+ cells, about 93 % CD 105+ cells to about 91 % CD 105+ cells, about 93 % CD 105+ cells to about 90 % CD 105+ cells, about 93 % CD 105+ cells to about 85 % CD105+ cells, about 93 % CD105+ cells to about 80 % CD105+ cells, about 93 % CD105+ cells to about 75 % CD105+ cells, about 93 % CD105+ cells to about 70 % CD105+ cells, about 92 % CD 105+ cells to about 91 % CD 105+ cells, about 92 % CD 105+ cells to about 90 % CD105+ cells, about 92 % CD105+ cells to about 85 % CD105+ cells, about 92 % CD105+ cells to about 80 % CD105+ cells, about 92 % CD105+ cells to about 75 % CD105+ cells, about 92 % CD 105+ cells to about 70 % CD 105+ cells, about 91 % CD 105+ cells to about 90 % CD105+ cells, about 91 % CD105+ cells to about 85 % CD105+ cells, about 91 % CD105+ cells to about 80 % CD105+ cells, about 91 % CD105+ cells to about 75 % CD105+ cells, about 91 % CD 105+ cells to about 70 % CD 105+ cells, about 90 % CD 105+ cells to about 85 % CD105+ cells, about 90 % CD105+ cells to about 80 % CD105+ cells, about 90 % CD105+ cells to about 75 % CD105+ cells, about 90 % CD105+ cells to about 70 % CD105+ cells, about 85 % CD 105+ cells to about 80 % CD 105+ cells, about 85 % CD 105+ cells to about 75 % CD105+ cells, about 85 % CD105+ cells to about 70 % CD105+ cells, about 80 % CD105+ cells to about 75 % CD105+ cells, about 80 % CD105+ cells to about 70 % CD105+ cells, or about 75 % CD105+ cells to about 70 % CD105+ cells. In some embodiments, the composition of MSCs may be comprised of at least about 100 % CD105+ cells, about 95 % CD105+ cells, about 94 % CD105+ cells, about 93 % CD105+ cells, about 92 % CD105+ cells, about 91 % CD 105+ cells, about 90 % CD 105+ cells, about 85 % CD 105+ cells, about 80 % CD105+ cells, about 75 % CD105+ cells, or about 70 % CD105+ cells. In some embodiments, the composition of MSCs may be comprised of at least at least about 100 % CD105+ cells, about 95 % CD 105+ cells, about 94 % CD 105+ cells, about 93 % CD 105+ cells, about 92 % CD105+ cells, about 91 % CD105+ cells, about 90 % CD105+ cells, about 85 % CD105+ cells, about 80 % CD105+ cells, or about 75 % CD105+ cells. In some embodiments, the composition of MSCs may be comprised of at least at most about 95 % CD 105+ cells, about 94 % CD 105+ cells, about 93 % CD 105+ cells, about 92 % CD 105+ cells, about 91 % CD 105+ cells, about 90 % CD105+ cells, about 85 % CD105+ cells, about 80 % CD105+ cells, about 75 % CD105+ cells, or about 70 % CD 105+ cells.
[0319] In some embodiments, the composition of MSCs may be comprised of at least 90% CD166+ cells. In some embodiments, the composition of MSCs may be comprised of at least about 70 % CD 166+ cells to about 100 % CD 166+ cells. In some embodiments, the composition of MSCs may be comprised of at least about 100 % CD 166+ cells to about 95 % CD 166+ cells, about 100 % CD 166+ cells to about 94 % CD 166+ cells, about 100 % CD 166+ cells to about 93 % CD 166+ cells, about 100 % CD 166+ cells to about 92 % CD 166+ cells, about 100 % CD 166+ cells to about 91 % CD 166+ cells, about 100 % CD 166+ cells to about 90 % CD 166+ cells, about 100 % CD 166+ cells to about 85 % CD 166+ cells, about 100 % CD166+ cells to about 80 % CD166+ cells, about 100 % CD166+ cells to about 75 % CD166+ cells, about 100 % CD 166+ cells to about 70 % CD 166+ cells, about 95 % CD 166+ cells to about 94 % CD166+ cells, about 95 % CD166+ cells to about 93 % CD166+ cells, about 95 % CD 166+ cells to about 92 % CD 166+ cells, about 95 % CD 166+ cells to about 91 % CD 166+ cells, about 95 % CD 166+ cells to about 90 % CD 166+ cells, about 95 % CD 166+ cells to about 85 % CD166+ cells, about 95 % CD166+ cells to about 80 % CD166+ cells, about 95 % CD166+ cells to about 75 % CD166+ cells, about 95 % CD166+ cells to about 70 % CD166+ cells, about 94 % CD 166+ cells to about 93 % CD 166+ cells, about 94 % CD 166+ cells to about 92 % CD166+ cells, about 94 % CD166+ cells to about 91 % CD166+ cells, about 94 % CD 166+ cells to about 90 % CD 166+ cells, about 94 % CD 166+ cells to about 85 % CD 166+ cells, about 94 % CD 166+ cells to about 80 % CD 166+ cells, about 94 % CD 166+ cells to about 75 % CD166+ cells, about 94 % CD166+ cells to about 70 % CD166+ cells, about 93 % CD 166+ cells to about 92 % CD 166+ cells, about 93 % CD 166+ cells to about 91 % CD 166+ cells, about 93 % CD 166+ cells to about 90 % CD 166+ cells, about 93 % CD 166+ cells to about 85 % CD166+ cells, about 93 % CD166+ cells to about 80 % CD166+ cells, about 93 % CD166+ cells to about 75 % CD166+ cells, about 93 % CD166+ cells to about 70 % CD166+ cells, about 92 % CD 166+ cells to about 91 % CD 166+ cells, about 92 % CD 166+ cells to about 90 % CD166+ cells, about 92 % CD166+ cells to about 85 % CD166+ cells, about 92 % CD166+ cells to about 80 % CD166+ cells, about 92 % CD166+ cells to about 75 % CD166+ cells, about 92 % CD 166+ cells to about 70 % CD 166+ cells, about 91 % CD 166+ cells to about 90 % CD166+ cells, about 91 % CD166+ cells to about 85 % CD166+ cells, about 91 % CD166+ cells to about 80 % CD166+ cells, about 91 % CD166+ cells to about 75 % CD166+ cells, about 91 % CD 166+ cells to about 70 % CD 166+ cells, about 90 % CD 166+ cells to about 85 % CD166+ cells, about 90 % CD166+ cells to about 80 % CD166+ cells, about 90 % CD166+ cells to about 75 % CD166+ cells, about 90 % CD166+ cells to about 70 % CD166+ cells, about 85 % CD 166+ cells to about 80 % CD 166+ cells, about 85 % CD 166+ cells to about 75 % CD166+ cells, about 85 % CD166+ cells to about 70 % CD166+ cells, about 80 % CD166+ cells to about 75 % CD166+ cells, about 80 % CD166+ cells to about 70 % CD166+ cells, or about 75 % CD166+ cells to about 70 % CD166+ cells. In some embodiments, the composition of MSCs may be comprised of at least about 100 % CD166+ cells, about 95 % CD 166+ cells, about 94 % CD 166+ cells, about 93 % CD 166+ cells, about 92 % CD 166+ cells, about 91 % CD 166+ cells, about 90 % CD 166+ cells, about 85 % CD 166+ cells, about 80 % CD166+ cells, about 75 % CD166+ cells, or about 70 % CD166+ cells. In some embodiments, the composition of MSCs may be comprised of at least at least about 100 % CD166+ cells, about 95 % CD 166+ cells, about 94 % CD 166+ cells, about 93 % CD 166+ cells, about 92 % CD166+ cells, about 91 % CD166+ cells, about 90 % CD166+ cells, about 85 % CD166+ cells, about 80 % CD166+ cells, or about 75 % CD166+ cells. In some embodiments, the composition of MSCs may be comprised of at least at most about 95 % CD 166+ cells, about 94 % CD 166+ cells, about 93 % CD 166+ cells, about 92 % CD 166+ cells, about 91 % CD 166+ cells, about 90 % CD 166+ cells, about 85 % CD 166+ cells, about 80 % CD 166+ cells, about 75 % CD 166+ cells, or about 70 % CD 166+ cells.
MSCs for Treating Medical Conditions
[0320] In some embodiments, the compositions described herein are administered to treat a medical condition. In some embodiments, the medical condition comprises a perianal fistula. In some embodiments, the composition of human MSCs provided for treating a medical condition may be administered in quantities of about 10 million to about 10 billion. In some embodiments, human MSCs may be administered in quantities of about 10 million to about 100 million, about 10 million to about 1 billion, about 10 million to about 10 billion, about 100 million to about 1 billion, about 100 million to about 10 billion, or about 1 billion to about 10 billion. In some embodiments, the human MSCs may be administered in quantities of about 10 million, about 100 million, about 1 billion, or about 10 billion. In some embodiments, human MSCs may be administered in quantities of at least about 10 million, about 100 million, or about 1 billion. In some embodiments, human MSCs may be administered in quantities of at most about 100 million, about 1 billion, or about 10 billion. In some embodiments, the human MSCs are isolated and processed as described herein.
[0321] In some embodiments the cell composition may aid in producing a state of mixed chimerism. In some embodiments, the administered cell composition within a subject of human MSCs may generate CD45-huCD73+huCD90+ cells within said subject. In some embodiments, the administered human MSCs may be derived from bone marrow (vBM-MSC), adherent vertebral body MSCs (vBA-MSCs), or both.
[0322] In another aspect of the present disclosure, a method and composition are provided for treating a medical condition by administering a composition of at least 10 million human MSCs and at least 500,000 nucleated BM cells or derivatives thereof.
[0323] In some embodiments, the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof into a subject may result in successful engraftment of at least a subset of the nucleated BM cells within the subject. In some embodiments, the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof into a subject may generate hematopoietic cells of a particular HLA haploid in a background of hematopoietic cells of another particular HLA haploid wherein the other particular HLA haploid is mis-matched with the former HLA haploid. In some embodiments, the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof into a subject may generate hematopoietic cells of a particular HLA haploid specific to the donor of the human MSCs and nucleated BM cells or derivatives thereof in a background of hematopoietic cells of a particular HLA haploid specific to the subject. In some embodiments, the hematopoietic cells generated in the subject following administration of human MSCs and nucleated BM cells or derivatives thereof are CD45+ cells. In some embodiments, the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof may generate CD45+H2d+ cells in a background of CD45+H2b+ cells. In some embodiments, the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof may generate CD45-huCD73+huCD90+ cells. In some embodiments, the method of administrating a composition of human MSCs and nucleated BM cells or derivatives thereof may generate a mixed chimerism within the subject. In some embodiments, the generated mixed chimerism within the subject may be maintained for at least 120 days from administration of the nucleated BM cells. In some embodiments, the generated mixed chimerism within the subject may be maintained for about 1 month to about 12 months. In some embodiments, the generated mixed chimerism within the subject may be maintained for about 1 month to about 2 months, about 1 month to about 3 months, about 1 month to about 4 months, about 1 month to about 5 months, about 1 month to about 6 months, about 1 month to about 7 months, about 1 month to about 8 months, about 1 month to about 9 months, about
1 month to about 10 months, about 1 month to about 11 months, about 1 month to about 12 months, about 2 months to about 3 months, about 2 months to about 4 months, about 2 months to about 5 months, about 2 months to about 6 months, about 2 months to about 7 months, about
2 months to about 8 months, about 2 months to about 9 months, about 2 months to about 10 months, about 2 months to about 11 months, about 2 months to about 12 months, about 3 months to about 4 months, about 3 months to about 5 months, about 3 months to about 6 months, about 3 months to about 7 months, about 3 months to about 8 months, about 3 months to about 9 months, about 3 months to about 10 months, about 3 months to about 11 months, about 3 months to about 12 months, about 4 months to about 5 months, about 4 months to about 6 months, about 4 months to about 7 months, about 4 months to about 8 months, about 4 months to about 9 months, about 4 months to about 10 months, about 4 months to about 11 months, about 4 months to about 12 months, about 5 months to about 6 months, about 5 months to about
7 months, about 5 months to about 8 months, about 5 months to about 9 months, about 5 months to about 10 months, about 5 months to about 11 months, about 5 months to about 12 months, about 6 months to about 7 months, about 6 months to about 8 months, about 6 months to about 9 months, about 6 months to about 10 months, about 6 months to about 11 months, about 6 months to about 12 months, about 7 months to about 8 months, about 7 months to about 9 months, about 7 months to about 10 months, about 7 months to about 11 months, about 7 months to about 12 months, about 8 months to about 9 months, about 8 months to about 10 months, about 8 months to about 11 months, about 8 months to about 12 months, about 9 months to about 10 months, about 9 months to about 11 months, about 9 months to about 12 months, about 10 months to about 11 months, about 10 months to about 12 months, or about 11 months to about 12 months. In some embodiments, the generated mixed chimerism within the subject may be maintained for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months. In some embodiments, the generated mixed chimerism within the subject may be maintained for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 11 months. In some embodiments, the generated mixed chimerism within the subject may be maintained for at most about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months.
[0324] In some embodiments, the generated mixed chimerism within the subject may be maintained for about 1 year to about 10 years. In some embodiments, the generated mixed chimerism within the subject may be maintained for about 1 year to about 2 years, about 1 year to about 3 years, about 1 year to about 4 years, about 1 year to about 5 years, about 1 year to about 6 years, about 1 year to about 7 years, about 1 year to about 8 years, about 1 year to about 9 years, about 1 year to about 10 years, about 2 years to about 3 years, about 2 years to about 4 years, about 2 years to about 5 years, about 2 years to about 6 years, about 2 years to about 7 years, about 2 years to about 8 years, about 2 years to about 9 years, about 2 years to about 10 years, about 3 years to about 4 years, about 3 years to about 5 years, about 3 years to about 6 years, about 3 years to about 7 years, about 3 years to about 8 years, about 3 years to about 9 years, about 3 years to about 10 years, about 4 years to about 5 years, about 4 years to about 6 years, about 4 years to about 7 years, about 4 years to about 8 years, about 4 years to about 9 years, about 4 years to about 10 years, about 5 years to about 6 years, about 5 years to about 7 years, about 5 years to about 8 years, about 5 years to about 9 years, about 5 years to about 10 years, about 6 years to about 7 years, about 6 years to about 8 years, about 6 years to about 9 years, about 6 years to about 10 years, about 7 years to about 8 years, about 7 years to about 9 years, about 7 years to about 10 years, about 8 years to about 9 years, about 8 years to about 10 years, or about 9 years to about 10 years. In some embodiments, the generated mixed chimerism within the subject may be maintained for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years. In some embodiments, the generated mixed chimerism within the subject may be maintained for at least about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, or about 9 years. In some embodiments, the generated mixed chimerism within the subject may be maintained for at most about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about
9 years, or about 10 years.
[0325] In some embodiments, the method of first administrating a composition of human MSCs and nucleated BM cells or derivatives thereof may further comprise a second administration of at least 10 million human MSCs to the subject in need thereof 1 day, 2 days, 3 days, 4 days, or any combination thereof after the first administration.
[0326] In some embodiments, the treated medical condition may be an immune response. In some embodiments, the medical condition may be a perianal fistula. In some embodiments, the treated medical condition may be an autoimmune disease. In some embodiments, the autoimmune disease may be Crohn’s Disease.
[0327] In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include human MSCs derived from BM (vBM-MSCs), adherent vertebral body MSCs (vBA-MSCs), or both. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include MSCs in quantities of about
10 million to about 10 billion. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include MSCs in quantities of about 10 million to about 100 million, about 10 million to about 1 billion, about 10 million to about 10 billion, about 100 million to about 1 billion, about 100 million to about 10 billion, or about 1 billion to about 10 billion. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include MSCs in quantities of about 10 million, about 100 million, about 1 billion, or about 10 billion. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include MSCs in quantities of at least about 10 million, about 100 million, or about 1 billion.
[0328] In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include nucleated BM in quantities about 1 million to about 2 million. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include nucleated BM in quantities about 1 million to about 1.5 million, about 1 million to about 2 million, or about 1.5 million to about 2 million. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include nucleated BM in quantities about 1 million, about 1.5 million, or about 2 million. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include nucleated BM in quantities at least about 1 million, or about 1.5 million. In some embodiments, the composition of human MSCs and nucleated BM cells or derivatives thereof may include nucleated BM in quantities at most about 1.5 million, or about 2 million.
[0329] In some embodiments, the human MSCs may be derived from the same origin as the nucleated BM cells. In some embodiments, the human MSCs may be derived from a different origin as the nucleated BM cells. In some embodiments, the composition may further comprise a human organ. In some embodiments, the human organ may be a heart, kidney, liver, lung, pancreases, intestine, thymus, or uterus. In some embodiments, the composition comprised of human MSCs and nucleated BM cells or derivative thereof may include MSCs with a matched human leukocyte antigen (HLA) haploid type as the human organ. In some embodiments, the composition comprised of human MSCs and nucleated BM cells or derivative thereof may include MSCs with mis-matched HLA haploid type as the human organ. In some embodiments, the composition comprised of human MSCs and nucleated BM cells or derivative thereof may include MSCs and nucleated BM cells or derivatives thereof with a mis-matched HLA haploid type as the human organ. In some embodiments, the composition comprised of human MSCs and nucleated BM cells or derivative thereof may include MSCs and nucleated BM cells or derivatives thereof with a matched HLA haploid type as the human organ.
[0330] In some embodiments, the nucleated BM cells or derivatives thereof may comprise hematopoietic stem cells (HSC). In some embodiments, the human MSCs may comprise a mismatched HLA haploid type as the nucleated BM cells or derivatives thereof. Compositions of MSCs and BM for Treating Perianal Fistulas
[0331] Another aspect of the present disclosure comprises compositions comprising one or more of the cell types described herein.
[0332] In some embodiments, described herein is a composition comprising vBA-MSCs. In some embodiments, the vBA-MSCs are . In some embodiments, the vBA-MSCs are isolated and processed as described herein. In some embodiments, the compositions described herein comprise at least about 500,000 vBA-MSCs to about 1,000,000,000 vBA-MSCs. In some embodiments, the compositions described herein comprise at least about 500,000 vBA-MSCs to about 1,000,000 vBA-MSCs, about 500,000 vBA-MSCs to about 10,000,000 vBA-MSCs, about 500,000 vBA-MSCs to about 100,000,000 vBA-MSCs, about 500,000 vBA-MSCs to about 1,000,000,000 vBA-MSCs, about 1,000,000 vBA-MSCs to about 10,000,000 vBA-MSCs, about 1,000,000 vBA-MSCs to about 100,000,000 vBA-MSCs, about 1,000,000 vBA-MSCs to about 1,000,000,000 vBA-MSCs, about 10,000,000 vBA-MSCs to about 100,000,000 vBA-MSCs, about 10,000,000 vBA-MSCs to about 1,000,000,000 vBA-MSCs, or about 100,000,000 vBA- MSCs to about 1,000,000,000 vBA-MSCs. In some embodiments, the compositions described herein comprise at least about 500,000 vBA-MSCs, about 1,000,000 vBA-MSCs, about 10,000,000 vBA-MSCs, about 100,000,000 vBA-MSCs, or about 1,000,000,000 vBA-MSCs. In some embodiments, the compositions described herein comprise at least at least about 500,000 vBA-MSCs, about 1,000,000 vBA-MSCs, about 10,000,000 vBA-MSCs, or about 100,000,000 vBA-MSCs. In some embodiments, the compositions described herein comprise at least at most about 1,000,000 vBA-MSCs, about 10,000,000 vBA-MSCs, about 100,000,000 vBA-MSCs, or about 1,000,000,000 vBA-MSCs. In some embodiments, the compositions described herein comprise vBA-MSCs and bone marrow-derived MSCs.
[0333] In some embodiments, described herein is a composition comprising Nucleated BM cells. In some embodiments, the nucleated BM cells are isolated and processed as described herein. In some embodiments, the compositions described herein comprise at least about 500,000 nucleated BM cells to about 2,000,000 nucleated BM cells. In some embodiments, the compositions described herein comprise at least about 500,000 nucleated BM cells to about 1,000,000 nucleated BM cells, about 500,000 nucleated BM cells to about 1,500,000 nucleated BM cells, about 500,000 nucleated BM cells to about 2,000,000 nucleated BM cells, about 1,000,000 nucleated BM cells to about 1,500,000 nucleated BM cells, about 1,000,000 nucleated BM cells to about 2,000,000 nucleated BM cells, or about 1,500,000 nucleated BM cells to about 2,000,000 nucleated BM cells. In some embodiments, the compositions described herein comprise at least about 500,000 nucleated BM cells, about 1,000,000 nucleated BM cells, about 1,500,000 nucleated BM cells, or about 2,000,000 nucleated BM cells. In some embodiments, the compositions described herein comprise at least at least about 500,000 nucleated BM cells, about 1,000,000 nucleated BM cells, or about 1,500,000 nucleated BM cells. In some embodiments, the compositions described herein comprise at least at most about 1,000,000 nucleated BM cells, about 1,500,000 nucleated BM cells, or about 2,000,000 nucleated BM cells.
[0334] In some embodiments, the compositions described herein comprise both vBA- MSCs and nucleated BM cells. In some embodiments, the vBA-MSCs and nucleated BM cells of a single composition are derived from a single donor. In some embodiments, the compositions described herein comprise both vBM-MSCs and nucleated BM cells. In some embodiments, the vBM-MSCs and nucleated BM cells of a single composition are derived from a single donor. In some embodiments, the compositions described herein comprise both vBM-MSCs and vBA-MSCs. In some embodiments, the vBM-MSCs and vBA-MSCs of a single composition are derived from a single donor. In some embodiments, the compositions described herein comprise vBM-MSCs, vBA-MSCs, and nucleated BM cells. In some embodiments, the vBM-MSCs, vBA-MSCs, and nucleated BM cells of a single composition are derived from a single donor.
[0335] In some embodiments, the nucleated bone marrow cells or derivatives thereof comprise hematopoietic stem cells (HSCs). In some embodiments, the HSCs comprise CD34+ cells.
METHODS OF TREATING
[0336] In certain aspects, described herein is a method for treating a perianal fistula in a subject, the method comprising injecting into the perianal fistula a first dose of a composition comprising more than about 10 million human mesenchymal stem cells (MSCs) obtained from a non-living source. In some embodiments, described herein are methods of administering a composition comprising the MSCs described herein to a subject with a perianal fistula. The specific rationale for MSCs in perianal fistulas is based upon 1) their anti-inflammatory properties; 2) published experience of MSC in this condition and perianal Crohn’s fistula demonstrating efficacy and safety; 3) existence of cGMP methods for their isolation and growth.
[0337] In some embodiments, the subject has an autoimmune disease. In some embodiments, the subject has Crohn’s disease. In some embodiments, the perianal fistula is refractory. In some embodiments, the subject is at least 18 years of age. In some embodiments, the subject has failed at least one medical treatment to treat the fistula. In some embodiments, the medical treatment to treat the fistula comprises treatment with azarthiprine, methotrexate, 6-mercaptopurine, anti-TNF, anti-integrin, or anti-interleukin. In some embodiments, the subject has undergone surgical repair to treat the fistula. In some embodiments, the surgical repair is selected from the list consisting of seton placement, glue or plug insertion, local tissue flaps, and ligation of intersphincteric fistula tract repair
[0338] In some embodiments, the perianal fistula has been present for at least about 1 month, about 2 months, about 3 months, about 4 months, about 5, about months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months. In some embodiments, the perianal fistula has been present for about 1 to 12 months, about 2 to 11 months, about 3 to 10 months, about 4 to 9 months, about 5 to 8 months, or about 6 to 7 months.
[0339] In some embodiments, the method comprises administering a first dose. In some embodiments, the method comprises administering a first dose of at least about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140 million, about 150 million, about 160 million, about 170 million, about 180 million, about 190 million, about 200 million or more than 200 million MSCs. In some embodiments, the method comprise administering a dose of from about 60 million to 200 million, 70 million to 200 million, 80 million to 200 million, 90 million to 200 million, 100 million to 200 million, 110 million to 200 million, 120 million to 200 million, 130 million to 200 million, 140 million to 200 million, 150 million to 200 million, 160 million to 200 million, 170 million to 200 million, 180 million to 200 million, or 190 million to 200 million MSCs. In some embodiments, the MSCs are administered by injecting into the perianal fistula. In some embodiments, the MSCs are obtained from a non-living source.
[0340] In some embodiments, the methods described herein comprise administering a first dose of MSCs. In some embodiments, the methods described herein comprise administering a second dose of MSCs. In some embodiments, the MSCs are administered at least one time, two times, three times, four times, five times or more than five times. In some embodiments, the MSCs are administered no more than one time, two times, three times, four times, or five times. In some embodiments, at least one month, two months, three months, four months, five months, six month, seven months, eight months, nine months, ten months, eleven months or a year separate the time between administration. In some embodiments, the MSCs are administered by injecting into the perianal fistula. In some embodiments, the MSCs are obtained from a non-living source. In some embodiments, the MSCs are obtained using the methods described herein. In some embodiments, the MSCs comprise vBA-MSCs. In some embodiments, the MSCs comprises vBM-MSCs.
[0341] In some embodiments, the method comprises administering a second dose of MSCs. In some embodiments, the method comprises administering a second dose of at least about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140 million, about 150 million, about 160 million, about 170 million, about 180 million, about 190 million, about 200 million or more than 200 million MSCs. In some embodiments, the method comprise administering a second dose of from about 60 million to 200 million, 70 million to 200 million, 80 million to 200 million, 90 million to 200 million, 100 million to 200 million, 110 million to 200 million, 120 million to 200 million, 130 million to 200 million, 140 million to 200 million, 150 million to 200 million, 160 million to 200 million, 170 million to 200 million, 180 million to 200 million, or 190 million to 200 million MSCs. In some embodiments, the MSCs are administered by injecting into the perianal fistula.
[0342] In some embodiments, the first and the second dose independently comprise more than about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140 million, about 150 million, about 160 million, about 170 million, about 180 million, about 190 million, about 200 million or more than 200 million MSCs. In some embodiments, the method comprise administering a dose of from about 60 million to 200 million, 70 million to 200 million, 80 million to 200 million, 90 million to 200 million, 100 million to 200 million, 110 million to 200 million, 120 million to 200 million, 130 million to 200 million, 140 million to 200 million, 150 million to 200 million, 160 million to 200 million, 170 million to 200 million, 180 million to 200 million, or 190 million to 200 million MSCs. In some embodiments, the MSCs are administered by injecting into the perianal fistula.
[0343] In some embodiments, the methods described herein comprise injecting a composition comprising MSCs into a perianal fistula. In some embodiments, the injection comprising injecting a composition comprising MSCs into the submucosal layer of the bowel wall. In some embodiments, the injection comprises injecting a composition comprising MSCs along the length or the fistula tract. In some embodiments, the methods comprise injecting a composition comprising MSCs in parallel of the wall of the fistula tract. In some embodiments, the methods comprise injecting a composition comprising MSCs no deeper than 2 mm from the fistula tract wall. In some embodiments, each fistula is injected at least one, two, three, four, five, or more than five times. In some embodiments, each half of the fistula is injected. In some embodiments, each quadrant of the fistula is injected.
[0344] In some embodiments, the MSCs are administered via injection. In some embodiments, the MSCs are administered via injection to the perianal fistula. In some embodiments, about at least about 1 mL, about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL, about 1.5 mL, about 1.6 mL, about 1.7 mL, about 1.8 mL, about 1.9. 2 mL, about 2.1 mL, about 2.2 mL, about 2.3 mL, about 2.4 mL, about 2.5 mL, about 2.6 mL, about 2.7 mL, about 2.8 mL, about 2.9 mL, about 3 mL, about 3.1,3.2 mL, about 3.3 mL, about 3.4 mL, about
3.5.3.6 mL, about 3.7 mL, about 3.8 mL, about 3.9 mL, about or 4 mLs of cells will be injected within the fistula opening. In some embodiments, each fistula tract will be injected. In some embodiments, a single fistula will be injected at least 1, 2, 3, 4, 5, 6 7, 8, 9, or 10 times. In some embodiments, at least about 1 mL, about 1.2 mL, about 1.4 mL, about 1.6 mL, about 1.8 mL, about 2 mL, about 2.2 mL, about 2.4 mL, about 2.5 mL, about 2.6 mL, about 2.8 mL, about 3 mL, about 3.2 mL, about 2.4 mL, about 3.6 mL, about 2.8 mL, about 4 mL, about 4.2 mL, about 4.4 mL, about 4.6 mL, about 4.8 mL, about 5 mL, about 5.2 mL, about 5.4 mL, about .5.6 mL, about 5.8 mL, about 6 mL, about 6.2 mL, about 6.4 mL, about 6.6 mL, about 6.8 mL, about 7 mL, about 7.2 mL, about 7.4 mL, about 7.6 mL, about 7.8 mL, about 8 mL, about 8.2 mL, about 8.4 mL, about 8.6 mL, about .8. mL, about 9 mL, about 9.2 mL, about 9.4 mL, about
9.6 mL, about 9.8 mL, about 9.8 mL, about 10 mL, about 10.2 mL, about 10.4 mL, about 10.6 mL, about 10.8 mL, about 11 mL, about 11.2 mL, about 11.4 mL, about 11.6 mL, about 11.8 mL, about 12 mL, about 12.2 mL, about 12.4 mL, about 12.5 mL, about 12.6 mL, about 12.8 mL, about 13 mL, about 13.2 mL, about 12.4 mL, about 13.6 mL, about 12.8 mL, about 14 mL, about 14.2 mL, about 14.4 mL, about 14.6 mL, about 14.8 mL, about 15 mL, about 15.2 mL, about 15.4 mL, about 1.5.6 mL, about 15.8 mL, about 16 mL, about 16.2 mL, about 16.4 mL, about 16.6 mL, about 16.8 mL, about 17 mL, about 17.2 mL, about 17.4 mL, about 17.6 mL, about 17.8 mL, about 18 mL, about 18.2 mL, about 18.4 mL, about 18.6 mL, about 1.8. mL, about 19 mL, about 19.2 mL, about 19.4 mL, about 19.6 mL, about 19.8 mL, about 19.8 mL, about or 20 mLs total will be injected into the fistula.
[0345] In some embodiments, the subject has at least a second perianal fistula. In some embodiments, the second perianal fistula is administered a first dose of a composition comprising MSCs. In some embodiments, the at least second perianal fistula is administered a dose of dose of at least about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140 million, about 150 million, about 160 million, about 170 million, about 180 million, about 190 million, about 200 million or more than 200 million MSCs. In some embodiments, the at least second perianal fistula is administered a second dose of from about 60 million to 200 million, 70 million to 200 million, 80 million to 200 million, 90 million to 200 million, 100 million to 200 million, 110 million to 200 million, 120 million to 200 million, 130 million to 200 million, 140 million to 200 million, 150 million to 200 million, 160 million to 200 million, 170 million to 200 million, 180 million to 200 million, or 190 million to 200 million MSCs. In some embodiments, the MSCs are administered by injecting into the perianal fistula.
[0346] In some embodiments, the subject has at least a third perianal fistula. In some embodiments, the third perianal fistula is administered a first dose of a composition comprising MSCs. In some embodiments, the at least third perianal fistula is administered a dose of dose of at least about 10 million, about 20 million, about 30 million, about 40 million, about 50 million, about 60 million, about 70 million, about 80 million, about 90 million, about 100 million, about 110 million, about 120 million, about 130 million, about 140 million, about 150 million, about 160 million, about 170 million, about 180 million, about 190 million, about 200 million or more than 200 million MSCs. In some embodiments, the at least third perianal fistula is administered a second dose of from about 60 million to 200 million, 70 million to 200 million, 80 million to 200 million, 90 million to 200 million, 100 million to 200 million, 110 million to 200 million, 120 million to 200 million, 130 million to 200 million, 140 million to 200 million, 150 million to 200 million, 160 million to 200 million, 170 million to 200 million, 180 million to 200 million, or 190 million to 200 million MSCs. In some embodiments, the MSCs are administered by injecting into the perianal fistula.
[0347] In some embodiments, the MSCs are allogeneic to the subject. In some embodiments, the MSCs are HLA-matched to the subject. In some embodiments, the MSCS are HLA-mismatched to the subject. In some embodiments, the MSCs and the subject have fewer than seven HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have fewer than six HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have fewer than five HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have fewer than four HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have fewer than three HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have fewer than two HLA matches out of eight HLA markers. In some embodiments, the MSCs and the subject have no HLA matches out of eight HLA markers. In some embodiments, the MSCs are obtained from a non-living source.
[0348] In some embodiments, the MSCs have been cultured for at least one passage prior to administration to the subject. In some embodiments the MSCs have been cultured for at least one, two, three, four, five, six, or seven passages before administration to the subject.
[0349] In some embodiments, the MSCs have been cryopreserved before administration to the subject using the methods described herein. In some embodiments, the MSCs have been thawed before administration. In some embodiments, the MSCs have been thawed to at least about 0°C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C or 10 °C. In some embodiments, the MSCs have been thawed to from about 0 °C to 10 °C, 0 °C to 9 °C, 0 °C to 8 °C, 0 °C to 7 °C,0 °C to 6 °C, 0 °C to 5 °C, 0 °C to 4 °C, 0 °C to 3°C, 0 °C to 2 °C or 0 °C to 1 °C. In some embodiments, the MSCs have been thawed at least about 1 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 19 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 or 24 hours prior to administration to the subject. In some embodiments, the MSCs have been thawed for at least about 1 days, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 6 days, 6.5 days, 7 days, or more than 7 days prior to administration to the subject.
[0350] In some embodiments the MSCs are warmed to room temperature before administration to a subject using the methods described herein. In some embodiments, the MSCs are warmed to about body temperature before administration to a subject. In some embodiments, the MSCs are warmed to about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, or 38°C before administration to a subject.
[0351] In some embodiments, administration of the MSCs results in a reduction in size of the fistula, absence of fluid collection, or a lack of edema. In some embodiments, administration of the MSCs result in greater than or equal to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or complete cessation of drainage of the fistula. In some embodiments, administration of the MSCs results in epithelization of the external fistula opening.
[0352] In some embodiments, the subject experiences a complete healing of the perianal fistula. In some embodiments, the subject experiences a complete healing of the perianal fistula after the first injection. In some embodiments the subject experiences a complete healing of the perianal fistula after the second injection. In some embodiments, complete healing is assessed b radiographic healing, clinical healing, or a combination thereof. In some embodiments, radiographic healing comprises a MRI with an absence of fluid collection greater than 2 cm in 2 of 3 dimensions, a lack of edema, lack of inflammation, or lack of signs of active inflammatory response, or a combination thereof. In some embodiments, clinical healing comprises 100% cessation of draining on both clinical exam with deep palpation and as per patient report, epithelization of the external fistula opening, or a combination thereof.
[0353] In some embodiments, the subject experiences a partial healing of the perianal fistula. In some embodiments, the subject experiences a partial healing of the perianal fistula after the first injection. In some embodiments the subject experiences a partial healing of the perianal fistula after the second injection. In some embodiments, partial health is assessed by radiographic healing, clinical healing, or a combination thereof. In some embodiments, radiographic healing comprises a MRI with an absence of fluid collection greater than 2 cm in 2 of 3 dimensions, a lack of edema, lack of inflammation, or lack of signs of active inflammatory response, or a combination thereof. In some embodiments, clinical healing comprises greater than or equal to 50% cessation of drainage on both clinical exam with deep palpation and as per patient report, epithelization of the external fistula opening, or a combination thereof.
[0354] In some embodiments, described herein is a method of reducing the size of a perianal fistula, the method comprising administering a composition comprising MSCs to a subject. In some embodiments, administration of the MSCs result in greater than or equal to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% reduction in size of the fistula. In some embodiments, the method comprises injecting the composition into the perianal fistula. In some embodiments, the method comprises injecting a first dose of a composition comprising more than about 10 million MSCs obtained from a non-living source.
[0355] In some embodiments, described herein is a method of reducing the amount of fluid collection in a perianal fistula, the method comprising administering a composition comprising MSCs to a subject. In some embodiments, administration of the MSCs result in greater than or equal to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% reduction in fluid collection of the fistula. In some embodiments, the method comprises injecting the composition into the perianal fistula. In some embodiments, the method comprises injecting a first dose of a composition comprising more than about 10 million MSCs obtained from a non-living source.
[0356] In some embodiments, the product administered comprises MSC formulated Plasma-Lyte ®. In some embodiments, the product administered comprises Human Serum Albumin (HSA). In some embodiments, the product administered comprises at least about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% HSA. In some embodiments, the product administered comprise DMSO. In some somebody, the product administered comprise at least about 1%, about 2%, about 3%. 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% DMSO. In some embodiments, the product administered comprises at least about 5M, about 10M, about 15M, about 20M, about 25M, about 30M, about 40M, about 45M, or about 50M cells/mL. In some embodiments, the product administered comprises MSC formulated Plasma-Lyte® A, 2.5% Human Serum Albumin (HSA), and 5% DMSO at 20M cells/mL. In some embodiments, the final product has an endotoxin dose of less than about 1, 2, 3, 4, or 5 Endotoxin Units (EU)/mL. [0357] In embodiments, Endotoxin testing is performed using the Endosafe®-PTS™ system according to SOP EPIC Limulus Amebocyte Lysate (LAL) Assay for Endotoxin Detection Using the Endosafe® - PTS™. The Endosafe®-PTS™ is a rapid, point-of-use test system that provides quantitative LAL results within 15 minutes after specimen preparation. The PTS™ utilizes LAL reagents in an FDA-licensed disposable test cartridge with a handheld reader for a completely contained, real-time endotoxin testing system. The PTS™ can be used to get a quick read on raw materials and STAT samples that require immediate analysis. The flexibility of the PTS™ allows it to be used in conventional, quality control testing laboratories as well as at the point of sample collection. Preferably cells for clinical use have < 2.5 EU/mL for release.
DEFINITIONS
[0358] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0359] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
[0360] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term “about” refers to ±10% of a stated number or value.
[0361] The term “from” as in “from 1 to 10” includes the initial and final number recited. Therefore, “from 1 to 10” includes the whole numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and includes fractions thereof, (e.g., about .1, .2, .3, .4, .5, .6, .7, .8, and about .9).
[0362] Use of absolute or sequential terms, for example, “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as exemplary.
[0363] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”
[0364] As used herein, the phrases “at least one”, “one or more”, and “and/or” are open- ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.
[0365] As used herein, “or” may refer to “and”, “or,” or “and/or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.
[0366] Terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts in a method of the present disclosure.
[0367] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
[0368] The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount. In some aspects, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase from about 10 to about 100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.
[0369] The terms, “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some aspects, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease from about 10 to about 100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease. [0370] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.
[0371] The term “zzz vivo" is used to describe an event that takes place in a subject’s body.
[0372] The term “ex vivo” is used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “in vitro” assay.
[0373] The term “z z vitro" is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
[0374] The term “ultimately” as in “MSCs [that] were ultimately obtained from a human cadaver” relates to the original source of the MSCs. In embodiments of the present disclosure, MSCs were directly obtained from deceased human donors, i.e., cadavers. In embodiments, MSCs directly obtained from deceased human donors are cultured for one or more passage; in these embodiments, a fraction of the MSCs present after one passage, will no have been obtained from a deceased from a donor. However, it remains true that the MSCs (even after culturing) were ultimately obtained from human cadaver. Thus, in some aspects, the ultimate source of a MSC was not a living donor.
[0375] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subj ect at risk of developing a particular disease, or to a subj ect reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
[0376] An "effective amount" or "therapeutically-effective amount" refers to that amount of a bone marrow product and/or HSCs contained in a bone marrow product as described herein which, when administered to a subject (e.g., human), that sufficient to promote treating a disease, e.g., a hematologic cancer. The amount of a bone marrow product and/or HSCs contained in the bone marrow product that constitutes a "therapeutically-effective amount" will vary depending on the cell preparations, the condition and its severity, the manner of administration, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
[0377] As used herein, the term “CD34” is used in reference to an antigen present on immature hematopoietic precursor cells and all hematopoietic colony-forming cells in bone marrow and blood. Certain populations of non-hematopoietic (i.e., CD45 negative) cells also express CD34. Of hematopoietic (i.e., CD45+ cells), the CD34 antigen expression is highest on early progenitor cells and decreases with the maturation of cells. The CD34 antigen is absent on fully differentiated hematopoietic cells. Normal peripheral blood lymphocytes, monocytes, granulocytes, and platelets do not express the CD34 antigen.
[0378] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
EXAMPLES
[0379] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Isolation of MSC
[0380] Described herein is an exemplary tissue processing protocol. In some cases, the tissue being processed can be vertebral bodies. In some cases, the tissue processing protocol can yield the bone marrow cells described herein.
A. Tissue Debriding
1. Spray down the surface of the exterior bag of fresh VBs with 70% isopropanol. In hood,
- I l l - remove outer nonsterile bag and dispose. Open inner bag and dispose of bag.
2. Unwrap specimen from blue towel and lap sponges. Record presence of packing materials and condition of the spine for: minimum 2 layers of sterile bas; blue towel; lap sponges; tissue moisture maintenance; and presence of pedicles.
3. Record the start time for tissue debriding.
4. Remove soft tissue surrounding pedicles to reveal correct sawing location. Scrape off exterior tissue with osteotomes.
5. If present, saw through pedicles. Retain anterior VBs and discard pedicles and posterior elements. Avoid exposing cancellous tissue.
6. Separate VBs by slicing through discs using the boning knife.
7. Remove remaining soft tissue from each individual VB surface, using a combination of scissors, knives, and osteotomes. Make note of any anatomical pathologies or injury during recovery (e.g. bone spurs, herniated discs, and degenerative discs, cuts into VBs from recovery, or others such as brittle bones).
8. Count the number of intact VBs and determine the levels recovered (e.g. T8-L5) Discard any VBs that were damaged during recovery and have cancellous tissue exposed.
9. Spray balance (CS-5000 model) with 70% IPA and place in a clean area inside the Biosafety cabinet (BSC). Tare balance with the sterile bag. Place VBs that will be processed further into the sterile bag, and record mass. Record the # of VBs used for BM extraction.
B. Surface Decontamination
1. Record the temperature of the VBs.
2. Place VBs into a sterile bag, then add 1 L of 10% bleach solution to the bag and ensure all VBs are submerged. Once bleach is added to the VB bag, immediately start a timer for 10 minutes. Allow 10 minutes of contact time before proceeding to B.4.
3. Remove all used processing equipment and drapes from the hood and remove soiled gloves. Clean BSC with 70% IPA and allow to dry before proceeding.
4. After 10 minutes of bleach solution contact time, immediately begin transfer of the VBs into a new sterile bag using a pair of sterile, long handled forceps.
5. Add 1 L of 3% hydrogen peroxide solution to the bag. Ensure VBs are completely submerged. Close the bag and shake briefly.
6. Transfer the VBs into a new sterile bag using new, sterile, long handled forceps.
7. Fill the bag with 1 L of Plasma-Lyte. Close the bag and shake briefly.
8. Transfer the VBs into a new sterile bag using new, sterile long handled forceps. 9. Fill the bag with 1 L of Plasma-Lyte. Close the bag and shake briefly.
10. Transfer the VBs to a sterile pan using long handled forceps. Use sterile gauze or lap sponges to absorb excessive liquid if needed.
11. Record the end time for surface decontamination.
C. Bone Grinding
1. Document the device used for grinding VBs and set up per Bone Grinder Operation and Maintenance or CCF Bone Grinder.
2. Record the grinding start time.
3. Obtain 1 L Grind media prepared at the beginning of the process.
4. Pour — 300mL of Grind Media into one sterile, stainless-steel pitcher. This pitcher will be called "Pitcher 1" and will contain cut VB pieces. Pour — 300 mL of Grind Media into another pitcher or catch pan named "Pitcher 2", to catch grindings. An additional — 300 mL will be used for rinsing through grinder while grinding. The remaining — 100 mL of Grind Media will be set aside for final rinsing of grinder and Pitcher 2 after all pieces are ground.
5. Place Pitcher 2 underneath the grinder head.
6. Using a clean drape and gloves donned, cut VBs into pieces of adequate size for the grinder using hand cutting tool. Cut pieces should immediately be submerged in Pitcher 1 with Grind Media.
7. Verify that IL of grind media was used and is in Pitcher 2. Turn off grinder and record the grinding end time.
D. Filtration
1. Open a Bone Marrow Collection Kit and record the mass of one empty, 600 mL TRANSFER-PACK using the VWR-3000P balance. Empty Mass of 600 mL TRANSFERPACK.
2. Assemble the bone marrow filtration kit and perform the bone marrow extraction following Bone Marrow Collection and Filtration using a total of 1000 mL of Rinse Media (2 x 500 mL). Note: Total media volume after is 2 L (IL Grind media and IL Rinse Media).
3. Document the mass (g) of each filled 600 mL TRANSFER-PACK and calculate the total mass of all 6 TRANSFER-PACKs.
4. Calculate the total mass of bone marrow (BM) extract: Total Mass (g) (D.3 [B]), Empty Mass (g) (D.l [A]), Empty Mass of all TRANSFER-PACKS (g) (A x 6), and Total Mass of BM Extract (g) (B-C).
5. Intermediate Accountability: Total Mass of BM Extract > 1800 g (if yes, proceed; if no alert supervisor).
6. Close the clamp on the extra 2000 mL TRANSFER-PACK and save for later use.
7. Visually inspect the bone marrow (BM) in each TRANSFER-PACK to confirm there are no visible grindings or soft tissue. If excessive clumping is observed during filtration, notify area management.
8. Identify the first TRANSFER-PACK filtered. Mix TRANSFER-PACK by inversion and then remove 0.3 mL of BM using a 1 mL syringe inside BSC. Place sample in a pre-labeled tube with the ISBT# and "QC 1 " along with the date and time. Submit sample to QC for testing on the Sysmex Hematology Analyzer. Record results below and calculate the TNC (use same number of significant figures from QC 1 Sysmex WBC concentration). Processing may proceed prior to obtaining this result. Note: Assume density of Ig/ml.
9. Seal the tubing near the connector on the end of each of the six TRANSFER-PACKs collected and label with the ISBT #.
10. Record the filtration end time.
E. Removal of Fat
1. Pair up TRANSFER-PACKs and use taring sticks so that the centrifuge is balanced prior to operation. Use volume compensating plates to prevent creasing of bags during centrifugation.
2. Set the centrifuge to 500 x g for 15 minutes at room temperature, with a brake setting of 4. Centrifuge TRANSFER-PACKs with tubing down.
3. While TRANSFER-PACKS are in the centrifuge, remove all drapes and supplies from the BSC nd clean all surfaces with 70% isopropyl alcohol (IP A).
4. Carefully remove TRANSFER-PACK, one at a time, from the centrifuge and hang on a ring stand.
5. Weld on an empty, new 600 mL Fenwal bag (post-fat intermediate bag) to the centrifuged TRANSFER-PACK. Label the new post-fat intermediate bag with the ISBT #. Inspect the weld prior to proceeding.
6. With the centrifuged TRANSFER-PACK hanging on one ring stand, place a bag clamp just below the fat, and open the weld on the tubing and drain pellet into new post-fat intermediate bag. Agitate the pellet and spike ports gently to resuspend all pellet. Allow at least half of the volume from the centrifuged bag to drain into the post-fat intermediate bag before proceeding. Note: It is best practice to not allow all the liquid to drain out from above the clamp. If liquid seems to be draining quickly, use one hand to press the clamp closed to slow the draining of liquid.
7. Close the tubing with a hemostat or tube sealer.
8. Weld the next centrifuged TRANSFER-PACK onto the same post-fat intermediate bag used to collect the pellet in E.6. Leave enough tubing on this bag for future welds.
9. Repeat E.6. -E.7.
10. For the next two centrifuged TRANSFER-PACKs, repeat E.4.-E.9. creating the second post-fat intermediate bag.
11. Repeat E.4.-E.9. for the final two centrifuged bags creating a third post-fat intermediate bag.
F. Concentrate
1. Set the centrifuge to 500 x g for 15 minutes at room temperature, with a brake setting of 4. Centrifuge post-fat intermediate bags with tubing up. Use volume compensating plates to prevent creasing of bags during centrifugation.
2. Carefully remove a post-fat intermediate bag from the centrifuge and hang on the plasma press. Only remove one bag at a time from the centrifuge.
3. For each bag centrifuged, weld on a 1000 mL waste bag (label as “Waste”) and use the plasma extractor to remove the supernatant into the waste bag. Use a hemostat to clamp tubing as soon as the pellet breaks or when the pellet rises close to the top.
4. Seal the tubing and cut through to remove the post-fat intermediate bag from the waste bag, leaving enough tubing attached for welding. Weld on a female lure extension.
5. Repeat F.2.-F.4. for each post-fat intermediate bag.
6. Discard waste bags in biohazard trash bag.
7. Label a new, empty 2 L bulk bag from the BM filtration kit with the ISBT #, then measure and record the mass. If the bag is removed from the BSC for weighting, clean the luer connection with sterile alcohol after returning to the BSC. Wait until dry before proceeding.
8. For the following materials, spray with 70% IP A, place inside the BSC and wait until dry before proceeding: 50 mL syringes (3), 30 mL syringe, 50 mL conical tube and rack, Rinse Media.
9. Combine pellets from each of the three small bags into the pre-weighed bulk bag using a new 50 mL syringe for each small bag. Note: Press down on the plunger of the syringe slowly and avoid creating bubbles.
10. Aseptically transfer 25 mL of rinse media into a 50 mL conical tube. Use a new 30 mL syringe to rinse each bag serially with - 20 mL of Rinse Media and add to the bulk bag. Note: A 50 mL syringe may be used to carry volume between bags if 30 mL syringe is too small.
G. Sampling and Accountability
1. On the bulk bag, open the clamp and drain BM extract in the tubing back into the bag. Invert bag three times minimum to mix, ensuring all pellet is resuspended. Remove about 0.5 mL of BM extract using a 1 mL syringe inside BSC. Place sample volume in a pre-labeled sterile sample tube with the ISBT # and "QC2" along with the date and time of sample collection. Submit sample to QC for testing, along with at least 50 mL of Rinse Media. Record the time samples were submitted for testing.
2. Measure and record the mass of the bulk bag of bone marrow extract. Subtract the empty mass from the filled mass to get the mass of BM extract (one decimal place), including empty mass [G] (g), filled mass [H] (g), and mass of BM extract [H-G] (g).
3. Record results from QC2 printout below and calculate QC2 concentration and the QC2 TNC Count (use the same number of significant figures from QC2 Sysmex WBC concentration for QC2 TNC Count). Note: Assume density of 1 g/mL, including QC2 Sysmex WBC Concentration (cells/ pL); QC2 Dilution Factor; QC2 Concentration (cells/mL) (K x L x 1000); and QC2 TNC Count [M x G.2 (J)].
4. Calculate the TNC % Yield to one decimal place for QC2 TNC Count (G.3 [N]); QC1 (D.8 [F]); and % Yield = (N - F) x 100.
H. Determining the number of bags
1. Record the QC2 TNC count from [G.3 (N)]. Calculate the total volume needed (one decimal place), Total Volume Needed (mL) [N (140 x 106)]. NOTE: If the volume is less than 228.9 mL, alert production supervisor.
2. Determine the number of bags and vials to prepare using the total volume needed previously.
3. Calculate the volume of freeze media needed, volume of Rinse Media, and volume of DMSO to add. Round calculated numbers to one decimal place (Total Volume Needed
(H.l [P]); Mass of BM Extract [G.2. J] (g is approximately ml); Total Vol. Freeze Media (Q-R); Vol. of DMSO (Q x 0.1); and Vol. of Rinse Media (S-T). Note: Assume density of BM extract is 1 g/ml.
I. Cryoprotectant Addition
1. Prepare the freeze media using rinse media prepared per B-6 of and 100% DMSO. Add the volume of rinse media calculated in H.3 [U] to a sterile bottle labeled "Freeze Media" with the date prepared. 2. Add the volume of DMSO calculated in H.3 [T] to the freeze media bottle. Gently invert the bottle once to mix.
3. Record the temperature of the Freeze Media.
4. If temperature of the Freeze Media is >25.0°C, wait until the temperature of the Freeze media decreases to < 25.0°C. Record the new temperature of the Freeze Media prior to use if applicable.
5. Inside BSC place the BM bulk bag on a rocker for mixing. Remove the plunger of a large syringe and connect to the lure port on the bulk bag. Keep the syringe upright during the entire addition.
6. Calculate the volume of Freeze media to add per minute to the bulk bag as determined by volume of Freeze Media (H.3.[S]) and volume to add per Minute (S x 0.1).
7. Set a timer for 10 minutes and begin adding freeze media through the syringe at a rate of 10% of the freeze media volume per minute, calculated in 1.6.
8. Record the start and end time of the DMSO addition. Aim for elapsed time from about 9 to about 11 minutes.
J. Cryopreservation
1. Refer to H.2. to determine the number of cryopreservation bags and surrogate vials needed. Close clamps and label cryopreservation bags with the prepared product labels, containing the ISBT number, product name, and date processed. Note: Label is placed inside the pocket on the top right of each bag. Use the tube sealer to tack the pocket so that the label will not fall out.
2. Use a 10 ml syringe to pull the entire volume for surrogate vials needed and fill with bone marrow (1 ml per vial).
3. For each cry opreservation bag, inside the BSC, use a new 100 ml syringe to fill the bag with 65 ml of bone marrow.
4. Unscrew the syringe to allow the tubing to drain back into the bag, then re-attach the syringe and draw air out of the bag while holding system upright.
5. Clamp tubing when bone marrow fills tubing, just before passing the Y connector. Discard syringe and replace cap.
6. Mix the bulk bag by inversion before removing more volume. Repeat J.3 -J.5 for each cry opreservation bag of product to prepare.
7. Record the actual number of bags prepared.
8. If there is bone marrow left in the bulk bag, vials for research use may be prepared. Label the required number of 5 mL cryovials and fill each one with 5 mL of BM by syringe or pipette.
9. Use the tube sealer to seal the tubing to create four segments on each product bag for cry opreservation.
10. Record the end time for bagging. Note: Product and samples must be frozen as quickly as possible after addition of DMSO.
11. Notify QC that bags are ready for cry opreservation. Note: QC will perform a packaging inspection prior to freezing product bags.
12. For each cry opreservation bag, cut through the seal in the tubing to remove 4 segments.
13. Document SmartCool boxes and Green CoolCells used.
14. Record the date and time the cassettes and samples were placed in the freezer.
15. Document Purple CoolCells used and freeze vials prepared for research use according to Cry opreservation (J) of Example 1. Record the date and time vials were placed in the freezer. K. Inventory
1. Enter donor material into Freezerworks in a Quarantine status, according to Freezerworks Inventory Management. Note: Ensure the numbers on each passive cooling container match the box number in Freezerworks.
Example 2. CD34 Selection from Fresh or Thawed BM from Deceased Donors Using CliniMACS plus
[0381] Described herein is a protocol for isolating cells expressing CD34 from fresh or thawed bone marrow (BM) from deceased donors. .
Buffer and bags preparation
Label five 600 ml Transfer-Pack bags as follows, and record the weight of each bag:
1) Cell Prep Bag 1 (can be more than 1 bag)
2) Plasma Waste
3) Waste 1
4) Waste 2
Buffers:
A. Prepare in Biosafety cabinet (BSC)
B. Labeling Buffer (2 bags):
1) Obtain 2 bags of Plasma Lyte (I L)
2) Obtain 2 30 cc syringes with 18 gauge needles affixed.
3) Using syringe and needle, inject 20 ml Benzonase (1000 U/ml) and 20 ml HSA (25%) to each 1 L Plasma Lyte bag.
4) Use a new syringe and needle for each injection.
5) Mix well by inverting at least 5 times.
6) Label each bag with “Labeling buffer”.
7) Final concentrations are 20U/ml Benzonase and 0.5% HSA.
C. Selection Buffer:
1) Obtain a 1 L bag of Plasma Lyte.
2) Obtain a 30 cc syringe with an 18 gauge needle affixed.
3) Using syringe and needle, inject 20 ml HSA (25%) into a 1 L Plasma Lyte bag.
4) Mix well by inverting at least 5 times.
5) Label the bag with “Selection buffer”.
6) Final concentration is 0.5% HSA.
Preparation for labeling of fresh (A) or frozen (B) bone marrow products
A. Protocol for fresh bone marrow product:
1) After grinding and removing fat, centrifuge bone marrow cell suspension in blood collection bags at 300xg for 15 minutes.
2) Perform following in a BSC.
3) Combine all bone marrow cell pellets into the Cell Prep Bag 1
4) Rinse all blood collection bags with 50 ml of Rinse media and transfer to Cell Prep Bag 1.
5) Weigh bag.
6) Determine total volume of cell suspension in the Cell Prep Bag 1 by subtracting original weight from that obtained in step 5 of this section. Use the following formula to convert weight to volume: Igram = 1 ml.
7) Gently mix Cell Prep Bag 1 with a rotating motion.
8) Use a 1.0 ml syringe to withdraw 0.5 ml bone marrow through a sampling site coupler and transfer to a 1.5 ml Eppendorf tube for CD34+ cell and T cell enumeration using flow cytometry.
9) Fill the Cell Prep Bag 1 with approximately 400 ml Labeling buffer and centrifuge at 300g for 15 minutes with a brake setting of 4 at room temperature.
10) Reduce volume in Cell Prep Bag 1 to desired volume based on total T cell and CD34+ cell counts as indicated in Table 6. Table 6. Optimal labeling volume and tubing set determination for the selection of CD34+ cells
Figure imgf000121_0001
B. Protocol for thawed bone marrow:
1) Thaw cells in 2 cryobags in a 37 °C water bath
2) Transfer all bags to a BSC.
3) Aseptically clean the ports and spike of each bag.
4) Using a 5 cc syringe with affixed needle, immediately inject Benzonase (lOOOU/ml) into each cryobag to achieve a final concentration of 20U/mL (e.g., for 70 ml of bone marrow product, inject 1.4 mL Benzonase) and mix well.
5) Combine contents from the 2 thawed cryobags into Cell prep Bag 1 by withdrawing using a 100 mL syringe attached to the transfer port.
6) Rinse each bag with 50 ml of Labeling buffer and slowly transfer to same Cell Prep Bag 1.
7) Record weight of Cell Prep Bag 1.
8) Record total volume of cell suspension in the Cell Prep Bag 1 (should no more than 200 mL) by subtracting the original weight from the weight obtained in step 7 (Igram = 1 mL).
9) Slowly fill Cell Prep Bag 1 with an equal volume of Labeling buffer by adding 10% of the volume per minute while shaking on a shaker.
10) Quickly add another volume of Labeling buffer to Cell Prep Bag 1.
11) After mixing well, remove 0.5 ml sample for T cell and CD34+ cell enumeration by flow cytometry.
12) Optional step: If clumps are present, insert standard blood filter, filter the cells and transfer to the second Cell Prep Bag.
13) Centrifuge at 300 g for 15 minutes with a brake setting of 4 at room temperature. 14) Express supernatant, gently mix cell pellet and combine all cells into one bag.
15) Wash bags and adjust volume to target volume with Labeling buffer according to
Table 6
Cell labeling and selection
A. Add human IVIG to Cell Prep Bag at final concentration 1.5mg/ml.
B. The calculated volume of IVIG added should be included in the final labeling weight, not to exceed 95 g or 190 g, depending on scale of preparation (Table 6).
C. Inject 100 ml of sterile air into the bag using a 100 ml syringe with affixed 0.2 micron filter.
D. Place the Cell Prep Bag on an orbital rotator and gently shake for 5 minutes at room temperature.
E. After 5 minutes, using a 20 ml syringe, inject 1 vial (7.5 ml) of CD34+ Reagent for Standard- scale or 2 vials (15 ml) for Large-scale into the Cell Prep Bag through the sampling site coupler.
F. Incubate bag on the orbital rotator for 30 minutes at room temperature.
G. In BSC, remove air in Cell Prep Bag using a 100 ml syringe. Add 500±10 ml (g) of Labeling buffer to the Cell Prep Bag. Centrifuge at 300g for 15 minutes, with a brake setting of 4 at room temperature.
H. Remove as much of the supernatant as possible (at least 500 ml for standardscale and 450 ml for Large-scale) from the Cell Prep Bag using a plasma press. Be careful not to remove cells.
I. Record the amount of supernatant removed.
J. Add 500±10 ml (g) of Labeling buffer to the Cell Prep Bag.
K. Centrifuge at 300g for 15 minutes, with a brake setting of 4 at room temperature.
L. Remove as much of the supernatant as possible (at least 500 ml for standardscale and 450 ml for Large-scale) from the Cell Prep Bag using a plasma press.
M. Gently mix cell pellet and resuspend pellet with Labeling buffer 1 to target volume 140 ml for standard-scale preparation or 265 ml for large-scale.
N. Inside the BSC, transfer 0.5 ml bone marrow using a 1 mL syringe to a 1.5 ml Eppendorf tube to perform pre-CliniMACS QC including cell count, T cell and CD34+ cell enumeration.
O. The product is ready to process on the CliniMACS plus instrument according to the Manufacture’s instruction with the exception that custom Selection buffer is used instead MACS buffer.
P. The volume of the selected cells at the end is expected to be -40-50 ml for the standard selection tubing set and -75-80 ml for large selection.
Q. Obtain samples for product QC.
R. Selected cells are ready for immediate infusion or cryopreservation.
Example 3: Thawing of Cryopreserved Bone Marrow Samples
[0382] Described herein is an exemplary procedure for thawing cryopreserved bone marrow (BM). In some cases, the procedure can be for thawing cryopreserved BM bags and vials for testing. In some instances, when thawing bags or vials (e.g. clinical process validations, clinical development, etc.), it can be used to thawing of bone marrow product or thawing of bone marrow vials.
Preparation
1) Prepare Rinse Media per Example 1. Obtain at least 5 mL of Rinse Media per sample.
2) Prepare a biosafety cabinet (BSC). Ensure all supplies are in the BSC prior to beginning the thaw. Label a 150 mL bottle or a 50 mL conical tube with ISBT donor #, bag #, and final dilution factor (DF=3).
3) Ensure a water bath has sufficient water (1 inch from top) and is prewarmed to 37±1°C. Place the water bath within a few feet of the storage location.
B. Thawing
1) Initiate and complete thawing of bone marrow product or bone marrow vials.
2) Locate the desired cryopreserved sample in the storage location and indicate which location samples will be removed from (-86°C or vapor nitrogen). Minimize the number of vials and bags thawed at once to ensure timely and accurate completion of the procedure.
3) Remove samples from inventory.
4) For bags, remove the sample from the cassette and immediately submerge in water bath.
5) For bags: Touch bag gently while submerged. Avoid forcibly cracking ice in bag. Continue just until a few small pieces of ice remain. Remove from the water bath. Record time removed from water bath and immediately take the temperature using an IR thermometer.
6) For vials: Swirl and mix sample while submerged to ensure even thawing. Continue until the last visible ice melts. Remove from the water bath. Never allow samples to sit in bath after thawing.
C. Dilution
1) Spray the sample container with ethanol and place in BSC. All of the remaining steps will be performed in a BSC.
2) For bags: Remove the cap from the bottle or conical tube. Spray and wipe scissors with 70% ethanol. Cut BM bag tubing and drain into bottle or tube. Remove a 1 mL sample using a micropipette and transfer to a 5 mL or 15 mL conical tube.
3) For vials: Transfer entire 1 mL sample volume using a micropipette and transfer to a 5 mL or 15 mL conical tube.
4) Start a timer. Using Rinse Media, add 10% of the 1 mL sample volume (100 pL) each minute for 10 minutes.
5) Add another 1 mL of Rinse Media all at once to the same sample above. The final dilute sample volume is 3 mL (DF=3).
D. Sampling
1) Flow Cytometry Sample
1. In a microcentrifuge tube, add 200 pl, of thawed dilute cells (DF=3) and 400 pl, of Rinse Media. Label with donor ID, sample ID, flow, non-sterile (NS), and DF=9.
2. Count NS sample per TNC Quantitation of Thawed Bone Marrow Using the
Sysmex XP-300 Hematology Analyzer to obtain counts on the Sysmex XP-300.
3. Record Sysmex result of thawing of bone marrow product or thawing of bone marrow vials.
4. Use the remaining NS sample for flow cytometry analysis for Bone Marrow
Staining for CD45, CD34, and CD3 markers using flow cytometry.
2) CFU Sample
1. In 15 mL conical tube, add 100 pL of cells and 9.9 mL of Rinse Media. Label with donor ID, sample ID, CFU, sterile, and DF=300.
2. Remove a 500 pL aliquot and label with donor ID, sample ID, CFU, NS, and
DF-300.
3. Count NS sample. Record count and calculate volumes. 4. Perform the CFU assay per Colony Forming Unit Assay for Cryopreserved Bone Marrow.
Example 4: Characterization of cadaver-bone marrow derived MSCs
[0383] The cadaver bone marrow derived MSCs have been compared in a series of standard, accepted assays to MSCs from a standard, live-donor bone marrow source. Cadaver- derived MSCs are bioequivalent to this reference source of MSCs, and are expected to have the same, remarkably safe safety profile as the numerous other sources of MSCs that have been administered to patients.
[0384] A comparison was performed between deceased donor (DD) and living donor (LD) BM-MSC (Figs. 1-4). Commercially available previously expanded live donor BM-MSC (Ex LD BM-MSC), obtained cryopreserved at passage 2, as well as MSC freshly isolated from live donor, aspirated whole BM (LD BM-MSC) were used. Deceased donor BM-MSC (DD BM- MSC) were isolated from freshly recovered whole bone marrow eluted from fragmented vertebral bodies (VBs). MSC from three donors for each source were expanded to passage 2 and cryopreserved. Upon subsequent thawing, cells were passaged once prior to performing the analyses. MSC from all 3 sources demonstrated essentially identical immunophenotypic cell surface marker profiles, with very low numbers of cells that expressed CD14, CD19, CD34, CD45 and HLA-DR, and, conversely, nearly all cells expressed CD73, CD90 and CD 105 (FIG. 2).
[0385] MSC from each source grew rapidly in culture through 5 passages (the longest period examined) with no differences in population doubling times (PDTs) between passages 4 and 5 (FIG. 3). The CFU-F potential at passage 3 of BM-MSC freshly isolated from living and deceased donors was the same, whereas significantly lower (P<0.05) CFU-F were present in Ex LD BM-MSC (FIG. 4). Finally, trilineage differentiation potentials were compared and it was found that each MSC population formed adipocytes, chondrocytes and osteocytes in vitro at qualitatively the same frequencies (FIG. 5).
Example 5: Illustrative methods for preparing a final MSC Product
[0386] Bone marrow cells, CD34+ isolates, or isolated MSCs (either or both of vB A-MSC and vBM-MSC) are obtained.
Passage 0 (Primary Cell Culture)
[0387] One bag of cryopreserved bone marrow is utilized for primary cell culture. The cryopreserved bag is taken out of inventory and an MSC Batch Record is initiated. A unit is thawed as per the batch record instruction. Approximately 4.5 billion mononuclear cells are plated in six CellBIND® 10-chamber CellSTACKS® and cultured for 10-14 days in MSC Culture Media (Alpha MEM with 10% human platelet lysate (hPL) and 2ng/mL each FGF-2 and EGF). No bovine or porcine components and no antibiotics and antimycotics are used in production. The cells are cultured for about 14 days with fresh media changes every 3 to 4 days. These are termed passage 0 (P0) cells.
Passage 1 (MCB)
[0388] When the P0 cells have reached greater than 75% confluence, cells are detached using TrypLETM Select, and about 410 million cells are immediately replated in MSC Culture Media in 16 CellBind 10-chamber CellSTACKS. These cells are termed Passage 1 (Pl) cells. The Pl cells are incubated for about 4 to about 5 days and once the flasks have reached greater than 75% confluence, cells are detached using TrypLE Select. Pl cells are resuspended in PlasmaLyte-A + 2.5% HSA + 5% DMSO at about 13 million cells/mL and packaged in about 220 x 2 mL cryovials at 2 mL per cryovial using an automatic filler for the CellSeal vials. Cells are then cryopreserved as described elsewhere herein and placed into the vapor phase above LN2 in alarm monitored cryogenic tanks for storage at <-140 °C. This constitutes the MCB.
[0389] A MCB cell population will have the following preferable features described in Table 7 and as shown in the column “Result” actual results have been obtained from MCB cells prepared according to the methods of the present disclosure.
Table 7
Figure imgf000126_0001
Figure imgf000127_0001
Passage 2 to Passage 3 (WCB)
[0390] One Pl vial from the MCB will be thawed and plated in one CellBIND 10-chamber CellSTACK in MSC Culture Media. These cells are now passage 2 (P2) cells. The P2 cells are cultured for about 4 to about 5 days and once the flasks have reached greater than 75% confluence, cells are detached using TrypLE. About 410 million cells are replated in MSC Culture Media in sixteen CellBIND 10-chamber CellSTACKS. These will be passage 3 (P3) cells. The P3 cells are cultured for about 4 to about 5 days and once the flasks reached greater than 75% confluence, cells are detached using TrypLE. P3 cells are resuspended in PlasmaLyte-A + 2.5% HSA + 5% DMSO at 5M cells/mL and packaged in about 14 CryoStore™ bags at about 65 mL per bag. Cells are then cryopreserved as described elsewhere herein. In short, cryopreservation comprises precooling/equilibration at 4°C followed by passive cry opreservation at a rate of -l°C/minute to <-80 °C and then moving into vapor phase above LN2 for storage at <-140 °C. This constitutes the WCB.
[0391] A WCB cell population will have the following preferable features described in Table 8 and as shown in the column “Result” actual results have been obtained from WCB cells prepared according to the methods of the present disclosure.
Table 8
Figure imgf000127_0002
Figure imgf000128_0001
Passage 4 (EOP)
[0392] One bag from the WCB will be thawed and plated in MSC Culture Media. These cells are now termed passage 4 (P4). The P4 cells are cultured for about 4 to about 5 days and once the flasks reached greater than 75% confluence, cells are detached, resuspended in PlasmaLyte-A + 2.5% HSA + 5% DMSO at about 20 million cells/mL and packaged in 5 mL CellSeal closed-system cryovials at 5 mL per cryovial using an automatic filler. Cells are then cryopreserved and placed into the vapor phase above LN2 in alarm monitored cryogenic tanks for storage at <-140 °C.
[0393] An EOP cell population will have the following preferable features described in Table 9 and as shown in the column “Result” actual results have been obtained from EOP cells prepared according to the methods of the present disclosure.
Table 9
Figure imgf000128_0002
Final Harvest
[0394] Dependent on the number of doses needed, one to ten P4 EOP aliquots will be thawed and plated in CellBIND 5-chamber or 10-chamber CellSTACK(s) in MSC Culture Media for cryo-recovery. The P4 cells are briefly cultured for about 20 to about 28 hours to allow metabolic activity to resume post-thaw. After this brief culturing, cells are detached using TrypLE, flasks are rinsed, and the pooled cells are then washed using PBS via centrifugation at 500xg. After centrifugation, the supernatant is aspirated, and cells are resuspended in PBS. An aliquot of this preparation is taken for a viable cell count via trypan blue or an equivalent assay using acridine orange/propidium iodide. This count dictates final concentration requirements for final formulation and packaging.
Process Timing and Intermediate Storage
[0395] The time elapsed through each culture step is variable, with greater than 75% confluence generally achieved in about 4 to about 5 days for passaged cells. Time elapsed from a culture harvest to the start of the cryopreservation is preferably less than or about 6 hours. Upon cryopreservation cells are theoretically stable indefinitely.
Final Formulation
[0396] When the cell count is known from Final Harvest, the cells are then centrifuged, supernatant is aspirated, and the pellet is resuspended in PlasmaLyte-A + 2.5% HSA at about 10 million cells/mL. The cells are then packaged in 5 mL CellSeal closed-system cryovials at 5 mL per cryovial. Two CellSeal vials at 10 million cells/mL equate to one dose for a patient , e.g., about 100 million cells. Cells may be shipped to clinical site at 2-8°C.
[0397] The vials can be kept in the 2-8°C shipper until use. Preferably, the cells will be administered to a subject in need less than 48 hours after receipt; in most cases, cells should not administered more than 72 hours after receipt.
[0398] A product suitable for administration to a subject in need, e.g., having a perianal fistula, will have the following preferable features described in Table 10.
Table 10
Figure imgf000129_0001
[0399] Experiments were performed to determine in process cryopreservation stability in 5% or 10% DMSO as a permeating cryopreservative. The preponderance of literature indicates cells stored below the glass transition temperature of water (-130 °C) are stable indefinitely, with estimates based on biophysical properties ranging from 200-30,000 years (see: Woods, E.J., et al., “Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use”. Cytotherapy 18(6):697-711. (2016)). The major potential source of damage is through thermocycling due to inappropriate storage. In methods of the present disclosure, all storage tanks are alarm monitored 24 hours per day and manually checked weekly to ensure temperature is maintained.
[0400] Preferably, all post-thaw testing will consist of viability (Cellometer, AO/PI); CFU- F (potency); and sterility. Cells should be greater than or equal to 70% viable; greater than or equal to 20,000 colonies per million cells plated; and no growth on sterility testing.
Example 6: Methods for treating a fistula
[0401] The current standard of care for virtually all patients with fistulizing perianal disease includes drainage of abscess with seton placement and maximizing medical therapy. Patients will be enrolled with refractory perianal fistula who have not responded to previous medical/surgical therapies. The next step in the management of these patients is a repeat operation including autologous tissue flap, or need for fecal diversion or proctectomy.
[0402] A clinical phase IB/IIA trial is being performed to assess treating perianal fistulas with MSCs. Patients with a single or multi tract fistula arising from the rectum or anal canal that travels through the internal/extemal sphincter complex to the perianal skin will be treated by direct injection of 1-2 doses of 100 million MSCs. The rationale for this dose comes from several other studies reporting safety and efficacy with 60-200 million cells injected.
[0403] The product administered is derived from a single, deceased healthy donor. There is evidence demonstrating safety in perianal Crohn’s fistula.
Primary objectives
[0404] The primary endpoint of this study is to determine the safety and feasibility of 1-2 treatments of 100 million allogeneic bone marrow derived MSCs for the treatment of perianal fistulizing Crohn’s disease.
Secondary objectives
[0405] The secondary objectives of this study include assessing complete or partial fistula healing induced by the delivery of 1-2 doses of 100 million allogeneic bone marrow derived MSCs on perianal fistulizing Crohn’s disease. Patients will be assessed as having complete healing; partial healing; lack of response; or worsening disease. Complete healing is assessed by radiographic healing (MRI with an absence of a fluid collection >2 cm in 3 of 3 dimensions, lack of edema, lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain) or clinical healing (100% cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening). Partial healing is assessed by radiographic healing (MRI with an absence of a fluid collection >2 cm in 2 of 3 dimensions, lack of edema, lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain) or clinical healing (greater than or equal to 50 % cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening). Lack of Response is assessed by radiographic and clinical healing which does not meet the threshold for Partial Healing. Worsening disease is assessed by radiographic (MRI with a fluid collection >2 cm in 2 of 3 dimensions, edema, inflammation or sign of active inflammatory response. An increased number of tracts may be seen, or increased branching from the primary tract) or clinical (increased drainage per patient report and on clinical exam) signs.
Subjects:
[0406] 20 subjects with medically refractory perianal Crohn’s disease and between 18 and
75 years of age are selected. The patients have a diagnosis of CD for at least 6 months. The patients enrolled in this study have perianal fistulas in the setting of Crohn’s disease and have failed at least one previous medical treatment and have had at least one surgical repair(s). Failure of conventional medical therapy is defined as a lack of response to systemic immune suppression (e.g. azathioprine, methotrexate, 6-mercaptopurine) or biologic (e.g. anti-TNF, anti-integrin, anti-interleukin) therapies to treat fistulizing CD for at least 3 months. Failure of surgical repair includes seton placement, glue or plug insertion, local tissue flaps, and/or ligation of intersphincteric fistula tract repair. The study team believes the risks of this study are manageable and minimal. The patients have no contraintradictions to MR evaluations, are available to comply with the protocol and provide written consent. Concurrent Crohn’ s-related therapies with stable doses corticosteroids, 5- ASA drugs, immunomodulators, anti-TNF therapy, anti-integrin and anti-interleukin therapies are permitted.
Exclusion Criteria
[0407] Exclusion criteria include inability to give informed consent; clinically significant medical conditions within the six months before administration of MSCs: e.g. myocardial infarction, active angina, congestive heart failure or other conditions that would, in the opinion of the investigators, compromise the safety of the patient; having Hepatitis B or C, HIV, abnormal AST or ALT at screening; a history of cancer including melanoma (with the exception of localized skin cancers) in the past five years; investigational drug within one month of treatment; pregnant or breast feeding or trying to become pregnant; presence of a rectovaginal or perineal body fistula; a change in Crohn’s immunosuppressive regimen within the 2 months prior to enrollment; uncontrolled intestinal Crohn’s disease which will require escalation for medical therapy or surgery within 2 months of enrollment; severe anal canal disease that is stenotic and requires dilation; currently taking corticosteroids; or allergy to DMSO.
Study product, dose and route
[0408] The investigational agent is comprised of culture-expanded mesenchymal stromal cells (MSC) isolated from the bone marrow of vertebral bodies of consented and disease- screened deceased organ and tissue donors. The final product is composed of MSC formulated Plasma-Lyte® A, 2.5% Human Serum Albumin (HSA), and 5% DMSO at 20M cells/mL.
[0409] The final product is certified to have an endotoxin level of <2 Endotoxin Units (EU)/mL. The total volume for injection is lOmL, for a maximum endotoxin of 20EU/dose. For the smallest patient contemplated (35Kg) this would result in a total dose of 35Kg 20EU = 1.75 EU/Kg.
[0410] Patients with refractory perianal fistulizing disease will be treated by direct inj ection of 100 million allogeneic bone marrow derived mesenchymal stem cells at baseline and possibly again after 3 months if not completely healed. (15 treatment and 5 controls).
[0411] Upon receipt of cells at the Case Western Reserve University National Center of Regenerative Medicine Cellular Therapy Lab (CTL), the vials may be transferred to cryogenic storage or kept in the dry shipper for up to 24 hours if preparation for administration is to occur within that time frame. The cells must be stored at <-140 °C until they are thawed.
[0412] Alternately, the cryogenic vials were thawed, contacted with a growth medium at a sufficient temperature and sufficient time, and shipped to the lab on wet ice. Here, the methods described in the section entitled “cryorecovery” will be employed.
[0413] MSC must be maintained in vapor phase liquid nitrogen at or colder than -135°C during storage Temperature readings must be monitored daily (excluding weekends and holidays) by either attaching a printout from the freezer’s electronic monitoring system or recording in the “LN2 Freezer Temperature Log”. Either log must be completed to document the method of temperature monitoring.
[0414] For thawing, a vial is immersed directly in a 37°C water bath with continuous, gentle agitation achieved through swirling the vial until the last of the ice just melts. The vial is then removed from the bath and accessed through the needle port and transferred to a 50 cc conical tube. 5 mL of rinse medium (one volume) consisting of Plasmalyte-A + 2.5% HSA is added to the cells at a rate of 0.5 mL per minute, gently agitating the tube by swirling after each addition. Once the last 0.5mL addition has taken place, another 5mL of Plasmalyte-A + 2.5% HSA is added abruptly, resulting in a final volume of 15 mL.
[0415] Next, the cells are washed via centrifugation, the supernatant is aspirated, and the pellet is resuspended in 10 mL of MSC Culture Medium. This 10 mL suspension is then pipetted directly into 615 mL of MSC Culture Medium in a IL bottle final volume of 625mL. [0416] The 625 mL suspension is then inoculated into a 5-chamber CellBIND CellSTACK and placed in a 37°C incubator in 5% CO2. This is termed Recovery Culture. This Recovery Culture can be harvested for final formulation within 22-28 hours.
[0417] Cryo-recovered P4 vBM-MSC cells are produced from human organ and tissue donors and packaged at 5 mL volumes of 10 * 106viable P4 vBM-MSCs/mL in Plasma-Lyte A + 2.5% HSA (Rinse Media) in CellSeal® 5 mL cryovials (Sexton Biotechnologies, Indianapolis, IN). This product and packaging configuration is designed for direct injection with no further manipulation required at the clinical site. After receipt at clinical site, store Cryo-recovered P4 vBM-MSC vials at hypothermic temperatures (2-8°C) in the Cryoport C3 shipper until ready for patient administration.
[0418] For administration of the dose, within 22-28 hours of the start of Recovery Culture, the CellSTACK is removed from the incubator, the media is removed, and the 125mL of TrypLE is added. Viable cells will be counted and resuspended in Plasmalyte + 2.5% HSA to 10M cells per mL concentration, and dispensed into 5 mL plastic syringes. The cells will be transported in a validated container at 20-25°C by trained personnel to the operating room where the Primary Investigator, Amy Lightner or designee will receive the cells. The exam under anesthesia will proceed with removal of setons and debridement and cleansing of the fistula tract. The internal opening of the fistula tract(s) will be ligated closed. At that time, the MSCs will delivered via direct injection through a 22G needle.
[0419] The internal opening (on the anal canal/rectal wall) will be closed with suture ligation (a stitch of the mucosa and submucosa) using absorbable 2-0 PDS suture. A total of 100 million cells in 10 mL of Plasma-Lyte® A supplemented with HSA (2.5%) will be injected via a 22 gauge needle will then be used for cell injection. 1.4 mL will be injected through the 22 gauge needle just deep to the suture ligation of the internal opening, into the submucosal layer of the bowel wall. The external opening of the fistula tract will be identified and 2.5 mL will be injected along the length of the fistula tract (in parallel to the wall of the tract no deeper than 2 mm from the fistula tract wall) in each of the four quadrants for a total of 10 mL injected around, and in parallel to the wall of the tract). If there are multiple tracts, these volumes will be equally divided between the fistula tracts. Patients may possibly be treated again after 3 months if the fistula is not completely healed and they did not have an allergic reaction after the first treatment.
Treatment Regimen
[0420] Patients will have refractory perianal Crohn’s fistulizing disease. The internal openings will be closed with suture ligation (a stitch of the mucosa and submucosa) using absorbable 2-0 PDS suture. A total of 100 million cells in 10 mL of Plasma-Lyte® A supplemented with human serum albumin (2.5%) and dimethyl sulfoxide (2.5%) will be prepared per herein disclosed methods. The solution will be drawn into three separate 5 mL syringes using a 16 gauge needle. A 22 gauge needle will then be used for cell injection. 1.4 mL will be injected through the 22 gauge needle just deep to the suture ligation of the internal opening, into the submucosal layer of the bowel wall. The external opening of the fistula tract will be identified and 2.5 mL will be injected along the length of the fistula tract (in parallel to the wall of the tract no deeper than 2 mm from the fistula tract wall) in each of the four quadrants for a total of 10 mL injected around, and in parallel to the wall of the tract). If there are multiple tracts, these volumes will be equally divided between the fistula tracts. Subjects may possibly be treated again after 3 months if the fistula is not completely healed and they did not have a study drug-related, SAE after the first treatment.
Duration of Administration
[0421] Patients in the treatment group (n=15) will have a direct injection of 100 million allogeneic bone marrow derived mesenchymal stem cells at baseline. If at 3 months post initial injection, complete clinical healing is not achieved, then the patient will receive a repeat injection of 100 million allogeneic bone marrow derived MSCs in the same fashion as the initial treatment. If healing has been achieved as 3 months, the patient will not be retreated. Control patients (n=5) without improvement will cross over at 6 months for receipt of mesenchymal stem cells and be followed for one year after treatment to a total duration of 18 months.
Regimen and follow up visits
[0422] Subjects will be screened at outpatient clinic visits in the colorectal and/or gastroenterology departments and interested qualified subjects will be offered participation in the trial and consented. After consent, patients will undergo general health assessment with laboratory workup including a CBC with differential and C-Reactive protein, AST/ALT, Acute Hepatitis Panel, HIV, and creatinine. All blood products and concomitant medications will be reviewed and recorded. If there are indications from the exam and MRI of active abscess at the time of consent, as part of standard of care for fistulizing perianal Crohn’s disease the patient would undergo an exam under anesthesia (EUA) by a colorectal surgeon for drainage of any abscess and placement of seton. If the patient has a seton already in place and there is no sign of abscess on MRI or exam in the prior 30 days, the patient will be offered the MSC injection procedure (Visit 2).
[0423] If there is evidence of active pelvic abscess from the physical exam or MRI, the patient will be scheduled for an exam under anesthesia (EUA) by a colorectal surgeon for drainage of any abscess and placement of seton (Visit 1.1). Four weeks after placement of seton, the patient will return for Visit 1.2 where a perianal exam to assess for active abscess will be performed. If no active abscess is found, the patient will be scheduled for the MSC injection procedure (Visit 2). If there is still active abscess found, the patient will screen fail from the trial.
[0424] Randomization to treatment with MSC versus control with normal saline will be assigned by an electronic data capture system. Fifteen patients with no active abscess and/or who have had successful alleviation of abscess and epithelialization of the fistula tract after seton placement will undergo EUA for placement of 100 million bone marrow derived allogeneic MSCs. The dominant tract is the tract with the largest diameter opening which is assessed at the time of exam under anesthesia with a fistula probe, or if the diameter is equivalent, the longest fistula tract length. The internal opening (on the anal canal/rectal side) will be closed with suture ligation (a stitch of the mucosa and submucosa) using absorbable 2- 0 PDS suture. A total of 100 million cells in 10 mL of Plasma-Lyte® A supplemented with human serum albumin (2.5%) and dimethyl sulfoxide (2.5%) will be prepared per MSC Instructions for Use (IFU). The solution will be drawn into three separate 5 mL syringes using a 14-gauge needle. A 20-gauge needle will then be used for cell injection. 1.4 mL will be injected through the 20-gauge needle just deep to the suture ligation of the internal opening, into the submucosal layer of the bowel wall. The external opening of the fistula tract will be identified and 2.5 mL will be injected along the length of the fistula tract (in parallel to the wall of the tract no deeper than 2 mm from the fistula tract wall) in each of the four quadrants for a total of 10 mL injected around, and in parallel to the wall of the tract). If there are multiple tracts, these volumes will be equally divided between the fistula tracts. If the patient is randomized into the control arm, the patients will still have a suture ligation of the internal opening and injection of normal saline rather than the MSC product.
[0425] If at 3 months post injection of 100 million allogeneic bone marrow derived MSCs, there is not complete clinical healing (100% cessation of drainage on both clinical exam with deep palpation and per patient report, clinical exam, MRI and epithelization of the external fistula opening), and the subject did not have an study-drug related SAE after the first treatment, a repeat injection of 100 million allogeneic bone marrow derived MSCs will be administered. If patients originally received placebo, they will be given the dose of 100 million bone marrow derived MSCs at the six month visit and be followed for one year in the same schema as the treatment group, for a total of 18 months.
[0426] Follow up visits may include collecting medical surgery history; a general and perianal exam with vital signs, including assessing clinical healing by physical exam of the perianal area and compression of the external opening of the fistula tract to assess for drainage; PDAI score; Wexner Incontinence Score; EQ-5D; IBD-PRTI; IBDQ, SF-36; laboratory studies including CBC with differential and CRP; and concomitant medications; and recording any adverse events.
[0427] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed is:
1. A method for treating a perianal fistula in a subject, the method comprising injecting into the perianal fistula a first dose of a composition comprising more than about 10 million human mesenchymal stem cells (MSCs) obtained from a non-living source.
2. The method of claim 1, wherein the subject is afflicted with an autoimmune disease.
3. The method of claim 2, wherein the autoimmune disease is associated with Crohn’s Disease.
4. The method of any one of claims 1 to 3, further comprising injecting into the fistula an at least second dose of a composition comprising more than about 10 million MSCs.
5. The method of any one of claims 1 to 4, wherein the first dose and/or the at least second dose independently comprise more than about 50 million MSCs.
6. The method of any one of claims 1 to 5, wherein the first dose and/or the at least second dose independently comprise more than about 100 million MSCs.
7. The method of any one of claims 1 to 6, wherein the at least second dose is administered about one month, two months, three months, four months, five months, or six months after the first dose.
8. The method of any one of claims 1 to 7, wherein the subject has an at least second perianal fistula.
9. The method of claim 8, wherein the at least second perianal fistula is injected with a first dose of a composition comprising more than about 10 million MSCs.
10. The method of claim 8, wherein the at least second perianal fistula is injected with an at least second dose of a composition comprising more than about 10 million MSCs.
11. The method of claim 8 or claim 9, wherein the at least second fistula is injected with a first dose of a composition comprising more than about 50 million MSCs and/or an at least second dose of a composition comprising more than about 50 million MSCs.
12. The method of any one of claims 1 to 11, wherein the MSCs are allogenic to the subject.
13. The method of any one of claims 1 to 12, wherein the MSCs are HLA matched to the subject. e method of any one of claims 1 to 12, wherein the MSCs are HLA mis-matched to the subject. e method of claim 14, wherein the MSCs and subject have fewer than six HLA matches out of eight HLA markers, fewer than four HLA matches out of six HLA markers, or fewer than five HLA matches out of ten HLA markers. e method of any one of claims 1 to 15, wherein the MSCs are obtained from one or more vertebral bodies. he method of any one of claims 1 to 16, wherein the MSCs are obtained from a single donor. e method of any one of claims 1 to 17, wherein the composition comprises from about
0.5 % CD45+ cells to about 10 % CD45+ cells. he method of claim 18, wherein the composition comprises less than about 5% CD45+ cells. e method of any one of claims 1 to 19, wherein the composition comprises from about
70 % CD 105+ cells to about 100 % CD 105+ cells. e method of claim 20, wherein the composition comprises at least 90% CD105+ cells. e method of any one of claims 1 to 21, wherein the composition comprises from about
70 % CD 166+ cells to about 100 % CD 166+ cells. e method of claim 22, wherein the composition comprises at least about 90% CD 166+ cells. he method of any one of claims 1 to 23, wherein the MSCs are vertebral body bone marrow MSCs (vBM-MSCs). e method of claim 24, wherein the MSCs are also vertebral body bone adherent MSCs
(vBA-MSCs). e method of claim 25, wherein the vBA-MSCs are separated from a vertebral body or a fragment thereof by incubation of the vertebral body or the fragment thereof in a collagenase solution. he method of any one of claims 1 to 23, wherein the MSCs are vertebral body bone adherent MSCs (vBA-MSCs). e method of any one of claims 1 to 27, wherein the MSCs are both vBA-MSCs and vBM-
MSCs. e method of any one of claims 1 to 28, wherein the MSCs have been cultured for at least one passage, at least two passages, at least three passages, at least four passages, or at least five passages. he method of any one of claims 1 to 29, wherein the MSCs have been cryopreserved before injecting into the perianal fistula. e method of claim 30, wherein the MSCs have been warmed to a temperature of about 0
°C to about 4 °C at least 12 hours before injecting into the perianal fistula. e method of claim 31, wherein the MSCs have been warmed to a temperature of about 0
°C to about 4 °C at least 24 hours before injecting into the perianal fistula. e method of any one of claims 30 to 32, wherein, after being cryopreserved, the MSCs are contacted with a growth medium at a suitable temperature and suitable time sufficient to provide an at least 25% to at least 250% increase in cell number. e method of claim 32, wherein the MSCs have been warmed to a temperature of about 0
°C to about 4 °C at least two days, three days, four days, five days, six days, or a week before injecting into the perianal fistula. he method of any one of claims 1 to 34, wherein the MSCs are warmed to about room temperature before injecting into the perianal fistula. he method of any one of claims 1 to 34, wherein the MSCs are warmed to about body temperature before injecting into the perianal fistula. e method of any one of claims 1 to 36, wherein the composition comprising the MSCs includes two or more of Plasma-Lyte® A, Human Serum Albumin (HSA), and DMSO. e method of claim 37, wherein the composition comprising the MSCs includes Plasma-
Lyte® A, HSA, and DMSO. e method of any one of claims 1 to 36, wherein the composition comprising the MSCs includes at least about 2 million cells per ml. he method claim 39, wherein the composition comprising the MSCs includes at least about 3 million cells per ml, 4 million cells per ml, 5 million cells per ml, 6 million cells per ml, 7 million cells per ml, 8 million cells per ml, 9 million cells per ml, 10 million cells per ml, 15 million cells per ml, 20 million cells per ml, 30 million cells per ml, 40 million cells per ml, or 50 million cells per ml. e method of any one of claims 1 to 40, wherein the composition comprising the MSCs includes at least about 2 million MSCs per ml. he method claim 41, wherein the composition comprising the MSCs includes at least about 3 million MSCs per ml, 4 million MSCs per ml, 5 million MSCs per ml, 6 million MSCs per ml, 7 million MSCs per ml, 8 million MSCs per ml, 9 million MSCs per ml, 10 million MSCs per ml, 15 million MSCs per ml, 20 million MSCs per ml, 30 million MSCs per ml, 40 million MSCs per ml, or 50 million MSCs per ml. e method of any one of claims 1 to 42, wherein the composition has an endotoxin level of less than about 1, 2, 3, 4, or 5 Endotoxin Units (EU)/ml. e method of claim 43, wherein the composition has an endotoxin level of less than about
2 EU/ml. e method of any one of claims 1 to 44, wherein the injecting is via a 22G needle. e method of any one of claims 1 to 45, wherein each dose comprises at least about 1 ml of the composition. e method of claim 46, wherein each dose comprises at least about 2 ml, 3 ml, 4 ml, 5 ml,
6 ml, 7 ml, 8 ml, 9 ml, or 10 ml of the composition. e method of any one of claims 1 to 47, wherein the injecting is into the submucosal layer of the bowel wall. e method of any one of claims 1 to 48, wherein the injecting is along the length of the fistula tract and/or in parallel to the wall of the tract and/or no deeper than 2 mm from the fistula tract wall. he method of any one of claims 1 to 49, wherein the subject has received a previous medical and/or surgical therapy for treating perianal fistula. e method of claim 50, wherein the subject has not responded fully to the previous medical and/or surgical therapy for treating perianal fistula. e method of claim 50 or claim 51, wherein previous medical therapy comprises one or more of systemic immune suppression (e.g. azathioprine, methotrexate, 6- mercaptopurine) or biologic (e.g. anti-TNF, anti-integrin, anti-interleukin) therapies. e method of any one of claims 50 to 52, wherein the previous surgical therapy comprises a surgical repair including seton placement, glue or plug insertion, local tissue flaps, and/or ligation of intersphincteric fistula tract repair. he method of any one of claims 1 to 52, wherein the non-living source from which the
MSCs were ultimately obtained from a human cadaver. he method of any one of claims 1 to 52, wherein the subject experiences a complete healing as assessed by radiographic healing (MRI with an absence of a fluid collection >2 cm in 3 of 3 dimensions, lack of edema, a lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain) and/or clinical healing (100% cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening). e method of any one of claims 1 to 52, wherein the subject experiences a partial healing as assessed by radiographic healing (MRI with an absence of a fluid collection >2 cm in 2 of 3 dimensions, lack of edema, lack of inflammation, or lack of sign of active inflammatory response, although a remnant scar of a fistula tract may remain) and/or clinical healing (Greater than or equal to 50 % cessation of drainage on both clinical exam with deep palpation and per patient report and epithelization of the external fistula opening). composition for use in the method of any one of claims 1 to 56. composition, comprising about at least 10 million human mesenchymal stem cells
(MSCs) obtained from a non-living source, wherein said composition is capable of treating a perianal fistula in a subject. he composition of claim 58, wherein the composition comprises less than 5% CD45+ cells. e composition of claim 58 or claim59, wherein the composition comprises at least 90%
CD 105+ cells. e composition of any one of claims 58 to 60, wherein the composition comprises at least
90% CD 166+ cells. he composition of any one of claims 58 to 61, wherein the human MSCs comprise vertebral body bone marrow MSCs (vBM-MSCs), adherent vertebral body MSCs (vBA- MSCs), or both. e composition of any one of claims 58 to 62, wherein the MSCs have been cultured for at least one passage, at least two passages, at least three passages, at least four passages, or at least five passages. e composition of any one of claims 58 to 63, wherein the MSCs are obtained from one or more vertebral bodies. he composition of any one of claims 58 to 64, wherein the MSCs are obtained from a single donor. e composition of any one of claims 58 to 65, wherein the MSCs have been cryopreserved.he composition of claim 66, wherein the MSCs have been warmed to a temperature of about 0 °C to about 4 °C at least 12 hour, 24 hours, two days, three days, four days, five days, six days, or a week before use in treating a perianal fistula in a subject. he composition of claim 66 or claim 67, wherein the MSCs are warmed to about room temperature before use in treating a perianal fistula in a subject. he composition of any one of claims 66 to 67, wherein the MSCs are warmed to about body temperature before use in treating a perianal fistula in a subject. e composition of any one of claims 58 to 69, wherein the composition comprises at least about 2 million cells per ml, 3 million cells per ml, 4 million cells per ml, 5 million cells per ml, 6 million cells per ml, 7 million cells per ml, 8 million cells per ml, 9 million cells per ml, 10 million cells per ml, 15 million cells per ml, 20 million cells per ml, 30 million cells per ml, 40 million cells per ml, or 50 million cells per ml. e composition of claim 70, wherein the at least about 2 million cells, 3 million cells, 4 million cells, 5 million cells, 6 million cells, 7 million cells, 8 million cells, 9 million cells, 10 million cells, 15 million cells, 20 million cells, 30 million cells, 40 million cells, or 50 million cells are MSCs. e composition of any one of claims 58 to 71, wherein the MSCs were ultimately obtained from a human cadaver.
- 141 -
PCT/US2021/064970 2020-12-23 2021-12-22 Methods and compositions for the treatment of perianal fistula in crohn's disease WO2022140613A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063130273P 2020-12-23 2020-12-23
US63/130,273 2020-12-23

Publications (1)

Publication Number Publication Date
WO2022140613A1 true WO2022140613A1 (en) 2022-06-30

Family

ID=82158393

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/064970 WO2022140613A1 (en) 2020-12-23 2021-12-22 Methods and compositions for the treatment of perianal fistula in crohn's disease

Country Status (1)

Country Link
WO (1) WO2022140613A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020214400A1 (en) * 2019-04-15 2020-10-22 Ossium Health, Inc. System and method for extraction and cryopreservation of bone marrow

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020214400A1 (en) * 2019-04-15 2020-10-22 Ossium Health, Inc. System and method for extraction and cryopreservation of bone marrow

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
AHRENS NORBERT; TORMIN ARIANE; PAULUS MARGIT; ROOSTERMAN DANIELA; SALAMA ABDULGABAR; KRENN VEIT; NEUMANN ULF; SCHEDING STEFAN: "Mesenchymal stem cell content of human vertebral bone marrow.", TRANSPLANTATION, WILLIAMS AND WILKINS, GB, vol. 78, no. 6, 27 September 2004 (2004-09-27), GB , pages 925 - 929, XP009137115, ISSN: 0041-1337, DOI: 10.1097/01.TP.0000133305.81823.2A *
ALBERT D DONNENBERG, VIJAY S GORANTLA, STEFAN SCHNEEBERGER, LINDA R MOORE, GERALD BRANDACHER, HEATHER M STANCZAK, EILEEN K KOCH, W: "Clinical implementation of a procedure to prepare bone marrow cells from cadaveric vertebral bodies", REGENERATIVE MEDICINE, FUTURE MEDICINE LTD., GB, vol. 6, no. 6, 1 November 2011 (2011-11-01), GB , pages 701 - 706, XP055749876, ISSN: 1746-0751, DOI: 10.2217/rme.11.89 *
ILSE MOLENDIJK, BERT A. BONSING, HELENE ROELOFS, KOEN C.M.J. PEETERS, MARTIN N.J.M. WASSER, GERARD DIJKSTRA, C. JANNEKE VAN DER WO: "Allogeneic Bone Marrow–Derived Mesenchymal Stromal Cells Promote Healing of Refractory Perianal Fistulas in Patients With Crohn’s Disease", GASTROENTEROLOGY, ELSEVIER INC., US, vol. 149, no. 4, 1 October 2015 (2015-10-01), US , pages 918 - 927.e6, XP055373007, ISSN: 0016-5085, DOI: 10.1053/j.gastro.2015.06.014 *
JOHNSTONE BRIAN H., MILLER HANNAH M., BECK MADELYN R., GU DONGSHENG, THIRUMALA SREEDHAR, LAFONTAINE MICHAEL, BRANDACHER GERALD, WO: "Identification and characterization of a large source of primary mesenchymal stem cells tightly adhered to bone surfaces of human vertebral body marrow cavities", CYTOTHERAPY, ISIS MEDICAL MEDIA, OXFORD,, GB, vol. 22, no. 11, 1 May 2020 (2020-05-01), GB , pages 617 - 628, XP055932776, ISSN: 1465-3249, DOI: 10.1016/j.jcyt.2020.07.003 *
MORAVCIKOVA ERIKA, MEYER E. MICHAEL, CORSELLI MIRKO, DONNENBERG VERA S., DONNENBERG ALBERT D.: "Proteomic Profiling of Native Unpassaged and Culture‐Expanded Mesenchymal Stromal Cells (MSC)", CYTOMETRY A, WILEY-LISS, vol. 93, no. 9, 1 September 2018 (2018-09-01), pages 894 - 904, XP055953940, ISSN: 1552-4922, DOI: 10.1002/cyto.a.23574 *
RACHELE CICCOCIOPPO, MARIA ESTER BERNARDO, ADELE SGARELLA, RITA MACCARIO, MARIA ANTONIETTA AVANZINI, CRISTINA UBEZIO, ANTONELLA MI: "Autologous bone marrow-derived mesenchymal stromal cells in the treatment of fistulising Crohn's disease", GUT, vol. 60, no. 6, 1 June 2011 (2011-06-01), UK , pages 788 - 798, XP009538539, ISSN: 0017-5749, DOI: 10.1136/gut.2010.214841 *
RAO, PRAKASH N., DEO, DAYANAND D., MARCHIONI, MISTY A., TAGHIZADEH, ROUZBEH R., CETRULO, KYLE, SAWCZAK, SHARYN, MYRICK, JACOB: "Structural and Functional Characterization of Deceased Donor Stem Cells: A Viable Alternative to Living Donor Stem Cells", 2019, STEM CELLS INTERNATIONAL, DOI: 10.1155/2019/5841587 *

Similar Documents

Publication Publication Date Title
US8481253B2 (en) Cryopreservation of adipose tissue for the isolation of mesenchymal stem cells
US11744243B2 (en) Systems and methods for extraction and cryopreservation of bone marrow
KR100779812B1 (en) Preservation of non embryonic cells from non hematopoietic tissues
US11697799B2 (en) System and method for extraction and cryopreservation of bone marrow
JP2023036882A (en) Method of isolating mesenchymal stem cells from umbilical cord amniotic membrane using cell culture medium
JP7401865B2 (en) Methods for obtaining enriched populations of functional mesenchymal stem cells, cells obtained thereby, and compositions comprising the cells
CA2719806A1 (en) Method, system, and apparatus for hypothermic collection, storage, transport and banking of birth tissue
JP2010530214A (en) Acquisition, isolation and cryopreservation of endometrial / menstrual cells
US20220186187A1 (en) Compositions and methods for extraction of mesenchymal stem cells
TW201512395A (en) Method for rapid mincing of adipose tissues to isolate alive cells in vitro
WO2022140613A1 (en) Methods and compositions for the treatment of perianal fistula in crohn&#39;s disease
WO2022226356A1 (en) Methods and compositions for the treatment of ileal anal anastomosis and ileal pouch fistulas in crohn&#39;s disease
CN116583594A (en) System and method for extraction and cryopreservation of bone marrow
WO2022221672A1 (en) Interferon gamma-primed mesenchymal stromal cells as prophylaxis for graft versus host disease
US20210317415A1 (en) Perfusion system for harvesting bone marrow
WO2023201371A1 (en) Synchronized mesenchymal stromal cells
WO2011109717A1 (en) Methods and processes for collecting, preserving, and culturing adult stem cells obtained from lochia and related compositions of matter

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21912190

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21912190

Country of ref document: EP

Kind code of ref document: A1