WO2018132594A1 - Methods of enhancing fibroblast therapeutic activity - Google Patents
Methods of enhancing fibroblast therapeutic activity Download PDFInfo
- Publication number
- WO2018132594A1 WO2018132594A1 PCT/US2018/013357 US2018013357W WO2018132594A1 WO 2018132594 A1 WO2018132594 A1 WO 2018132594A1 US 2018013357 W US2018013357 W US 2018013357W WO 2018132594 A1 WO2018132594 A1 WO 2018132594A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- fibroblasts
- cell
- disc
- regeneration
- Prior art date
Links
Classifications
-
- 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
- C12N5/0656—Adult fibroblasts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- 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
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- 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
- A61K35/33—Fibroblasts
-
- 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
- C12N2501/113—Acidic fibroblast growth factor (aFGF, FGF-1)
-
- 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
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
- C12N2502/115—Platelets, megakaryocytes
-
- 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
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
Definitions
- Embodiments of the disclosure concern at least the fields of cell biology, molecular biology, biochemistry, and medicine.
- Musculoskeletal disorders of the spine are an extremely common occurrence associated with debilitating back pain, leading to enormous psychosocial and economic ramifications.
- Lower-back pain is the leading source of disability in people under 45 years of age, and it results in significant economic losses [1].
- 80% of people in the United States will experience back pain at some point in their lifetime [2], and it is the second most common reason for symptomatic physician visits [3].
- causes of back pain range from injury -induced, which presents as a minor problem, accelerating to a chronic disorder, as well as degenerative spine diseases that lead to degenerative spondylolisthesis and spinal stenosis.
- the vast majority of chronic back pain is associated with degeneration of the intervertebral disc, which can manifest in many different clinical conditions including spinal stenosis and instability, radiculopathy, myelopathy, and disc herniation.
- disc degeneration occurs as a natural process, in many individuals asymptomatically.
- the origin of pain has therefore been termed "discogenic” not necessarily because of the disc degenerative process, but in part due to the granulation tissue that invades the disc space and causes inflammation and nociception [4].
- disc degeneration begins as early as the first decade of life when various biochemical changes become apparent in the endplate and the nucleus pulposus [11]. Subsequently, in the second decade notochordal cells begin undergoing replacement by chondrocytes. In the third decade loss of the fine fibrous connective tissue network and replacement by hyalinized collagen fibers occurs, with concurrent initiation of fissures in the annulus fibrosis. Beginning in the fourth decade and almost always present in the fifth is replacement of the nucleus pulposus jelly-like substance with a fibrous collagen type II structure that resembles the annulus fibrosis.
- nucleus pulposus is completely devoid of the water carrying proteoglycan mass and instead is empty or filled with amorphous material.
- cartilage endplate which contains blood vessels for the annular fibrosis and nucleus pulposus becomes replaced with fibrocartilage and blood flow progressively diminishes. Since the only 2 routes for the exchange of solutes with the blood vessels outside the disk are via the periphery of the annulus, and through the end-plates, the natural degeneration of the endplate blood supply causes a decrease ability of nucleus pulposus cells to function/survive, thus leading to decreased proteoglycan synthesis and disc degeneration [12].
- proteoglycans in the nucleus pulpous occurs naturally by the cellular component of the nucleus pulposus.
- Specific growth factors such as TGF-b and EGF are involved in the stimulation of proteoglycan synthesis.
- TGF-b and EGF are involved in the stimulation of proteoglycan synthesis.
- Another reason for inhibition of proteoglycan synthesis is lower pH caused by ischemia of the lumbar area [14].
- matrix metalloproteases are involved in cleaving proteoglycans, and that upregulation of matrix metalloprotease activity is associated with disc degeneration [15].
- Activation of matrix metalloproteases is known to be induced by inflammatory cytokines such as T F and IL-1 [16]. Additionally, animal studies have demonstrated that hyperphysiological loading of the disc segment induces upregulation of matrix metalloproteases [17]. [0008] Therefore it appears that lower back pain, at least in a large proportion of patients, is caused by an inflammatory event that occurs in conjunction with lumbar disc degeneration. The link between inflammation and pain is established in studies showing that radiologically degenerated discs are not associated with main in a large number of subjects. In contrast, the presence of localized inflammation associated with disc degeneration, as exemplified by the presence of granulation tissue which is believed to be causative of nociception and the symptoms of chronic, intractable, lower back pain [10, 18].
- matrix loss is a balance between matrix synthesis and degradation, it is possible to increase disc matrix by increasing synthesis or by decreasing degradation.
- One approach is to prevent matrix loss by inhibiting the degradative enzymes.
- MMPs matrix metalloproteinases
- TIMPs matrix metalloproteinases
- Chondrogenic morphogens are cytokines that not only possess mitogenic capability but are characterized by their ability to increase the chondrocyte-specific phenotype of the target cell. Most of the research in chondrogenic morphogens has been with transforming growth factor-b (TGF-b), bone morphogenetic proteins (BMPs) or growth and differentiation factors (GDFs). Chondrogenic morphogens are particularly attractive because they may reverse the fibrotic phenotype of disc cells to the more chondrocyte phenotype of disc nucleus cells in younger and more "normal" discs. By definition, these molecules are secreted molecules and hence can potentially act in autocrine, paracrine and endocrine fashion.
- TGF-b transforming growth factor-b
- BMPs bone morphogenetic proteins
- GDFs growth and differentiation factors
- TGF-b 1 is one of the first disc morphogenic molecules to be studied. Thompson et al. reported that TGF-b 1 was a mitogen but also showed that it was a highly anabolic molecule leading to significantly increased proteoglycan synthesis per cell. Gene transfer of TGF-b using an adenoviral/CMV vector was capable of reversing radiological signs of disc degeneration in a rabbit model [28].
- BMP -2 is another prototypic chondrogenic morphogen [29].
- Yoon et al. reported that recombinant human BMP -2 increased production of rat disc cell proteoglycan and significantly increased the chondrocyte phenotype of the disc cells as shown by increased aggrecan and Type II collagen gene expression, whereas there was no change in Type I collagen gene expression [30].
- Kim et al. reported that BMP-2 can partially reverse the inhibitory effect of nicotine on the synthesis of disc cell proteoglycan [31].
- BMP-7 also known as OP-1 (osteogenic protein-1), is another disc cell morphogen that has demonstrated potent in vitro activity in terms of enhancing matrix formation in disc cells [32-34].
- GDF-5 is also known as CDMP-1 is also considered for regeneration of disc cells, although only in vitro experimentation has occurred [35].
- the present disclosure concerns methods and compositions that satisfy a long-felt need in the art for degenerative disc repair.
- the present disclosure is directed to methods and compositions related to certain cells useful for therapy in an individual, such as therapy in one or more discs of a mammal.
- the cells are fibroblasts that in particular cases have been modified upon exposure to one or more biologically active substance and/or one or more conditions.
- the exposure improves one or more therapeutic activities compared to fibroblasts that lack the exposure.
- any particular therapeutic activity of the fibroblasts may be enhanced upon one or more exposures to one or more biologically active substance and/or one or more conditions, in some cases the activity is anti-inflammatory, angiogenic, regenerative and/or disc-regenerating properties, as examples.
- the fibroblasts are directly or indirectly the cause of amelioration of at least one symptom of a medical condition related to one or more discs of an individual.
- methods of the disclosure directly or indirectly result in an increase disc matrix, including by increasing synthesis in the disc, by decreasing degradation, and/or by preventing matrix loss by inhibiting degradative enzymes.
- methods for augmenting efficacy of fibroblasts for regeneration of cells and/or tissues, comprising the steps of (optionally) obtaining fibroblast cells; contacting fibroblasts with one or more biologically active substances; and/or culturing the fibroblasts under conditions to enhance efficacy of the fibroblasts for regeneration of the cells and/or tissues.
- the one or more biologically active substances comprise one or more cytokines, such as growth factors (for example, FGF-alpha, FGF-beta, and/or a member of the TGF-beta family).
- the one or more biologically active substances comprise platelet rich plasma.
- regeneration of cells and/or tissue by the fibroblasts comprises immune modulation, angiogenesis, regeneration of spinal discs, a combination thereof, and so forth.
- the fibroblast cells being utilized may be selected from the group consisting of (a) fibroblasts obtained by biopsy, cultured and proliferated; (b) subsets thereof having greater ability to differentiate; and (c) a combination thereof.
- the fibroblasts express stage specific embryonic antigen 3 (SSEA3).
- the fibroblasts are comprised in a pharmaceutically acceptable carrier selected from the group consisting of sterile solutions, hydrogels, implantable cell matrices, devices and a combination thereof.
- Embodiments of the disclosure include methods for increasing PD-1L expression in fibroblasts, suppressing T-cell activation by fibroblasts, and/or suppressing T-cell production of one or more factors, such as interferon gamma, by fibroblasts.
- FIG. 1 shows the effects of increasing amounts of platelet rich plasma on PD-1L expression in fibroblasts
- FIG. 2 shows the effects of increasing amounts of platelet rich plasma on suppression of T cell activation by fibroblasts
- FIG. 3 demonstrates the effects of increasing amounts of platelet rich plasma on suppression of T cell production of interferon gamma by fibroblasts.
- Biocompatible polymers used in the present disclosure are selected from the group consisting of carbomers (acrylic acid polymers crosslinked with a polyalkenyl polyether), polyalkylene glycols (for example, polyethylene glycols and polypropylene glycols), poloxamers (polyoxyethylene-polyoxypropylene block copolymers), polyesters, polyethers, polyanhydrides, polyacrylates, polyvinyl acetates, polyvinyl pyrrolidones, and polysaccharides such as, for example, hyaluronic acid, derivatives of hyaluronic acid, in particular crosslinked hyaluronic acid and esters of hyaluronic acid (for example, benzyl ester of hyaluronic acid),
- carbomers acrylic acid polymers crosslinked with a polyalkenyl polyether
- polyalkylene glycols for example, polyethylene glycols and polypropylene glycols
- poloxamers polyoxyethylene-pol
- hydroxyalkylcelluloses for example, hydroxymethylcellulose and hydroxyethylcellulose
- carboxyalkylcelluloses for example, carboxymethylcellulose
- exemplary growth factors include, but are not limited to, platelet-derived growth factor (PDGF), platelet-derived angiogenesis factor (PDAF), vascular endothelial growth factor (VEGF), platelet-derived epidermal growth factor (PDEGF), platelet factor 4 (PF-4), transforming growth factor beta.
- PDGF platelet-derived growth factor
- PDAF platelet-derived angiogenesis factor
- VEGF vascular endothelial growth factor
- PEGF platelet-derived epidermal growth factor
- PF-4 platelet factor 4
- TGF-B acidic fibroblast growth factor (FGF -A), basic fibroblast growth factor (FGF-B), transforming growth factor A (TGF-A), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), B thromboglobulin-related proteins (BTG), thrombospondin (TSP), fibronectin, von Wallinbrand's factor (vWF), fibropeptide A, fibrinogen, albumin, plasminogen activator inhibitor 1 (PAI-1), osteonectin, regulated upon activation normal T cell expressed and presumably secreted (RANTES), gro-A, vitronectin, fibrin D-dimer, factor V, antithrombin III, immunoglobulin-G (IgG),
- immunoglobulin-M immunoglobulin-A (IgA), a2-macroglobulin, angiogenin, Fg-D, elastase, keratinocyte growth factor (KGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), tumor necrosis factor (TNF), fibroblast growth factor (FGF) and interleukin-1 (IL- 1), Keratinocyte Growth Factor-2 (KGF-2), and combinations thereof.
- KGF keratinocyte growth factor
- EGF epidermal growth factor
- FGF fibroblast growth factor
- TNF tumor necrosis factor
- FGF fibroblast growth factor
- IL-1 interleukin-1
- KGF-2 Keratinocyte Growth Factor-2
- regeneration refers to the growth of new cells and/or tissue in an area, such as a damaged area, including a spinal disc.
- terapéuticaally effective amount refers to that amount which, when administered to an individual for treating a disease, is sufficient to effect such treatment for the disease, including to ameliorate at least one symptom of the disease.
- the disclosure concerns means of augmenting therapeutic activity of fibroblasts, such as fibroblasts that are used at least for anti-inflammatory, angiogenic, regenerative and/or disc-regenerating properties at one or more sites in vivo.
- fibroblasts are cultured with cytokines, growth factors, peptides, or combinations thereof prior to administration to an individual, such as a mammal, including humans, horses, dogs, cats, and so forth.
- the disclosure encompasses augmentation of regenerative activities for fibroblasts to be used as therapeutic agents, for example through culture (before and/or during administration to an individual) with one or more agents, such as platelet rich plasma (PRP).
- PRP platelet rich plasma
- the disclosure provides methods for enhancing one or more fibroblast activities for therapeutic activity by co-administering one or more agents and/or PRP, for example together with the fibroblasts.
- the enhanced fibroblasts are delivered to an individual for the purpose of treating a disc medical condition in the individual.
- an individual is determined to be in need of the enhanced fibroblasts, such as because of a damaged disc or risk thereof.
- An individual at risk is one that is over the age of about 40, 45, 50, 55, 60, 65, 70, 75, 80, and so forth; an individual that is or was an athlete; an individual with a vocation that requires physical activity; an individual with a spinal injury; or a combination thereof).
- the fibroblasts are exposed to platelet-rich plasma and such exposure directly or indirectly results in enhanced fibroblasts.
- Numerous growth factors, cytokines and peptides are released from activated platelets, and one approach to therapeutically leverage this is to utilize an autologous platelet concentrate suspended in plasma, also known as platelet-rich plasma (PRP).
- PRP platelet-rich plasma
- Several means of preparing PRP are known in the art, some of which are described in the following and incorporated by reference herein [36, 37]. Examples of devices used for generation of PRP include SmartPReP, 3iPCCS, Sequestra, Secquire, CATS, Interpore Cross, Biomet GPS, and Harvest's BMAC [38], for example. Other means of generating PRP are described in U.S.
- fibroblasts are delivered systemically or locally to an individual in need thereof, including an individual in need of treatment, including by using a carrier (for example, hydrogel) comprising platelet rich plasma (PRP) and/or hyaluronic acid (HA); in particular cases PRP and/or HA are blended with batroxobin (BTX) as gelling agent.
- a carrier for example, hydrogel
- PRP platelet rich plasma
- HA hyaluronic acid
- BTX batroxobin
- the fibroblasts may be encapsulated in a hydrogel, such as PRP/HA/BTX hydrogel, and cultured, for example in both growing medium and/or medium with or without TGF- ⁇ (for example) for a certain duration of time, such as from one minute (min) up to 21 days.
- TGF- ⁇ for example
- a range of culture duration for any cells may be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 (or more minutes or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or more hours) to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.
- the range of time may be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more minutes to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
- the hydrogel jellifies at a certain temperature in a certain period of time.
- the hydrogel may jellify in 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes or more at 18, 19, 20, 21, 22, 23, 24, 25, or higher °C or in 1, 2, 3, 4, 5, or more minutes at 35, 36, 37, 38, 39, or 40 or more °C in a manner such that the fibroblasts maintain high cell viability and proliferation.
- the disclosure encompasses the use of fibroblasts for local delivery (such as by intra-disc injections) in individuals with degenerative disc disease.
- the fibroblasts are cultured in suitable conditions to enhance GAG production, which in at least some cases is achieved by culture with one or more cytokines, such as TGF- beta.
- cytokines such as TGF- beta.
- the disclosure encompasses the use of activation of fibroblasts prior to therapeutic use, including administration of one or more biologically active substances that act as "regenerative adjuvants" for the fibroblasts.
- the cells in the formulation may display typical fibroblast morphologies when growing in cultured monolayers. Specifically, cells may display an elongated, fusiform or spindle appearance with slender extensions, or cells may appear as larger, flattened stellate cells that may have cytoplasmic leading edges. A mixture of these
- the cells may express one or more proteins characteristic of normal fibroblasts including the fibroblast-specific marker, CD90 (Thy-1), a 35 kDa cell-surface glycoprotein, and the extracellular matrix protein, collagen, as examples.
- the fibroblast dosage formulation in specific embodiments may be an autologous, allogeneic, or xenogeneic cell therapy product comprising a suspension of fibroblasts, including grown from skin using standard tissue culture procedures as examples.
- fibroblasts of any kind are utilized in methods for regeneration, and in preparation for (or as part of) these methods, the fibroblasts may be harvested, cultured, and expanded using certain techniques.
- the fibroblasts utilized in the disclosure are generated, in one embodiment, by outgrowth from a biopsy of the recipient individual's own skin (in the case of autologous preparations), or skin of one or more healthy donors (for allogeneic preparations), or a combination thereof may be employed.
- fibroblasts are used from young donors, although in other cases the fibroblasts are not from young donors and may be adults including middle-aged, for example.
- fibroblasts are transfected with polynucleotides that encode one or more gene products, for example to allow for enhanced growth and overcoming of the Hayflick limit.
- the starting material comprises multiple (such as two, three, four, or more) 3-mm punch skin biopsies collected using standard aseptic practices.
- the biopsies are collected by an individual and placed into a vessel (such as a vial) comprising standard medium, such as comprising sterile phosphate buffered saline (PBS).
- PBS sterile phosphate buffered saline
- the biopsy after arrival at a manufacturing facility, the biopsy is inspected and, upon acceptance, transferred directly to the manufacturing area. Upon initiation of the process, the biopsy tissue is then washed prior to enzymatic digestion. After washing, a Liberase Digestive Enzyme Solution is added without mincing, and the biopsy tissue is incubated for a suitable time and using a suitable temperature, such as at 37.0 ⁇ 2° C for one hour. Time of biopsy tissue digestion is a process parameter that can affect the viability and growth rate of cells in culture and be optimized routinely.
- An example of the process is as follows: Liberase may be used that is a collagenase/neutral protease enzyme cocktail obtained formulated from Lonza Walkersville, Inc. (Walkersville, Md.) and
- IMDM Initiation Growth Media
- GA 10% Fetal Bovine Serum
- FBS Fetal Bovine Serum
- IMDM Initiation Growth Media
- cells are pelleted by centrifugation and resuspended in 5.0 mL Initiation Growth Media.
- centrifugation is not performed, with full inactivation of the enzyme occurring by the addition of Initiation Growth Media only.
- Initiation Growth Media is added prior to seeding of the cell suspension into a T-175 cell culture flask for initiation of cell growth and expansion.
- a T-75, T-150, T-185 or T-225 flask can be used in place of the T-75 flask.
- Cells are incubated at 37 ⁇ 2.0° C. with 5.0 ⁇ 1.0% CO2 and fed with fresh Complete Growth Media every three to five days. All feeds in the process are performed by removing half of the Complete Growth Media and replacing the same volume with fresh media. Alternatively, full feeds can be performed. Cells should not remain in the T-175 flask greater than 30 days prior to passaging. Confluence is monitored throughout the process to ensure adequate seeding densities during culture splitting.
- T-175 flask When cell confluence is greater than or equal to 40% in the T-175 flask, they are passaged by removing the spent media, washing the cells, and treating with Trypsin-EDTA to release adherent cells in the flask into the solution. Cells are then trypsinized and seeded into a T-500 flask for continued cell expansion. Alternately, one or two T-300 flasks, One Layer Cell Stack (1 CS), One Layer Cell Factory (1 CF) or a Two Layer Cell Stack (2 CS) can be used in place of the T-500 Flask. Morphology is evaluated at each passage and prior to harvest to monitor the culture purity throughout the culture purity throughout the process.
- Morphology is evaluated by comparing the observed sample with visual standards for morphology examination of cell cultures.
- the cells display typical fibroblast morphologies when growing in cultured monolayers. Cells may display either an elongated, fusiform or spindle appearance with slender extensions, or appear as larger, flattened stellate cells which may have cytoplasmic leading edges. A mixture of these morphologies may also be observed. Fibroblasts in less confluent areas can be similarly shaped, but randomly oriented. The presence of keratinocytes in cell cultures is also evaluated. Keratinocytes appear round and irregularly shaped and, at higher confluence, they appear organized in a cobblestone formation.
- keratinocytes are observable in small colonies.
- Cells are incubated at 37 ⁇ 2.0° C. with 5.0. +-.1.0%) CO2 and passaged every three to five days in the T-500 flask and every five to seven days in the ten layer cell stack (IOCS). Cells should not remain in the T-500 flask for more than 10 days prior to passaging.
- Quality Control (QC) release testing for safety of the Bulk Drug Substance includes sterility and endotoxin testing. When cell confluence in the T-500 flask is 95%), cells are passaged to a 10 CS culture vessel.
- two Five Layer Cell Stacks (5 CS) or a 10 Layer Cell Factory (10 CF) can be used in place of the 10 CS.
- IOCS Passage to the 10 CS is performed by removing the spent media, washing the cells, and treating with Trypsin- EDTA to release adherent cells in the flask into the solution. Cells are then transferred to the 10 CS. Additional Complete Growth Media is added to neutralize the trypsin and the cells from the T-500 flask are pipetted into a 2 L bottle containing fresh Complete Growth Media. The contents of the 2 L bottle are transferred into the 10 CS and seeded across all layers. Cells are then incubated at 37 ⁇ 2.0° C.
- the passaged dermal fibroblasts are rendered substantially free of immunogenic proteins present in the culture medium by incubating the expanded fibroblasts for a period of time in protein free medium, Primary Harvest When cell confluence in the 10 CS is 95% or more, cells are harvested. Harvesting is performed by removing the spent media, washing the cells, treating with Trypsin-EDTA to release adherent cells into the solution, and adding additional Complete Growth Media to neutralize the trypsin. Cells are collected by centrifugation, resuspended, and in-process QC testing performed to determine total viable cell count and cell viability.
- an additional passage into multiple cell stacks (up to four 10 CS) is performed.
- cells from the primary harvest are added to a 2 L media bottle containing fresh Complete Growth Media.
- Re-suspended cells are added to multiple cell stacks and incubated at 37 ⁇ 2.0° C. with 5.0. +-.1.0% CO2.
- the cell stacks are fed and harvested as described above, except cell confluence must be 80% or higher prior to cell harvest.
- the harvest procedure is the same as described for the primary harvest above.
- a mycoplasma sample from cells and spent media is collected, and cell count and viability performed as described for the primary harvest above.
- the method decreases or eliminates immunogenic proteins be avoiding their introduction from animal-sourced reagents.
- cells are cryopreserved in protein-free freeze media, then thawed and washed prior to prepping the final injection to further reduce remaining residuals.
- additional Drug Substance is needed after the harvest and cryopreservation of cells from additional passaging is complete, aliquots of frozen Drug Substance—Cry ovial are thawed and used to seed 5 CS or 10 CS culture vessels.
- a four layer cell factory (4 CF), two 4 CF, or two 5 CS can be used in place of a 5 CS or 10 CS.
- a frozen cryovial(s) of cells is thawed, washed, added to a 2 L media bottle containing fresh Complete Growth Media and cultured, harvested and
- the cell suspension is added Cell confluence must be 80% or more prior to cell harvest.
- the cells are harvested and washed, then formulated to contain 1.0-2.7xl0 7 cells/mL, with a target of 2.2xl0 7 cells/mL.
- the target can be adjusted within the formulation range to accommodate different indication doses.
- the drug substance consists of a population of viable, autologous human fibroblast cells suspended in a cryopreservation medium consisting of Iscove's Modified Dulbecco's Medium (EVIDM) and Profreeze-CDM.TM. (Lonza, Walkerville, Md.) plus 7.5% dimethyl sulfoxide (DMSO).
- a lower DMSO concentration may be used in place of 7.5% or
- CryoStor.TM. CS5 or CryoStor.TM. CS10 may be used in place of IMDM/Profreeze/DMSO.
- purity/identity of the Drug Substance is performed and must confirm the suspension contains 98% or more fibroblasts.
- the usual cell contaminants include keratinocytes.
- the purity/identify assay employs
- CD90 and CD 104 cell surface markers for fibroblast and keratinocyte cells, respectively
- CD90 Thy-1
- Antibodies against CD90 protein have been shown to exhibit high specificity to human fibroblast cells.
- CD 104, integrin beta4 chain is a 205 kDa transmembrane glycoprotein which associates with integrin alpha6 chain (CD49f) to form the alpha6/beta4 complex. This complex has been shown to act as a molecular marker for keratinocyte cells (Adams and Watt 1991).
- Antibodies to CD 104 protein bind to 100% of human keratinocyte cells.
- Cell count and viability is determined by incubating the samples with Viacount Dye Reagent and analyzing samples using the Guava PCA system.
- the reagent is composed of two dyes, a membrane-permeable dye which stains all nucleated cells, and a membrane-impermeable dye which stains only damaged or dying cells.
- the use of this dye combination enables the Guava PCA system to estimate the total number of cells present in the sample, and to determine which cells are viable, apoptotic, or dead.
- the method was custom developed specifically for use in determining purity/identity of autologous cultured fibroblasts.
- cells can be passaged from either the T-175 flask (or alternatives) or the T-500 flask (or alternatives) into a spinner flask containing microcarriers as the cell growth surface.
- Microcarriers are small beadlike structures that are used as a growth surface for anchorage dependent cells in suspension culture. They are designed to produce large cell yields in small volumes.
- a volume of Complete Growth Media ranging from 50 mL-300 mL is added to a 500 mL, IL or 2 L sterile disposable spinner flask. Sterile microcarriers are added to the spinner flask.
- the culture is allowed to remain static or is placed on a stir plate at a low RPM (15-30 RRM) for a short period of time (1-24 hours) in a 37 ⁇ 2.0° C with 5.0 ⁇ 1.0% CO2 incubator to allow for adherence of cells to the carriers.
- the speed of the spin plate is increased (30-120 RPM).
- Cells are fed with fresh Complete Growth Media every one to five days, or when media appears spent by color change.
- Cells are collected at regular intervals by sampling the microcamers, isolating the cells and performing cell count and viability analysis.
- the concentration of cells per carrier is used to determine when to scale-up the culture.
- cells are washed with PBS and harvested from the microcamers using trypsin-EDTA and seeded back into the spinner flask in a larger amount of microcamers and higher volume of Complete Growth Media (300 mL-2 L).
- additional microcamers and Complete Growth Media can be added directly to the spinner flask containing the existing microcarrier culture, allowing for direct bead-to-bead transfer of cells without the use of trypsinization and reseeding.
- the cells can be directly seeded into the scale-up amount of microcamers. After the attachment period, the speed of the spin plate is increased (30-120 RPM).
- Microcamers used within the disposable spinner flask may be made from poly blend such as BioNOC II ® (Cesco Bioengineering, distributed by Bellco Biotechnology, Vineland, N.J.) and FibraCel® (New Brunswick Scientific, Edison, N.
- gelatin such as Cultispher-G (Percell Biolytica, Astrop, Sweden)
- cellulose such as CytoporeTM (GE Healthcare, Piscataway, N.J.) or coated/uncoated polystyrene, such as 2D MicroHexTM (Nunc, Weisbaden, Germany), Cytodex® (GE Healthcare, Piscataway, N.J.) or Hy- Q SphereTM (Thermo Scientific Hyclone, Logan, Utah).
- cells can be processed on poly blend 2D microcamers such as BioNOC II.RTM. and FibraCel® using an automatic bellow system, such as
- Bioengineering distributed by Bellco Biotechnology, Vineland, N.J.) in place of the spinner flask apparatus.
- Cells from the T-175 (or alternatives) or T-500 flask (or alternatives) are passaged into a bellow bottle containing microcamers with the appropriate amount of Complete Growth Media, and placed into the system.
- the system pumps media over the microcamers to feed cells, and draws away media to allow for oxygenation in a repeating fixed cycle.
- Cells are monitored, fed, washed and harvested in the same sequence as described above.
- cells can be processed using automated systems. After digestion of the biopsy tissue or after the first passage is complete (T-175 flask or alternative), cells may be seeded into an automated device.
- ACE Automated Cellular Expansion
- the ACE system can be a scaled down, single lot unit version comprised of a disposable component that consists of cell growth surface, delivery tubing, media and reagents, and a permanent base that houses mechanics and computer processing capabilities for heating/cooling, media transfer and execution of the automated programming cycle.
- a disposable component that consists of cell growth surface, delivery tubing, media and reagents, and a permanent base that houses mechanics and computer processing capabilities for heating/cooling, media transfer and execution of the automated programming cycle.
- each sterile irradiated ACE disposable unit Upon receipt, each sterile irradiated ACE disposable unit will be unwrapped from its packaging and loaded with media and reagents by hanging pre-filled bags and connecting the bags to the existing tubing via aseptic connectors.
- the process continues as follows: a) Inside a biological safety cabinet (BSC), a suspension of cells from a biopsy that has been enzymatically digested is introduced into the "pre-growth chamber" (small unit on top of the cell tower), which is already filled with Initiation Growth Media containing antibiotics. From the BSC, the disposable would be transferred to the permanent ACE unit already in place; b) After approximately three days, the cells within the pre-growth chamber are trypsinized and introduced into the cell tower itself, which is pre-filled with Complete Growth Media.
- BSC biological safety cabinet
- the "bubbling action" caused by CO2 injection force the media to circulate at such a rate that the cells spiral downward and settle on the surface of the discs in an evenly distributed manner; c) For approximately seven days, the cells are allowed to multiply. At this time, confluence will be checked (method unknown at time of writing) to verify that culture is growing. Also at this time, the Complete Growth Media will be replaced with fresh Complete Growth Media. CGM will be replaced every seven days for three to four weeks. At the end of the culture period, the confluence is checked once more to verify that there is sufficient growth to possibly yield the desired quantity of cells for the intended treatment; d) If the culture is sufficiently confluent, it is harvested. The spent media (supernatant) is drained from the vessel.
- PBS will then is pumped into the vessel (to wash the media, FBS from the cells) and drained almost immediately. Trypsin- EDTA is pumped into the vessel to detach the cells from the growth surface. From the spin separator, the cells will be sent through an inline automated cell counting device or a sample collected for cell count and viability testing via laboratory analyses. Once a specific number of cells has been counted and the proper cell concentration has been reached, the harvested cells are delivered to a collection vial that can be removed to aliquot the samples for cryogenic freezing.
- automated robotic systems may be used to perform cell feeding, passaging, and harvesting for the entire length or a portion of the process.
- Cells can be introduced into the robotic device directly after digest and seed into the T-175 flask (or alternative).
- the device may have the capacity to incubate cells, perform cell count and viability analysis and perform feeds and transfers to larger culture vessels.
- the system may also have a computerized cataloging function to track individual lots. Existing technologies or customized systems may be used for the robotic option.
- a party that obtains the fibroblasts may or may not be a party that manipulates the fibroblasts to produce their enhancement and/or deliver them to an individual.
- the fibroblasts may be manipulated, including to enhance one or more activities useful for a therapeutic purpose.
- the fibroblasts are exposed to one or more biologically active agents and/or conditions prior to (and/or during) delivery to an individual in need thereof, and in some cases the exposure to one or more biologically active agents and/or conditions prior to (and/or during) delivery may or may not occur during a culturing step.
- fibroblasts are pre-activated by contact with a composition or mixture of compositions comprising a biologically active agent that is at least one growth factor, and the growth factor(s) may be selected from the group consisting of transforming growth factors (TGF), fibroblast growth factors (FGF), platelet-derived growth factors (PDGF), epidermal growth factors (EGF), vascular endothelial growth factors (VEGF), insulin-like growth factors (IGF), platelet-derived endothelial growth factors (PDEGF), platelet-derived angiogenesis factors (PDAF), platelet factors 4 (PF-4), hepatocyte growth factors (HGF) and a combination thereof.
- TGF transforming growth factors
- FGF fibroblast growth factors
- PDGF platelet-derived growth factors
- EGF epidermal growth factors
- VEGF vascular endothelial growth factors
- IGF insulin-like growth factors
- PEGF platelet-derived endothelial growth factors
- PDAF platelet-derived angiogenesis factors
- the growth factors are transforming growth factors (TGF), platelet-derived growth factors (PDGF) fibroblast growth factors (FGF) and a combination thereof.
- the growth factors are selected from the group consisting of transforming growth factors beta (TGF-beta), platelet-derived growth factors BB (PDGF-BB), basic fibroblast growth factors (bFGF) and a combination thereof.
- TGF-beta transforming growth factors beta
- PDGF-BB platelet-derived growth factors BB
- bFGF basic fibroblast growth factors
- the growth factor-comprising compositions are delivered to an individual simultaneously with, or subsequent to, delivery of fibroblasts. The delivery may occur by injection, in certain embodiments.
- the fibroblasts may be autologous, allogeneic, or xenogeneic with respect to the recipient individual.
- a platelet plasma composition is administered together with the fibroblasts or subsequent to administration of the fibroblasts, and the platelet plasma composition may comprise, consist essentially of, or consist of platelets and plasma and may be derived from bone marrow and/or peripheral blood.
- the present disclosure may use platelet plasma composition(s) from either or both of these sources, and either platelet plasma composition may be used to regenerate either a nucleus or annulus or both in need thereof. Further, the platelet plasma composition may be used with or without concentrated bone marrow (BMAC).
- BMAC concentrated bone marrow
- platelets are non-nucleated blood cells that as noted above may be found in bone marrow and peripheral blood.
- a platelet plasma composition may be obtained by sequestering platelets from whole blood and/or bone marrow through centrifugation, for example into three strata: (1) platelet rich plasma; (2) platelet poor plasma; and (3) fibrinogen.
- platelets from one of the strata e.g., the platelet rich plasma (PRP) from blood
- PRP platelet rich plasma
- one may use the platelets whole or their contents may be extracted and concentrated into a platelet lysate through a cell membrane lysis procedure using thrombin and/or calcium chloride, for example.
- the lysate will act more rapidly than the PRP (or platelet poor plasma from bone marrow).
- PRP platelet poor plasma from bone marrow
- platelet poor plasma that is derived from bone marrow has a greater platelet concentration than platelet rich plasma from blood, also known as platelet poor/rich plasma, ("PP/RP" or "PPP").
- PP/RP or PPP may be used to refer to platelet poor plasma derived from bone marrow, and in some embodiments, preferably PP/RP is used or PRP is used as part of the composition for disc regeneration.
- PRP refers only to compositions derived from peripheral blood
- PPP or PP/RP refers to compositions derived from bone marrow.
- the platelet plasma composition which may or may not be in the form of a lysate, may serve one or more of the following functions: (1) to release/provide growth factors and/or cytokines for tissue regeneration; (2) to reduce inflammation; (3) to attract/mobilize cell signaling; (4) to initiate fibroblast repair of damaged annulus through fibroblast growth factors (FGF); (5) to stabilize disc annulus; (6) to repair annulus disc tears; (7) to stimulate revascularization to a disc; and (8) to stimulate stem cell activation.
- FGF fibroblast growth factors
- the cytokine(s) may be concentrated in order to optimize their functional capacity. Concentration may be accomplished in two steps. First, blood may be obtained and concentrated to a volume that is 5-15% of what it was before concentration. Devices that may be used include but are not limited to a hemofilter or a hemoconcentrator. For example, 60 cc of blood may be concentrated down to 6 cc. Next, the concentrated blood may be filtered to remove water. This filtering step may reduce the volume further to 33%-67% (e.g., approximately 50%) of what it was prior to filtration.
- a concentration product of 6 cc one may filter out water so that one obtains a product of approximately 3 cc.
- the platelet rich plasma, platelet poor plasma and fibrinogen are obtained from blood, they may for example be obtained by drawing 20-500 cc of peripheral blood, 40-250 cc of peripheral blood or 60-100 cc of peripheral blood.
- the amount of blood that one should draw will depend on the number of discs that have degenerated and the size of the discs. As persons of ordinary skill in the art will appreciate, a typical disc has a volume of 2-5 cc or 3-4 cc.
- fibroblasts are treated, or administered together with activated PRP. The method of generation of activated PRP may be used according to U.
- separating the PRP from whole blood wherein the separating step further comprises the steps of: collecting 10 ml of the whole blood from an animal or patient into a vacuum test tube containing 3.2% sodium citrate, and primarily centrifuging the collected whole blood at 1,750-1,900 g for 3 to 5 minutes; collecting a supernatant liquid comprising a plasma layer with a buffy coat obtained from said centrifugation; transferring the collected supernatant liquid to a new vacuum test tube by a blunt needle, and secondarily centrifuging the collected supernatant liquid at 4,500-5,000 g for 4 to 6 minutes; and collecting the PRP concentrated in a bottom layer by another blunt needle; mixing 1 mL of the PRP collected from the separating step with a calcium chloride solution with a concentration of 0.30-0.55 mg/mL by a three-way connector; and mixing a mixture of the PRP and the calcium chloride solution with
- fibroblasts administration of fibroblasts is performed together with biocompatible polymers and growth factors or PRP, or Platelet Gel.
- allogeneic fibroblasts are obtained from a screened donor(s) using similar methods as described above.
- a screened donor provides tissue for expansion of fibroblasts and generation of a master cell bank (MCB).
- MCB master cell bank
- WBC working cell bank
- Reprogrammed cells possess a normal karyotype, differentiate into beating cardiomyocytes in vitro and differentiate into representatives of all three germ layers in vivo.
- a subpopulation of human dermal fibroblasts that express the pluripotency marker SSEA3 demonstrates enhanced iPSC generation efficiency as described by Bryne, et al, PLoS One, 4(9):e7118 (2009).
- SSE A3 -positive and SSEA3 -negative populations were transduced with the same retroviral vectors, under identical experimental conditions, and seeded onto inactivated mouse embryonic fibroblasts (MEFs). After three weeks of culture under standard hESC conditions, plates were examined in a double-blind analysis by three independent hESC biologists for iPSC colony formation. Colonies with iPSC morphology were picked and expanded.
- hESCs which included alkaline phosphatase, Nanog, SSEA3, SSEA4, TRA160 and TRA181.
- the SSEA3- selected iPSCs also demonstrated a normal male karyotype (46, XY), the ability to differentiate into functional beating cardiomyocytes in vitro and differentiate into representatives of all three germ layers in vivo. Because no iPSC colony formation or line derivation from the transduced SSEA3 -negative cells was observed, this indicates that these cells possess significantly lower or even no reprogramming potential relative to the SSE A3 -expressing cells.
- a 10-fold enrichment of primary fibroblasts that strongly express SSEA3 results in a significantly greater efficiency (8-fold increase) of iPSC line derivation compared to the control derivation rate (p ⁇ 0.05).
- the SSE A3 -positive cells appeared indistinguishable, morphologically, from the SSE A3 -negative fibroblasts; furthermore, expression of the SSEA3 antigen is not considered a marker of other cell types such as mesenchymal or epidermal adult stem cells.
- platelet rich plasma is used to activate a rare subpopulation of SSEA3 -expressing cells that exists in the dermis of adult human skin and may be isolated accordingly.
- SSEA3 -expressing cells undergo a significant increase in cell number in response to injury, indicating a role in regeneration.
- SSEA3 -expressing regeneration-associated (SERA) cells were derived through primary cell culture, purified by fluorescence activated cell sorting (FACS) and characterized.
- FACS fluorescence activated cell sorting
- the SERA cells demonstrated a global transcriptional state most similar to bone marrow and fat derived mesenchymal stem cells (MSCs) and the highest expressing SSEA3 expressing cells co-expressed CD 105.
- MSCs bone marrow and fat derived mesenchymal stem cells
- these cells cannot differentiate into adipocytes, osteoblasts or chondrocytes.
- Treatment of individuals with lower back pain may be accomplished through one embodiment of the disclosure, such as through the administration of fibroblast cells that have been genetically modified to upregulate expression of angiogenic stimuli or anti-inflammatory activities.
- genes may be introduced by a wide range of approaches including adenoviral, adeno-associated, retroviral, alpha-viral, lentiviral, Kunjin virus, or HSV vectors, liposomal, nano-particle mediated as well as electroporation and Sleeping Beauty transposons.
- Genes with angiogenic stimulatory function that may be transfected include but are not limited to: VEGF, FGF-1, FGF-2, FGF-4, EGF, HGF, and a combination thereof.
- transcription factors that are associated with upregulating expression of angiogenic cascades may also be transfected into cells used for treatment of lower back pain, including: HIF-lalpha, HTF-2, NET, NF-kB, or a combination thereof.
- Genes inhibitory to inflammation may be used such as: TGF-a, TGF-b, IL-4, IL-10, IL-13, IL-20 thrombospondin, or a
- Transfection may also be utilized for administration of genetic manipulation means in a manner to substantially block transcription or translation of genes which inhibit angiogenesis.
- Antisense oligonucleotides, ribozymes or short interfering RNA may be transfected into cells for use for treatment of lower back pain in order to block expression of antiangiogenic proteins such as: canstatin, IP-10, kringle 1-5, and collagen
- gene inhibitory technologies may be used for blocking ability of cells to be used for treatment of lower back pain to express inflammatory proteins including: IL-1, TNF- alpha, IL-2, IL-6, IL-8, IL-9, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN- alpha, IFN-beta, and IFN-gamma.
- inflammatory proteins including: IL-1, TNF- alpha, IL-2, IL-6, IL-8, IL-9, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN- alpha, IFN-beta, and IFN-gamma.
- Globally acting transcription factors associated with inflammation may also be substantially blocked using not only the genetic means described but also decoy oligonucleotides.
- Suitable transcription factors for blocking include various subunits of the NF-kB complex such as p55, and/or p60, STAT family members, particularly STAT1, STAT5, STAT4, and members of the Interferon Regulatory Factor family such as IRF-1, IFR-3, and IFR-8, for example.
- Enhancement of angiogenic stimulation ability of the cells useful for the treatment of back pain can be performed through culturing under conditions of restricted oxygen. It is known in the art that stem cells in general, and ones with angiogenesis promoting activity specifically, tend to reside in hypoxia niches of the bone marrow. When stem cells differentiate into more mature progeny, they progressively migrate to areas of the bone marrow with higher oxygen tension. [40].
- hypoxia is induced through induction of one or more agents that cause the upregulation of the HIF-1 transcription factor.
- the oxygen levels may be between 0.1%-5%, 0.1%-4%, 0.1%-3%, 0.1%-2%, 0.1%-1%, 0.1%-0.75%, 0.1%-0.5%, 0.1%-0.25%, 0.2%-5%, 0.2%-4%, 0.2%-3%, 0.2%-2%, 0.2%-l%, 0.2%-0.75%, 0.2%-0.5%, 0.5%-5%, 0.5%-4%, 0.5%-3%, 0.5%-2%, 0.5%-l%, 0.5%-0.75%, 0.75%-5%, 0.75%-4%, 0.75%-3%, 0.75%-2%, 0.75%-l%, l%-5%, l%-5%, l%-5%, l%-4%, l%-3%, l%-2%, 2%-5%, 2%-4%, 2%-3%, 3%-5%, 3%-4%, or 4%-5%% oxygen, in specific embodiments.
- the duration of exposure of the cells to hypoxic conditions may be for a duration of 30 minutes (min)-3 days, 30 min-2 days, 30 min-1 day, 30 min- 12 hours (hrs), 30 min-8 hrs, 30 min-6 hrs, 30 min-4 hrs, 30 min-2 hrs, 30 min-1 hour (hr), 1 hr-3 days, lhr-2 days, 1 hr-1 day, 1-12 hrs, 1-8 hrs, 1-6 hrs, 1-4 hrs, 1-2 hrs, 2 hrs-3 days, 2hrs-2 days, 2 hrs-1 day, 2 hrs- 12 hrs, 2-10hrs, 2-8hrs, 2-6 hrs, 2-4 hrs, 2-3 hrs, 6 hrs-3 days, 6 hrs-2 days, 6 hrs-1 day, 6-12 hrs, 6-8 hrs, 8hrs-3 days, 8 hrs-2 days, 8 hrs-1 day, 8-16 hrs, 8-12 hrs, 8-10 hrs, 12hrs-3 days, 12 hrs-2 days,
- cells generated may be tested for angiogenic and/or anti-inflammatory activity before use in clinical conditions, in specific embodiments. Testing may be performed by various means known to one skilled in the art. In terms of assessing angiogenic potential the means include, but are not limited to: a) Ability to support endothelial cell proliferation in vitro using human umbilical vein endothelial cells or other endothelial populations (Assessment of proliferation may be performed using tritiated thymidine incorporation or by visually counted said proliferating endothelial cells.
- a viability dye such as MTT or other commercially available indicators may be used); b) Ability to support cord formation in subcutaneously implanted matrices (The matrices, which may include Matrigel or fibrin gel, for example, are loaded with cells generated as described above and implanted subcutaneously in an animal.
- the animal may be an immunodeficient mouse such as a SCID or nude mouse in order to negate immunological differences. Subsequent to implantation formation of endothelial cords may be assessed visually by microscopy.
- a species-specific marker may be used in order to distinguish cells stimulating angiogenesis versus host cells responding to said cells stimulating angiogenesis; c) Ability to accelerate angiogenesis occurring in the embryonic chicken chorioallantoic membrane assay (Cells may be implanted directly, or via a matrix, into the chicken chorioallantoic membrane on embryonic day 9 and cultured for a period of approximately 2 days. Visualization of angiogenesis may be performed using in vivo microscopy); and/or d) Ability to stimulate neoangiogenesis in the hind limb ischemia model described above. [0061] Assessment of the anti-inflammatory abilities of fibroblast cells generated or isolated for potential clinical use may also be performed.
- Putative anti-inflammatory fibroblast cells may be co-cultured at various ratios with an immunological cell.
- the immunological cell may be stimulated with an activatory stimulus.
- the ability of the putative anti-inflammatory cell to inhibit, in a dose-dependent manner, production of inflammatory cytokines or to augment production of anti-inflammatory cytokines may be used as an output system of assessing antiinflammatory activity. Additional output parameters may include: proliferation, cytotoxic activity, production of inflammatory mediators, or upregulation of surface markers associated with activation.
- Cytokines assessed may include: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,IL-27, TNF, IFN and/or RANKL.
- Specific immunological cells may be freshly isolated or may be immortalized cell lines.
- the immunological cells may be: T cells, B cells, monocytes, macrophages, neutrophils, eosinophils, basophils, dendritic cells, natural killer cells, natural killer T cells, gamma delta-T cells, or a combination thereof.
- immunological stimuli may include an antibody, a ligand, a protein, or another cells. Examples including: crosslinking antibodies to T cell receptor, to costimulatory molecules such as CD28, to activation associated molecules such as CD69 or to receptors for stimulatory cytokines such as IL-2. Additional examples of inflammatory stimuli may include co-culture with allogeneic stimulator cells such as in mixed lymphocyte reactions, or may include stimulation with a nonspecific activator such as a lectin. Specific lectins may include conconavalin-A,
- phytohemagluttinin, or wheat germ agglutinin may be activators of the toll like receptor pathway such as lipopolysaccharide, CpG DNA motifs or bacterial membrane fractions.
- the methods described in the above two paragraphs are shown only as examples that may be used to determine, before entry into clinical use, whether a cell population generated as described in the present invention is capable of producing the desired angiogenic stimulatory or anti-inflammatory effects. These examples are only provided as guides which one skilled in the art can optimize upon using routine experimentation.
- cells to be used for treatment of lower back pain may be cryopreserved for subsequent use, as well as for
- cryopreservation requires attention be paid to three main concepts, these are: 1) the
- cryoprotective agent 2) the control of the freezing rate, and 3) the temperature at which the cells will be stored.
- Cryoprotective agents are well known to one skilled in the art and can include but are not limited to dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidine, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, or choline chloride as described in U.S. Patent No. 6,461,645 (incorporated by reference herein in its entirety), for example.
- DMSO dimethyl sulfoxide
- glycerol polyvinylpyrrolidine
- polyethylene glycol albumin
- dextran sucrose
- ethylene glycol i-erythritol
- D-ribitol D-ribitol
- a method for cryopreservation of cells that is utilized by some skilled artisans comprises DMSO at a concentration not being immediately cytotoxic to cells under conditions which allow it to freely permeate the cell and to protect intracellular organelles; the DMSO combines with water and prevents cellular damage induced from ice crystal formation. Addition of plasma at concentrations between 20-25% by volume can augment the protective effect of DMSO. After addition of DMSO, cells should be kept at temperatures below 4 C, in order to prevent DMSO-mediated damage. Methods of actually inducing the cells in a state of suspended animation involve utilization of various cooling protocols. While cell type, freezing reagent, and concentration of cells are important variables in determining methods of cooling, it is generally accepted that a controlled, steady rate of cooling is optimal.
- cryopreservation There are numerous devices and apparatuses known in the field that are capable of reducing temperatures of cells for optimal cryopreservations.
- One such apparatus is the Thermo Electro Cryomed FreezerTM manufactured by Thermo Electron Corporation. Cells can also be frozen in CryoCyteTM containers as made by Baxter.
- cryopreservation is as follows: 2 x 10 6 CD34 cells/ml are isolated from cord blood using the Isolex SystemTM as per manufacturer's instructions (Baxter). Cells are incubated in DMEM media with 10% DMSO and 20%) plasma. Cooling is performed at 1 Celsius. /minute from 0 to -80 Celsius.
- fibroblasts may be derived from tissues comprising skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, adipose tissue, foreskin, placental, and/or umbilical cord, for example. In specific embodiments, the fibroblasts are placental, fetal, neonatal or adult or mixtures thereof.
- the number of administrations of cells to an individual will depend upon the factors described herein at least in part and may be optimized using routine methods in the art. In specific embodiments, a single administration is required. In other embodiments, a plurality of administration of cells is required. It should be appreciated that the system is subject to variables, such as the particular need of the individual, which may vary with time and circumstances, the rate of loss of the cellular activity as a result of loss of cells or activity of individual cells, and the like. Therefore, it is expected that each individual could be monitored for the proper dosage, and such practices of monitoring an individual are routine in the art.
- the cells are subjected to one or more media compositions that comprises, consists of, or consists essentially of Roswell Park Memorial Institute (RPMI- 1640), Dublecco's Modified Essential Media (DMEM), Eagle's Modified Essential Media (EMEM), Optimem, Iscove's Media, or a combination thereof.
- RPMI- 1640 Roswell Park Memorial Institute
- DMEM Dublecco's Modified Essential Media
- EMEM Eagle's Modified Essential Media
- Optimem Iscove's Media
- the fibroblasts are recombinantly manipulated to encode SSEA3, VEGF, FGF-1, FGF-2, FGF-4, EGF, HGF, HIF-1 alpha, HIF-2, NET, NF-kB, TGF-a, TGF-b, IL-4, IL-10, IL-13, IL-20 thrombospondin, canstatin, IP-10, kringle 1-5, collagen XVIII/endostatin, IL-1, TNF- alpha, IL-2, IL-6, IL-8, IL-9, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, IFN- alpha, IFN-beta, IFN-gamma, p55, p60, STAT1, STAT5, STAT4, IRF-1, IFR-3, IFR-8, or a combination thereof.
- one or more types of the fibroblast cells are manipulated to harbor one or more expression vectors that each encode one or more gene products of interest.
- a recombinant expression vector(s) can be introduced as one or more DNA molecules or constructs, where there may be at least one marker that will allow for selection of host cells that contain the vector(s).
- the vector(s) can be prepared in conventional ways, wherein the genes and regulatory regions may be isolated, as appropriate, ligated, cloned in an appropriate cloning host, and analyzed by sequencing or other convenient means.
- individual fragments including all or portions of a functional unit may be isolated, where in some cases one or more mutations may be introduced using "primer repair", ligation, in vitro mutagenesis, etc. as appropriate.
- the vector(s) once completed and demonstrated to have the appropriate sequences may then be introduced into the host cell by any convenient means.
- the constructs may be integrated and packaged into non-replicating, defective viral genomes like lentivirus, Adenovirus, Adeno- associated virus (AAV), Herpes simplex virus (HSV), or others, including retroviral vectors, for infection or transduction into cells.
- the vector(s) may include viral sequences for transfection, if desired.
- the construct may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like.
- the host cells may be grown and expanded in culture before introduction of the vector(s), followed by the appropriate treatment for introduction of the vector(s) and integration of the vector(s).
- the cells are then expanded and screened by virtue of a marker present in the construct.
- markers that may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.
- any of the genes or gene products described herein, or active portions thereof, may be cloned into mammalian expression constructs comprising one or more promoter sequences enabling expression in cells such as the CMV promoter [Artuc et al., Exp. Dermatol. 1995, 4:317-21].
- suitable constructs include, but are not limited to pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available, or the pSH expression vector which enables a regulated polynucleotide expression in human foreskin cells [Ventura and Villa, 1993, Biochem.
- Retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif, USA, including Retro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the transgene is transcribed from CMV promoter.
- Retro-X vectors pLNCX and pLXSN which permit cloning into multiple cloning sites and the transgene is transcribed from CMV promoter.
- Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.
- fibroblasts are harvested by a means allowing for detachment from tissue culture plates, for example, by trypsinization and transferred to either a 6-well (Nunc, Denmark) or a 24-well plate (Nunc) for proliferation. Approximately 3 days post-transfection, the cell media is changed to media allow for proliferation and expansion of modified fibroblasts.
- Neurobasal A (NBA) proliferation medium comprising Neurobasal- A (Invitrogen), 1% D-glucose (Sigma Aldrich), 1% Penicillin/Streptomycin/Glutamine (Invitrogen), 2% B27 supplement with Retinoic acid (Invitrogen), 0.2% EGF (Peprotech, USA), 0.08% FGF-2 (Peprotech), 0.2% Heparin (Sigma Aldrich, USA) and Valproic acid (Sigma-Aldrich) to a concentration of 1 DM.
- the media is then subsequently changed thrice weekly, and cells are re-plated regularly (for example, 2-8 times up to a maximum of weekly re-plating, becoming more regular as colonies began to develop) to remove non-reprogrammed cells until widespread colony formation is achieved.
- RNA can be delivered to any cells, including any modified cells, of the disclosure by various means including microinjection, electroporation, and lipid- mediated transfection, for example.
- introduction of vector(s) into cells may occur via transposons.
- An example of a synthetic transposon for use is the Sleeping Beauty transposon that comprises an expression cassette including the angiogenic agent gene thereof.
- one may have a target site for homologous recombination, where it is desired that vector(s) be integrated at a particular locus using materials and methods as are known in the art for homologous recombination.
- OMEGA O-vectors. See, for example, Thomas and Capecchi, 1987; Mansour, et al., 1988; and Joyner, et al, 1989.
- the vector(s) may be introduced as a single DNA molecule encoding at least one agent (including one or more tumor inhibitory agents or functional fragments thereof) and optionally another polynucleotide (such as genes), or different DNA molecules having one or more polynucleotides (such as genes).
- the vector(s) may be introduced simultaneously or consecutively, each with the same or different markers.
- one vector would contain one or more agents (such as angiogenic agent(s)) under the control of particular regulatory sequences.
- Vector(s) comprising useful elements such as bacterial or yeast origins of replication, selectable and/or amplifiable markers, promoter/enhancer elements for expression in prokaryotes or eukaryotes, etc. that may be used to prepare stocks of vector DNAs and for carrying out transfections are well known in the art, and many are commercially available.
- RNAs or proteinaceous sequences may be co-expressed with other selected RNAs or proteinaceous sequences in the same host cell. Co-expression may be achieved by co-transfecting the host cell with two or more distinct recombinant vectors. Alternatively, a single recombinant vector may be constructed to include multiple distinct coding regions for RNAs, which could then be expressed in host cells transfected with the single vector. [0072] In some situations, it may be desirable to kill the modified cells, such as when the object is to terminate the treatment, the cells become neoplastic, in research where the absence of the cells after their presence is of interest, and/or another event.
- Suicide genes are known in the art, e.g. the iCaspase9 system in which a modified form of caspase 9 is dimerizable with a small molecule, e.g. API 903. See, e.g., Straathof et al. , Blood 105:4247-4254 (2005).
- Fibroblasts are cultured with platelet rich plasma (PRP) to produce enhanced fibroblasts for therapeutic use.
- Foreskin fibroblasts, human bone marrow MSC and human adipose MSC were obtained from ATCC and cultured in DMEM media containing 10% fetal calf serum in the concentrations of PRP noted in FIG. 1 that were generated from a healthy donor. Cells were cultured for 48 hours.
- MFI Mean Fluorescent Intensity
- Fibroblasts are cultured with PRP to produce enhanced fibroblasts for therapeutic use.
- Foreskin fibroblasts, human bone marrow MSC and human adipose MSC were obtained from ATCC and cultured in DMEM media containing 10% fetal calf serum in the following concentrations of platelet rich plasma (PRP) generated from a healthy donor. Cells were cultured for 48 hours. Subsequently cells were plated with allogeneic peripheral blood mononuclear cells stimulating with 0.5 micrograms per ml of phytohemagglutinin. Cells were co-cultured for 72 hours with 1 microCurie of tritiated thymidine added in the last 12 hours of culture. Proliferation was assessed by scintillation counting. As observed, addition of PRP uniquely enhanced immune suppressive activity of fibroblasts.
- Fibroblasts are cultured with platelet rich plasma (PRP) to produce enhanced fibroblasts for therapeutic use.
- Foreskin fibroblasts, human bone marrow MSC and human adipose MSC were obtained from ATCC and cultured in DMEM media containing 10% fetal calf serum in the following concentrations of platelet rich plasma (PRP) generated from a healthy donor. Cells were cultured for 48 hours. Subsequently, cells were plated with allogeneic peripheral blood mononuclear cells stimulating with 0.5 micrograms per ml of
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Rheumatology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Physical Education & Sports Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Epidemiology (AREA)
Abstract
Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2019558339A JP7333272B2 (en) | 2017-01-11 | 2018-01-11 | Methods for enhancing fibroblast therapeutic activity |
CA3049768A CA3049768A1 (en) | 2017-01-11 | 2018-01-11 | Methods of enhancing fibroblast therapeutic activity |
CN201880013348.0A CN110337490A (en) | 2017-01-11 | 2018-01-11 | Enhance the method for fibroblast therapeutic activity |
AU2018207541A AU2018207541B2 (en) | 2017-01-11 | 2018-01-11 | Methods of enhancing fibroblast therapeutic activity |
EP18738916.8A EP3568465A4 (en) | 2017-01-11 | 2018-01-11 | Methods of enhancing fibroblast therapeutic activity |
JP2021184431A JP7426368B2 (en) | 2017-01-11 | 2021-11-12 | Methods to enhance fibroblast therapeutic activity |
JP2024007119A JP2024041996A (en) | 2017-01-11 | 2024-01-22 | Methods to enhance fibroblast therapeutic activity |
AU2024201176A AU2024201176A1 (en) | 2017-01-11 | 2024-02-22 | Methods of enhancing fibroblast therapeutic activity |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762445133P | 2017-01-11 | 2017-01-11 | |
US62/445,133 | 2017-01-11 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2018132594A1 true WO2018132594A1 (en) | 2018-07-19 |
Family
ID=62782706
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2018/013357 WO2018132594A1 (en) | 2017-01-11 | 2018-01-11 | Methods of enhancing fibroblast therapeutic activity |
Country Status (7)
Country | Link |
---|---|
US (2) | US11034934B2 (en) |
EP (1) | EP3568465A4 (en) |
JP (3) | JP7333272B2 (en) |
CN (1) | CN110337490A (en) |
AU (2) | AU2018207541B2 (en) |
CA (1) | CA3049768A1 (en) |
WO (1) | WO2018132594A1 (en) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019125996A1 (en) | 2017-12-20 | 2019-06-27 | Figene, Llc | Augmentation of fibroblast regenerative activity |
JP2022506683A (en) * | 2018-11-04 | 2022-01-17 | フィジーン、エルエルシー | Treatment and Composition of Type 1 Diabetes Using Fibroblasts as Facilitator for Pancreas Transplantation |
WO2022016184A1 (en) * | 2020-07-14 | 2022-01-20 | Figene, Llc | Augmentation of fibroblast therapeutic activity by complement blockade and/or inhibition |
JP2022512923A (en) * | 2018-11-04 | 2022-02-07 | フィジーン、エルエルシー | Treatment of cachexia with fibroblasts and their products |
JP2022512963A (en) * | 2018-11-09 | 2022-02-07 | フィジーン、エルエルシー | Regenerative Abscopal effect |
EP3908297A4 (en) * | 2019-01-11 | 2022-06-29 | Figene, LLC | Fibroblast regenerative cells |
EP3962504A4 (en) * | 2019-04-27 | 2022-09-14 | Figene, LLC | Enhancement of fibroblast therapeutic activity by t cell modulation |
EP3911328A4 (en) * | 2019-01-17 | 2022-11-02 | Figene, LLC | Fibroblasts and microvesicles thereof for reduction of toxicity associated with cancer immunotherapy |
EP3873496A4 (en) * | 2018-11-04 | 2022-11-09 | Figene, LLC | Treatment of cerebral hypoxia including stroke, chronic traumatic encephalopathy, and traumatic brain injury |
EP4031167A4 (en) * | 2019-09-16 | 2023-07-19 | Figene, LLC | Treatment of disc degenerative disease and stimulation of proteoglycan synthesis by fibroblast conditioned media and formulations thereof |
EP4045065A4 (en) * | 2019-10-15 | 2023-08-30 | Figene, LLC | Fibroblast-based immunotherapy of graves disease |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110337490A (en) * | 2017-01-11 | 2019-10-15 | 脊核细胞有限责任公司 | Enhance the method for fibroblast therapeutic activity |
CN109136169A (en) * | 2018-08-09 | 2019-01-04 | 上海交通大学医学院附属第九人民医院 | A kind of skin fibroblasts are changed into the system and its application method of artificial intervertebral disk |
JP2022538176A (en) * | 2019-06-28 | 2022-08-31 | フィジーン、エルエルシー | Administration of fibroblasts and their derivatives for the treatment of type 2 diabetes |
US20220370506A1 (en) * | 2019-12-26 | 2022-11-24 | Figene, Llc | Augmentation of fibroblast mediated regeneration of intravertebral discs |
US20230193204A1 (en) * | 2020-04-13 | 2023-06-22 | Figene, Llc | Treatment of erectile dysfunction by fibroblast administration |
US20230181647A1 (en) * | 2020-05-14 | 2023-06-15 | Figene, Llc | Treatment of ovarian failure using regenerative cells |
CN111560346A (en) * | 2020-05-21 | 2020-08-21 | 福建省海西细胞生物工程有限公司 | Method for efficiently extracting and proliferating autologous fibroblasts by explant adhesion method |
CN113755430B (en) * | 2021-09-09 | 2023-08-18 | 北京益华生物科技有限公司 | Method for high expression of AFGF |
CN117660325B (en) * | 2024-01-31 | 2024-04-19 | 苏州科为康生物医药科技有限公司 | Culture medium for preparing umbilical cord blood MSC and method thereof |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5165928A (en) | 1988-11-14 | 1992-11-24 | Cornell Research Foundation, Inc. | Biological control of phytophthora by gliocladium |
US5585007A (en) | 1994-12-07 | 1996-12-17 | Plasmaseal Corporation | Plasma concentrate and tissue sealant methods and apparatuses for making concentrated plasma and/or tissue sealant |
US5599558A (en) | 1989-09-15 | 1997-02-04 | Curative Technologies, Inc. | Selecting amounts of platelet releasate for efficacious treatment of tissue |
US5614204A (en) | 1995-01-23 | 1997-03-25 | The Regents Of The University Of California | Angiographic vascular occlusion agents and a method for hemostatic occlusion |
US6010627A (en) | 1995-06-06 | 2000-01-04 | Quantic Biomedical Partners | Device for concentrating plasma |
US6303112B1 (en) | 1998-06-22 | 2001-10-16 | Cytomedix Inc | Enriched platelet wound healant |
US20010038848A1 (en) * | 2000-02-18 | 2001-11-08 | Donda Russell S. | Implantable tissues infused with growth factors and other additives |
US6461645B1 (en) | 1987-11-12 | 2002-10-08 | Pharmastem Therapeutics, Inc. | Isolation and preservation of fetal and neonatal hematopoietic stem and progenitor cells of the blood |
US6649072B2 (en) | 2001-11-16 | 2003-11-18 | Robert Brandt | Method for producing autologous platelet-rich plasma |
US20100209404A1 (en) * | 2009-02-10 | 2010-08-19 | University Of Dayton | Enhanced method for producing stem-like cells from somatic cells |
US9011929B2 (en) | 2009-10-23 | 2015-04-21 | Sewon Cellontech Co., Ltd. | Composition for inducing tissue regeneration by activating platelet-rich plasma (PRP) |
Family Cites Families (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6328762B1 (en) * | 1999-04-27 | 2001-12-11 | Sulzer Biologics, Inc. | Prosthetic grafts |
US20090074729A2 (en) * | 1999-11-05 | 2009-03-19 | Donald Kleinsek | Augmentation and repair of spincter defects with cells including fibroblasts |
ITMI20021917A1 (en) * | 2002-09-10 | 2004-03-11 | New Dawn Consultores E Servicos L Da | ACTIVATOR FOR THE FORMATION OF PLASTIC GEL, PLASMA GEL POOR OF PLATES OR PLASMA GEL RICH IN PLATES. |
JP2006230418A (en) | 2003-05-23 | 2006-09-07 | M D Bio Inc | Filling material |
US20070122906A1 (en) * | 2003-12-29 | 2007-05-31 | Allan Mishra | Method of culturing cells |
JP4364696B2 (en) | 2004-03-30 | 2009-11-18 | ニプロ株式会社 | Tissue or organ regeneration material |
WO2007001016A1 (en) * | 2005-06-29 | 2007-01-04 | National University Corporation Nagoya University | Dermal tissue improving material and use thereof |
EP3146939B1 (en) | 2006-02-07 | 2018-09-05 | Spinalcyte, LLC | Composition for repair of cartilage using an in vivo bioreactor |
BRPI0710174A2 (en) | 2006-04-19 | 2011-08-23 | Univ Nagoya Nat Univ Corp | composition for periodontal soft tissue regeneration and method for producing it |
US20100239556A1 (en) * | 2006-12-13 | 2010-09-23 | TGR BioScience Pty Ltd. | Promoting ecm production by fibroblast cells and/or promoting migration of fibroblast cells in a biological system |
CN102131529A (en) * | 2008-06-30 | 2011-07-20 | 国立大学法人东京大学 | Bone defect filler not adsorbing bone growth factor and not inhibiting the activity of the same |
ES2653679T3 (en) * | 2011-11-09 | 2018-02-08 | Spinalcyte, Llc | Dermal fibroblasts for the treatment of degenerative disc disease |
CA2881126A1 (en) | 2012-08-10 | 2014-02-13 | Advanced Medical Technologies Llc | Generation of cartilage ex vivo from fibroblasts |
US20140356893A1 (en) * | 2013-06-04 | 2014-12-04 | Allan Mishra | Compositions and methods for using platelet-rich plasma for drug discovery, cell nuclear reprogramming, proliferation or differentiation |
US10206954B2 (en) | 2013-06-19 | 2019-02-19 | Spinalcyte, Llc | Adipose cells for chondrocyte applications |
CA2923857A1 (en) | 2013-09-09 | 2015-03-12 | Figene, Llc | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
CN104611289A (en) * | 2015-01-13 | 2015-05-13 | 广州赛奕德生物技术有限公司 | Method for simultaneously preparing autologous epidermal cells and fibroblasts, and biological beauty product comprising autologous epidermal cells and fibroblasts |
AU2017207445B2 (en) * | 2016-01-14 | 2023-12-07 | Spinalcyte, Llc | Cellular blend for the regeneration of chondrocytes or cartilage type cells |
CN110337490A (en) * | 2017-01-11 | 2019-10-15 | 脊核细胞有限责任公司 | Enhance the method for fibroblast therapeutic activity |
US20200338133A1 (en) | 2017-04-19 | 2020-10-29 | Figene, Llc | Stimulation of angiogenesis by fibroblast derived exosomes |
AU2018375151A1 (en) | 2017-11-29 | 2020-06-11 | Figene, Llc | Interaction of fibroblasts and immune cells for activation and uses thereof |
AU2018388979A1 (en) | 2017-12-20 | 2020-07-09 | Figene, Llc | Augmentation of fibroblast regenerative activity |
-
2018
- 2018-01-11 CN CN201880013348.0A patent/CN110337490A/en active Pending
- 2018-01-11 WO PCT/US2018/013357 patent/WO2018132594A1/en unknown
- 2018-01-11 EP EP18738916.8A patent/EP3568465A4/en active Pending
- 2018-01-11 CA CA3049768A patent/CA3049768A1/en active Pending
- 2018-01-11 JP JP2019558339A patent/JP7333272B2/en active Active
- 2018-01-11 AU AU2018207541A patent/AU2018207541B2/en active Active
- 2018-01-11 US US15/868,420 patent/US11034934B2/en active Active
-
2021
- 2021-05-20 US US17/303,133 patent/US20210309971A1/en active Pending
- 2021-11-12 JP JP2021184431A patent/JP7426368B2/en active Active
-
2024
- 2024-01-22 JP JP2024007119A patent/JP2024041996A/en active Pending
- 2024-02-22 AU AU2024201176A patent/AU2024201176A1/en active Pending
Patent Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6461645B1 (en) | 1987-11-12 | 2002-10-08 | Pharmastem Therapeutics, Inc. | Isolation and preservation of fetal and neonatal hematopoietic stem and progenitor cells of the blood |
US5165928A (en) | 1988-11-14 | 1992-11-24 | Cornell Research Foundation, Inc. | Biological control of phytophthora by gliocladium |
US5599558A (en) | 1989-09-15 | 1997-02-04 | Curative Technologies, Inc. | Selecting amounts of platelet releasate for efficacious treatment of tissue |
US5585007A (en) | 1994-12-07 | 1996-12-17 | Plasmaseal Corporation | Plasma concentrate and tissue sealant methods and apparatuses for making concentrated plasma and/or tissue sealant |
US6214338B1 (en) | 1994-12-07 | 2001-04-10 | Plasmaseal Llc | Plasma concentrate and method of processing blood for same |
US5614204A (en) | 1995-01-23 | 1997-03-25 | The Regents Of The University Of California | Angiographic vascular occlusion agents and a method for hemostatic occlusion |
US6010627A (en) | 1995-06-06 | 2000-01-04 | Quantic Biomedical Partners | Device for concentrating plasma |
US6303112B1 (en) | 1998-06-22 | 2001-10-16 | Cytomedix Inc | Enriched platelet wound healant |
US20010038848A1 (en) * | 2000-02-18 | 2001-11-08 | Donda Russell S. | Implantable tissues infused with growth factors and other additives |
US6649072B2 (en) | 2001-11-16 | 2003-11-18 | Robert Brandt | Method for producing autologous platelet-rich plasma |
US20100209404A1 (en) * | 2009-02-10 | 2010-08-19 | University Of Dayton | Enhanced method for producing stem-like cells from somatic cells |
US9011929B2 (en) | 2009-10-23 | 2015-04-21 | Sewon Cellontech Co., Ltd. | Composition for inducing tissue regeneration by activating platelet-rich plasma (PRP) |
Non-Patent Citations (44)
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3727319A4 (en) * | 2017-12-20 | 2021-08-25 | Figene, LLC | Augmentation of fibroblast regenerative activity |
US11959102B2 (en) | 2017-12-20 | 2024-04-16 | Figene, Llc | Augmentation of fibroblast regenerative activity |
WO2019125996A1 (en) | 2017-12-20 | 2019-06-27 | Figene, Llc | Augmentation of fibroblast regenerative activity |
EP4249586A3 (en) * | 2017-12-20 | 2023-11-29 | Figene, LLC | Augmentation of fibroblast regenerative activity |
EP3873496A4 (en) * | 2018-11-04 | 2022-11-09 | Figene, LLC | Treatment of cerebral hypoxia including stroke, chronic traumatic encephalopathy, and traumatic brain injury |
JP2022506683A (en) * | 2018-11-04 | 2022-01-17 | フィジーン、エルエルシー | Treatment and Composition of Type 1 Diabetes Using Fibroblasts as Facilitator for Pancreas Transplantation |
JP2022512923A (en) * | 2018-11-04 | 2022-02-07 | フィジーン、エルエルシー | Treatment of cachexia with fibroblasts and their products |
JP2022512963A (en) * | 2018-11-09 | 2022-02-07 | フィジーン、エルエルシー | Regenerative Abscopal effect |
EP3908297A4 (en) * | 2019-01-11 | 2022-06-29 | Figene, LLC | Fibroblast regenerative cells |
EP3911328A4 (en) * | 2019-01-17 | 2022-11-02 | Figene, LLC | Fibroblasts and microvesicles thereof for reduction of toxicity associated with cancer immunotherapy |
EP3962504A4 (en) * | 2019-04-27 | 2022-09-14 | Figene, LLC | Enhancement of fibroblast therapeutic activity by t cell modulation |
EP4031167A4 (en) * | 2019-09-16 | 2023-07-19 | Figene, LLC | Treatment of disc degenerative disease and stimulation of proteoglycan synthesis by fibroblast conditioned media and formulations thereof |
US11732239B2 (en) | 2019-09-16 | 2023-08-22 | Figene, Llc | Treatment of disc degenerative disease and stimulation of proteoglycan synthesis by fibroblast conditioned media and formulations thereof |
EP4045065A4 (en) * | 2019-10-15 | 2023-08-30 | Figene, LLC | Fibroblast-based immunotherapy of graves disease |
WO2022016184A1 (en) * | 2020-07-14 | 2022-01-20 | Figene, Llc | Augmentation of fibroblast therapeutic activity by complement blockade and/or inhibition |
Also Published As
Publication number | Publication date |
---|---|
EP3568465A4 (en) | 2021-01-27 |
JP7426368B2 (en) | 2024-02-01 |
US20210309971A1 (en) | 2021-10-07 |
CA3049768A1 (en) | 2018-07-19 |
JP7333272B2 (en) | 2023-08-24 |
US20180195044A1 (en) | 2018-07-12 |
US11034934B2 (en) | 2021-06-15 |
AU2018207541A1 (en) | 2019-08-01 |
JP2024041996A (en) | 2024-03-27 |
EP3568465A1 (en) | 2019-11-20 |
AU2018207541B2 (en) | 2023-12-21 |
CN110337490A (en) | 2019-10-15 |
JP2020503901A (en) | 2020-02-06 |
JP2022024047A (en) | 2022-02-08 |
AU2024201176A1 (en) | 2024-03-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210309971A1 (en) | Methods of enhancing fibroblast therapeutic activity | |
US10675307B2 (en) | Compositions comprising perivascular stem cells and nell-1 protein | |
Coleman et al. | Mesenchymal stem cells and osteoarthritis: remedy or accomplice? | |
US9598673B2 (en) | Treatment of disc degenerative disease | |
US20100015104A1 (en) | Generation of adipose tissue and adipocytes | |
Neumann et al. | Chondrogenesis of human bone marrow-derived mesenchymal stem cells is modulated by complex mechanical stimulation and adenoviral-mediated overexpression of bone morphogenetic protein 2 | |
US9011840B2 (en) | Activated mesenchymal stem cells for wound healing and impaired tissue regeneration | |
US20200078411A1 (en) | Biological Scaffolds, Products Containing Biological Scaffolds and Methods of Using the Same | |
KR101389852B1 (en) | Method for Culture of Skeletal Muscle Stem Cells and Uses Therefor | |
Shi et al. | Therapeutic effects of cell therapy with neonatal human dermal fibroblasts and rabbit dermal fibroblasts on disc degeneration and inflammation | |
EP2845898B1 (en) | Method for culturing neural crest stem cells, and use thereof | |
Moncada-Saucedo et al. | A Bioactive Cartilage Graft of IGF1‐Transduced Adipose Mesenchymal Stem Cells Embedded in an Alginate/Bovine Cartilage Matrix Tridimensional Scaffold | |
O'HEERON et al. | Patent 3049768 Summary | |
Zhang et al. | Lentivirus is an efficient and stable transduction vector for intervertebral disc cells | |
WO2021039882A1 (en) | Method for culturing tie2-positive stem/progenitor cell-containing cell population using culture substrate, and utilization thereof | |
US20230372402A1 (en) | Cytokine primed regenerative cells for treatment of ovarian failure | |
Yang et al. | Application of human umbilical cord mesenchymal stem cells in spinal cord injury | |
Zhu et al. | Reconstruction of rabbit degenerated intervertebral discs using SOX9 gene-modified bone marrow mesenchymal stem cells | |
CA3156239A1 (en) | Cannabidiol adjuvant therapy for treatment of disc degenerative disease | |
Tierney | The effect of peroneal nerve relocation on skeletal muscle regeneration within an extracellular matrix seeded with mesenchymal stem cell populations derived from bone marrow and adipose tissue | |
KR20210046196A (en) | Mesenchymal stem cell originated from equine amniotic membrane and its use |
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: 18738916 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 3049768 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2019558339 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2018207541 Country of ref document: AU Date of ref document: 20180111 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2018738916 Country of ref document: EP Effective date: 20190812 |