WO2015171144A1 - Produits dérivés d'une membrane amniotique immunocompatible - Google Patents

Produits dérivés d'une membrane amniotique immunocompatible Download PDF

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Publication number
WO2015171144A1
WO2015171144A1 PCT/US2014/037208 US2014037208W WO2015171144A1 WO 2015171144 A1 WO2015171144 A1 WO 2015171144A1 US 2014037208 W US2014037208 W US 2014037208W WO 2015171144 A1 WO2015171144 A1 WO 2015171144A1
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Prior art keywords
membrane
cells
tissue
wound
amniotic membrane
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PCT/US2014/037208
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English (en)
Inventor
Samson Tom
Alla Danilkovitch
Dana Yoo
Timothy Jansen
Jin-Qiang Kuang
Jennifer Michelle Marconi
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Osiris Therapeutics, Inc.
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Priority to SG11201609255XA priority Critical patent/SG11201609255XA/en
Priority to CA2948133A priority patent/CA2948133A1/fr
Priority to EP14891554.9A priority patent/EP3139936A4/fr
Priority to KR1020167034202A priority patent/KR20170002572A/ko
Priority to AU2014393404A priority patent/AU2014393404A1/en
Priority to PCT/US2014/037208 priority patent/WO2015171144A1/fr
Priority to JP2016567041A priority patent/JP2017514879A/ja
Publication of WO2015171144A1 publication Critical patent/WO2015171144A1/fr

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    • 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
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • 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

  • the present technology relates to methods and products that facilitate or improve wound healing including, for example placenta membrane-derived products and cryopreserved products, and methods that promote healing in a wound or near or at the site of a wound such as combinations of one or more of the following: stimulation of angiogenesis, secretion of growth factors, inhibition of proteases and free radical oxidation.
  • the present technology relates to products to protect injured or damaged tissue, or as a covering to prevent adhesions, to exclude bacteria, to inhibit bacterial activity, or to promote healing or growth of tissue.
  • An example of such a placental membrane is an amniotic membrane.
  • the field also relates to methods of manufacturing and methods of use of such membrane-derived products.
  • the human amniotic membrane is the innermost of the fetal membranes deriving from the amniotic sac and constituting the lining of the amniotic cavity. It is approximately 0.02 to 0.5 mm thick.
  • the AM consists of five layers: a single layer of epithelial cells rests on the basement membrane and contacts the amniotic fluid.
  • An underlying layer of connective tissue is attached to the basement membrane.
  • the connective tissue is comprised of three structural layers: a compact layer, a fibroblast layer (sometimes referred to as a mesenchymal layer), and a spongy layer.
  • the spongy layer is adjacent to the chorion.
  • the amnion is essentially devoid of vasculature.
  • the AM contains a number of factors that can contribute to wound healing such as, for example, extracellular matrix, growth factors, and viable cells. While some preserving methods can maintain some level of factors such as matrix or growth factors, preserving levels of viable cells presents a challenge.
  • fresh AM When fresh AM is used, there is increased risk of disease transmission. According to published reports, fresh amniotic tissue exhibits high cell viability (e.g., greater than 70%), however cell viability rapidly diminishes during storage (e.g., within 28 days at temperatures above 0°C), dropping the amount of viable cells to ranges of about 15%-35%. Freezing for a period of time (e.g., 3 weeks) was also shown to reduce cell viability to ranges of about 13% to 18%, regardless of the temperature or medium.
  • Gajiwala and Gajiwala report the preservation of AM by freeze-drying (lyophilisation) and gamma-irradiation sterilization.
  • AM is pasteurized at 60 °C, treated with 70% ethanol, and freeze-dried to remove most of the remaining moisture.
  • the AM is sterilized by exposure of 25 kGy gamma-radiation in a Cobalt 60 Gamma chamber unit or at an ISO-certified radiation plant.
  • the sterilized AM can be stored at room temperature for up to 6 months.
  • Apligraf and Dermagraft comprise cultured-expanded cells.
  • Apligraf and Dermagraft comprise detectable levels of certain factors such as, for example, Insulin-like Growth Factor Binding Protein-1 (IGFBP-1 ) and adiponectin, which are factors associated with the natural wound healing process.
  • IGFBP-1 Insulin-like Growth Factor Binding Protein-1
  • TIMPS tissue inhibitor of matrix metalloproteinase
  • wound healing is a multi-factorial biological process, many factors are needed to properly treat a wound; products having lower amounts of viable cells and limited diversity compared to the cells present in skin are less capable of healing wounds relative to a product having an increased population of viable cells and increased number of types of cells found in tissues. It would represent an advance in the art to provide an amnion-derived product that can be used in applications such as wound healing, wound dressings, cosmetic uses, or as a biologic skin substitute comprising a diverse population of cells representing a higher percentage of viable cells and an increased amount of factors, including, for example, growth factors, antioxidant agents, antiinflammatory agents, agents that promote angiogenesis and cytokines.
  • Apligraf is a living, bi-layered skin substitute manufactured using neonatal foreskin keratinocytes and fibroblasts combined with bovine Type I collagen. As used in this application, Apligraf refers to the product available for commercial sale as approved by the FDA in 1998.
  • Dermagraft is cryopreserved human fibroblasts derived from newborn foreskin tissue seeded on a synthetic extracellular matrix, and a bioabsorbable polyglactin mesh scaffold. According to its product literature, Dermagraft requires three washing steps before use which limits the practical implementation of Dermagraft as a wound dressing relative to products that require less than three washing steps. As used in this application, Dermagraft refers to the product available for commercial sale as approved by the FDA in 2001 .
  • Engineered wound dressings such as Apligraf and Dermagraft do not provide the best potential for wound healing because they comprise sub-optimal cellular compositions and therefore do not provide proper wound healing.
  • Apligraf and Dermagraft do not comprise MSCs or inherent tissue extracellular matrix and, as a result, the ratio between different factors secreted by cells does not enable efficient wound healing.
  • some factors that are important for wound healing including EGF, IGFBP1 , and adiponectin, are not detectable or are absent from both Apligraf and Dermagraft.
  • the matrix composition in these bioengineered products includes only Collagen type I and may also include hyaluronic acid. This differs from the complex structural matrix of skin which includes components such as various collagens (e.g., collagens I, III, IV, V, VI, etc.), elastin, glycoproteins and proteoglycans. Skin also includes mesenchymal stem cells in the dermis, which are lacking in the representative examples of bioengineered products, Apligraf and Dermagraft.
  • MMPs and TIMPs are among the factors that are important for wound healing. However, expression of these proteins must be highly regulated and coordinated. Excess of MMPs versus TIMPs is a marker of poor chronic wound healing (Liu et al, Diabetes Care, 2009, 32: 1 17; Mwaura et al, Eur J Vase Endovasc Surg, 2006, 31 : 306; Trengove et al, Wound Rep Reg, 1999, 7: 442; Vaalamo et al, Hum Pathol, 1999, 30: 795).
  • a2-macroglobulin and/or its receptor is known as a plasma protein that inactivates proteases from all four mechanistic classes: serine proteases, cysteine proteases, aspartic proteases, and metalloproteases (Borth et al., FASEB J, 1992,6: 3345; Baker et al., J Cell Sci, 2002, 1 15:3719).
  • Another important function of this protein is to serve as a reservoir for cytokines and growth factors, examples of which include TGF, PDGF, and FGF (Asplin et al, Blood, 2001 , 97: 3450; Huang et al, J Biol Chem, 1988; 263: 1535).
  • An in vitro cell migration assay is important for assessing the wound healing potential of a product.
  • the process of wound healing is highly complex and involves a series of structured events controlled by growth factors (Goldman, Adv Skin Wound Care, 2004, 1 :24). These events include increased vascularization, infiltration by inflammatory immune cells, and increases in cell proliferation.
  • the beginning stages of wound healing revolve around the ability of individual cells to polarize towards the wound and migrate into the wounded area in order to close the wound area and rebuild the surrounding tissue.
  • An assay capable of evaluating the wound healing potential of wound therapies by examining the correlation between cell migration and wound healing would represent an advance in the art.
  • aspects of the present technology represent a significant advance in the art as they relate to products and methods that promote angiogenesis, promote anti-inflammatory activity, promote antioxidant activity, and provide for increased amounts and varieties of growth factors. As discussed in more detail below, these products and methods can be used in any number of wound healing applications, soft tissue repair, or osteogenic repair.
  • the disclosure provides a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than about 40% of said tissue cells are viable;
  • the disclosure provides a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • C) depleted amounts of one or more types of functional immunogenic cells, wherein greater than 40% of the combined cells in the stromal layer and epithelial layer are viable cells.
  • the disclosure provides a membrane comprising cryopreserved amniotic membrane having one or more tissue components, wherein after cryopreservation and subsequent thawing the one or more tissue components comprise:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a membrane comprising cryopreserved amniotic membrane having one or more tissue components, wherein after cryopreservation and subsequent thawing the one or more tissue components comprise:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • amniotic membrane has depleted amounts of functional immunogenic cells, and wherein the one or more tissue components is present in an amount effective to:
  • the disclosure provides a membrane comprising cryopreserved placental membrane, wherein after cryopreservation and subsequent thawing the placental membrane comprises:
  • tissue cells wherein said tissue cells are native to the placental membrane and greater than 40% of said tissue cells are viable;
  • the placental membrane comprises amniotic membrane and chorionic membrane.
  • the depleted amounts of functional immunogenic cells produce immunogenic factors in amounts that are below levels sufficient to produce an immune response. In some embodiments, the depleted amounts of functional immunogenic cells produce immunogenic factors in amounts below detectable limits.
  • the membrane comprises tissue cells wherein about 50% to about 100% of said tissue cells are viable. In some embodiments, about 60% to about 100% of said tissue cells are viable. In further embodiments, about 70% to about 100% of said tissue cells are viable.
  • the membranes provide one or more tissue components (e.g., viable cells, one or more therapeutic factors, and/or extracellular matrix) in an amount that is effective to promote any of the activities of (i) - (viii) in vitro or in vivo.
  • tissue components e.g., viable cells, one or more therapeutic factors, and/or extracellular matrix
  • Various embodiments of the above-described aspects may further comprise a delivery substrate, such that the membrane is fixed to the delivery substrate.
  • the membrane may be stored for an extended period of time prior to subsequent thawing. In some embodiments the extended period of time is from about 6 months to about 25 months or more. In these embodiments, the viability of the tissue cells is substantially maintained upon thawing. In some embodiments, the viability of the tissue cells is substantially maintained for at least about 25 months or more when stored frozen.
  • the membrane can be thawed and ready for use within 30 minutes of the start of a thawing method.
  • the membrane can be stored in saline up to an hour after thawing and still maintain about 70% viable cells.
  • the disclosure provides a method of treating a wound on a subject comprising administering a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a method for accelerating wound healing comprising administering a membrane according to any of the aspects and embodiments described herein.
  • the administering is effective to promote wound closure by 12 weeks after an initial administering step.
  • the administering is effective to promote wound closure by 5-6 weeks after an initial administering step.
  • the administering is effective to promote reduction in wound size by 50% or more 28 days after an initial administering step.
  • the administering is effective to improve wound closure rate by at least about 44% relative to standard wound treatment.
  • the disclosure provides method of treating a subject for a wound that is refractory to a prior wound healing treatment, the method comprising administering to the site of the wound a membrane according to any of the aspects and embodiments described herein.
  • the administering is effective to promote wound closure by 12 weeks after an initial administering step.
  • the administering is effective to promote wound closure by 5-6 weeks after an initial administering step.
  • the administering is effective to promote reduction in wound size by 50% or more 28 days after an initial administering step.
  • the disclosure provides a method for treating a chronic wound comprising administering a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises: A) tissue cells, wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a method for treating an acute wound comprising administering a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the wound may be selected from the group consisting of lacerations, scrapes, burns, incisions, punctures, wound caused by a projectile, an epidermal wound, skin wound, chronic wound, acute wound, external wound, internal wound, congenital wound, ulcer, pressure ulcer, diabetic ulcer, tunnel wound, wound caused during or as an adjunct to a surgical procedure, venous skin ulcer, and avascular necrosis.
  • the membrane may either directly or may indirectly promote one or more of: (i) a reduction of the amount and/or activity of pro-inflammatory cytokines; (ii) an increase in the amount and/or activity of antiinflammatory cytokines; (iii) a reduction of the amount and/or activity of reactive oxygen species; (iv) an increase in the amount and/or activity of antioxidant agents; (v) a reduction of the amount and/or activity of proteases; (vi) an increase in cell proliferation; (vii) an increase in angiogenesis; and/or (viii) an increase in cell migration.
  • the disclosure provides a method of treating an inflammatory ocular condition in a subject comprising administering to the subject a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the inflammatory ocular condition is a wound or a disease characterized by inflammation.
  • the disclosure provides a method of promoting tissue repair and/or tissue regeneration in a subject comprising administering to the subject a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the method is used in combination with a surgical procedure selected from the group consisting of a tissue graft procedure, tendon surgery, ligament surgery, bone surgery, and spinal surgery.
  • the tissue is human tissue.
  • the human tissue is cartilage, skin, ligament, tendon, or bone.
  • the membrane may directly or indirectly stimulate tissue regeneration.
  • the disclosure provides a method of modulating inflammatory response comprising administering to the wound a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a method of modulating protease activity comprising administering to the wound a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a method of reducing the amount and/or activity of reactive oxygen species (ROS) and increasing the amount and/or activity of antioxidant agents comprising administering to the wound a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • ROS reactive oxygen species
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable; B) one or more therapeutic factors that are native to the amniotic membrane;
  • the disclosure provides a method of increasing angiogenesis comprising administering to wound a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a method of increasing cell migration comprising administering to wound a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a method of increasing cell proliferation comprising administering to wound a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a method of preventing or reducing scar or contracture formation in a subject comprising administering to the subject a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a method of treating or preventing tissue adhesion associated with a surgical procedure comprising administering a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the membrane is administered to the tissue and provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to treat or prevent tissue adhesion.
  • any of the above aspects relating to methods of modulating inflammatory response, modulating protease activity, reducing the amount and/or activity of reactive oxygen species, increasing the amount and/or activity of antioxidant agents, increasing/promoting angiogenesis, increasing cell migration, increasing cell proliferation, or preventing or reducing scar or contracture formation the administration of the membrane may directly or indirectly stimulate or induce the method.
  • kits for treating a wound or a tissue defect comprising:
  • the kit may further comprise an additive.
  • the additive may be selected from one or more antibiotics, emollients, keratolytic agents, humectants, antioxidants, preservatives, therapeutics, bandages, tools, cutting device, buffer, thawing medium, handling media, forceps, container and combinations thereof.
  • FIG. 1 depicts freezing rates of various freezing methods and various amounts of cryopreservation solution.
  • FIG. 1 A-1 D involved a 30 minute refrigeration step at 4°C before freezing at -80 °C.
  • FIG. 1 E-1 H show direct freezing at -80 °C.
  • FIG. 2 depicts cell process recovery (A) and cell viability (B) of amniotic membrane when stored with various amounts of cryopreservation solution.
  • FIG. 3 depicts cell process recovery (A) and cell viability (B) of the chorionic membrane when stored with various amounts of cryopreservation solution.
  • FIG. 4 shows the effect of cryopreservation on cell viability of chorioamniotic membrane.
  • FIG. 5 depicts the effects of refrigeration time and freezing parameters on process (cryopreservation) cell recovery of amniotic (A) and chorionic (B) membrane.
  • FIG. 6 depicts the effects of refrigeration time and freezing parameters on cell viability for amniotic membrane.
  • FIG. 7 shows representative images of fresh amniotic (A and C) and chorionic (E) membranes as well as cryopreserved amniotic (B and D) and chorionic (F) membranes stained with live/dead stain. Live cells appear green, while dead cells appear red.
  • FIG. 8 depicts results from Mixed Lymphocyte Reaction (MLR) assay which measures expression of IL-2Ra from T-cells stimulated by various placental membranes from manufacturing intermediates - trophoblast, choriotrophoblast (CT), amnion with choriotrophoblast (ACT), amnion (AM), chorion (CM), and amnion with chorion (A/C).
  • MLR Mixed Lymphocyte Reaction
  • FIG. 9 depicts results from MLR assay which measures expression of IL-2Ra from T-cells stimulated by various placental membranes from manufacturing intermediates and placental membranes after cryopreservation - Amnion (AM), Chorion (CM), Amnion with Chorion (A/C).
  • AM Amnion
  • CM Chorion
  • A/C Amnion with Chorion
  • FIG. 10A and B depict lipopolysaccharide (LPS) stimulated TNF-a released from various membrane preparations - Amnion+Chorion+Trophoblast (ACT), Chorion+Trophoblast (CT), Trophoblast (T), Amnion (AM), and Chorion (CM).
  • LPS lipopolysaccharide
  • FIG. 1 1 shows expression of IL-2Ra from T-cells stimulated by choriotrophoblast (CT) which secreted high levels of TNF-a.
  • CT choriotrophoblast
  • FIG. 12 shows images of cultured cells isolated from amniotic (A) and chorionic membranes (B), demonstrating plastic adherence. MSC isolated from human bone marrow aspirate are shown for comparison (C). Osteogenic potential of placental-derived cells is illustrated by purple stain for alkaline phosphatase (D).
  • FIG. 13 depicts expression of VEGF in fresh amniotic membrane as compared to cryopreserved amniotic membrane.
  • FIG. 14A depicts expression of IFN-2a and TGF-33 in amniotic membrane homogenates.
  • FIG. 14 B depicts expression of bFGF in amniotic membrane (AM) and chorionic membrane (CM) extracts.
  • FIG. 15 depicts expression of BMP-2, BMP-4, PLAB, PIGF (A), and IGF-1 (B) in amniotic membrane homogenates.
  • FIG. 16 depicts expression of EGF (A), IGFBP1 (B), and Adiponectin (C) in amniotic (AM), chorionic membranes (CM), and commercially available products.
  • FIG. 17 depicts the ratio of MMPs to TIMPs in amniotic, chorionic, and commercially available products.
  • FIG. 18 depicts expression of EGF in amniotic and chorionic membranes measured by ELISA in two separate placenta donors.
  • FIG. 19 shows that cryopreserved amniotic membranes produce high levels of anti-inflammatory cytokine PGE-2 when exposed to TNF-a.
  • FIG. 20 shows that cryopreserved amniotic membranes inhibit release of soluble pro-inflammatory cytokines such as IL-1 a (A) and TNF-a (B) and upregulate the release of anti-inflammatory IL-10 (C) when co-cultured with activated immune cells.
  • soluble pro-inflammatory cytokines such as IL-1 a (A) and TNF-a (B)
  • C anti-inflammatory IL-10
  • FIG. 21 shows that amniotic membrane products exhibit a statistically significant ( * p ⁇ 0.05) ability to inhibit MMP activity as seen by the reduction in purple dye released.
  • FIG. 22 depicts inhibition of elastase by cryopreserved amniotic membranes.
  • FIG. 23 shows antioxidant capacity of cryopreserved amniotic membrane and ascorbic acid (a potent antioxidant).
  • FIG. 24 shows that cryopreserved amniotic membranes are able to rescue early- stage apoptotic Human Dermal Fibroblasts (HDFs). Apoptotic cells appear as bright blue dots due to their condensation of chromatin and nuclear fragmentations.
  • HDFs Human Dermal Fibroblasts
  • FIG. 25 shows that cryopreserved amniotic membranes promote Human Umbilical Vein Endothelial Cells (HUVECs) to form tubes and the number of formed tubes is significantly greater than negative control.
  • HUVECs Human Umbilical Vein Endothelial Cells
  • FIG. 26 depicts the Cell Biolabs 24-well Cytoselect wound healing assay.
  • FIG. 27 depicts representative images of Human Dermal Microvascular Endothelial Cells (HMVECs) treated with 5% conditioned media from amniotic, chorionic, or a combination of amniotic/chorionic preparations.
  • HMVECs Human Dermal Microvascular Endothelial Cells
  • FIG. 28 depicts the promotion of endothelial cell migration by cryopreserved amniotic membrane (Amnion).
  • FIG. 29 depicts the promotion of fibroblast cell migration by cryopreserved amniotic membrane (Amnion).
  • FIG. 30 depicts the promotion of diseased keratinocyte migration by cryopreserved amniotic membrane (Amnion).
  • FIG. 31 A-E depicts the remarkable efficacy of placental products for treating diabetic foot ulcers in patient 1 .
  • FIG. 32A-D depicts the remarkable efficacy of placental products for treating diabetic foot ulcers in patient 2.
  • FIG. 34 depicts a Kaplan-Meier analysis showing a statistically greater probability of complete wound healing during the 12 week evaluation period for placental product compared to control (p ⁇ 0.0001 ).
  • FIG. 37A-F depicts a general overview of a method for surgical repair of a tendon that incorporates a cryopreserved membrane disclosed herein.
  • FIG. 38A-C depicts a general overview of a method for surgical repair of tendinosis that incorporates a cryopreserved membrane disclosed herein.
  • Choonic tissue or “Chorionic membrane” means the chorion or a portion thereof from placental tissue, e.g. the trophoblast, the somatic mesoderm, or combinations thereof.
  • Amniotic tissue or “Amniotic membrane” means the amnion or a portion thereof from placental tissue, e.g., the epithelium layer; the basement membrane; the compact layer; the fibroblast layer; and the intermediate (spongy) layer.
  • Placental membrane means the instant placental products and membranes disclosed and claimed herein. The term includes, and can be used interchangeably with, terms including placental membrane, cryopreserved placental membrane, cryopreserved chorioamniotic membrane, cryopreserved chorionic membrane, and cryopreserved amniotic membrane. Placental products may be used for tissue regeneration and wound repair.
  • Membranes and placental products that are "depleted of immunogenicity,” “depleted of immunogenic cells” or “depleted of immunogenic factors” or membranes and placental products that contain “depleted amounts of functional immunogenic cells” or “depleted amounts of one or more types of functional immunogenic cells” generally refer to a one or more placental product of the present technology that retains live therapeutic cells and/or retains therapeutic efficacy for the treatment of tissue injury (or defect) yet is free, substantially free, or depleted of at least one immunogenic cell type (e.g.
  • CD14+ macrophages, trophoblasts, and/or maternal blood cells and/or immunogenic factor that are otherwise present in a native placenta, amniotic membrane, or chorionic membrane.
  • a membrane (like those of the presently described technology) that is free, substantially free, or depleted of immunogenic cell types and/or immunogenic factors includes membranes that may retain some amount of immunogenic cells/factors but the retained amount is at a level that is insufficient to produce a functional response (e.g., below detectable amounts, in negligible amounts, in amounts insufficient to produce a functional immune response).
  • ECM Extracellular matrix
  • tissue such as, for example, placental tissues including amniotic membrane, chorionic membrane, and/or chorioamniotic membrane.
  • the term can include structural components of the ECM, such as collagens, laminins, fibronectin, hyaluronan, dermatan sulfate, heparin sulfate, chondroitin sulfate, decorin, and elastin, as well as soluble/functional therapeutic factors that may be present in the ECM (e.g., including proteins and fragments thereof).
  • MSC means mesenchymal stem cells and include fetal, neonatal, adult, or postnatal.
  • MSCs include amniotic MSCs (AMSCs) and chorionic MSCs (CMSCs).
  • MSCs generally express one or more of CD73, CD70, CD90, CD105, and CD166; and generally do not express CD45 and CD34.
  • MSCs differentiate into mesodermal lineages (osteogenic, chondrogenic, and adipogenic).
  • tissue cells means cells that are native, resident, or endogenous to the placental membrane, i.e. cells that are not exogenously added to the placental membrane, including amniotic and chorionic membranes.
  • Native factors means placental membrane factors that are native, resident, or endogenous to the placental membrane, i.e. factors that are not exogenously added to the placental membrane.
  • Therapeutic cells, " or “beneficial cells” include cells and components present in the stromal layer and/or the epithelial layer of the placental membrane, and include, for example, MSCs, fibroblasts, and/or epithelial cells.
  • Therapeutic factors means placenta-, chorionic membrane-, or amniotic membrane-derived factors that promote wound healing.
  • Therapeutic factors also encompass molecules that may be classified as cell growth factors/proteins, tissue repair factors/proteins, as well as other factors and proteins that generally promote wound healing.
  • Non-limiting examples of therapeutic factors include antimicrobial factors, chemoattractants, remodeling proteins such as proteases and protease inhibitors, immunoregulatory factors, chemokines, cytokines, growth factors and other factors.
  • Therapeutic factors also include factors that promote angiogenesis, cell proliferation and epithelialization.
  • Non-limiting examples of such factors include TGFa, TGFpi , TGFp2, TGFp3, EGF, HB-EGF, VEGF, VEGF-C, VEGF-D, HGF, PDGF-AA, PDGF-AB, PDGF-BB, PLGF, PEDF, Ang-2, IGF, IGFBP1 , IGFBP2, IGFBP3, adiponectin, a2-macroglobulin, FGFs (e.g., FGF-2/bFGF, KGF, KDG/FGF-7), matrix metalloproteinases (e.g., MMP-1 , MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13), tissue inhibitors of metalloproteinases (e.g., TIMP1 , TIMP2), thrombospodins (e.g., TSP1 , TSP2), fibronectin, IL-1 RA, NGAL, defen
  • Nemal cells refers to a mixed population of cells present (optionally in native proportions) composed of neonatal mesenchymal stem cells and neonatal fibroblasts. Both neonatal mesenchymal stem cells and neonatal fibroblasts are immunoprivileged; neither express surface proteins present on immunogenic cell types that trigger an immune response.
  • “Stromal layer” refers to the layers in the placental membrane that do not contain the epithelial layer.
  • cryopreservation agent or “cryopreservative” or “cryoprotectant” are used interchangeably herein and are substances that help to prevent damage (e.g., cellular damage) during the freezing process.
  • Suitable cryopreservation agents include, but are not limited to, Dimethyl Sulfoxide (DMSO), a glycerol, a glycol, a propylene glycol, an ethylene glycol, propanediol, polyethylene glycol (PEG), 1 ,2-propanediol (PROH) or a combination thereof.
  • cryopreservation agents include, for example, one or more non-cell permeating cryopreservatives selected from, for example, polyvinyl pyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide, an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinyl pyrrolidone, hydroxyethyl strarch, autologous plasma or a combination thereof.
  • cryopreservatives selected from, for example, polyvinyl pyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide, an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinyl pyrrolidone, hydroxyethyl strarch, autologous plasma or a combination thereof.
  • cryopreservation solution refers to a composition comprising at least one cryopreservation agent.
  • a cryopreservation solution or media may contain further components, for example, serum albumin, pharmaceutically acceptable carriers, buffers, electrolyte solutions, or saline (e.g., phosphate buffer saline).
  • the cryopreservation solution or media may be a solution, a slurry, suspension, etc.
  • the technology described herein provides for placental products comprising manipulated placental tissues.
  • the placental products can include cryopreserved amniotic membranes, cryopreserved chorionic membranes, and/or cryopreserved chorioamniotic membranes.
  • the cryopreservation methods retain high amounts of viable placental cells (i.e., cells that are native to the placental tissue(s)) and provide for the depletion of immunogenic cells and factors associated with immunogenic cells.
  • the disclosure relates to placental products, and particularly membranes comprising cryopreserved amniotic, chorionic, and/or chorioamniotic membranes that comprise a combination of viable cells, therapeutic factors, extracellular matrix, and reduced immunogenicity, which find use in any number of beneficial therapeutic methods.
  • the membranes can be applied to a wound or a tissue defect, and provide amounts of viable cells, therapeutic factors, extracellular matrix that can directly or indirectly induce a change in the region to which the membrane is applied (e.g., an adaptive medicine).
  • the membranes can provide for improved healing of wounds, such as chronic wounds by providing viable cells, therapeutic factors, and extracellular matrix in amounts that can provide or promote the normal stages of wound healing by any of or more of promoting: (i) a reduction of the amount and/or activity of proinflammatory cytokines; (ii) an increase in the amount and/or activity of anti-inflammatory cytokines; (iii) a reduction of the amount and/or activity of reactive oxygen species; (iv) an increase in the amount and/or activity of antioxidant agents; (v) a reduction of the amount and/or activity of proteases; (vi) an increase in cell proliferation; (vii) an increase in angiogenesis; and/or (viii) an increase in cell migration.
  • a chronic wound environment can include any one or more of 1 ) high levels of proinflammatory cytokines, 2) low levels of anti-inflammatory cytokines, 3) high levels of proteases and low levels of their inhibitors, as well as 4) high levels of oxidants and low levels of antioxidant to counter balance, the characteristics and functionality of the cryopreserved membranes disclosed herein are well suited to such applications.
  • the present technology discloses placental products for clinical use, including use in wound healing such as diabetic foot ulcers, venous leg ulcers, and burns.
  • the manufacturing process optionally eliminates essentially all potentially immunogenic cells from the placental membrane while preserving specific cells that play an important role in wound healing.
  • the present technology discloses a placental product that is selectively devitalized.
  • the placental product may be selectively depleted of substantially all immunogenic cells.
  • the membranes do not contain culture expanded cells.
  • the present technology provides a placental product that comprises at least one therapeutic factor, or a combination of any two or more therapeutic factors that are disclosed herein or otherwise known such as, for example, Insulin-like Growth Factor Binding Protein 1 (IGFBP1 ) or adiponectin.
  • the placental product may comprise Epidermal Growth Factor (EGF) and/or IGFBP1 .
  • the disclosure provides a placental product that can be used to increase the anti-inflammatory activity.
  • the disclosure provides a placental product that can be used to increase the antioxidant activity.
  • the disclosure provides a placental product that can be used to reduce adhesion and fibrosis and generally promote anti- scarring activity.
  • the disclosure provides a placental product that can be used to increase cell migration.
  • the disclosure provides a placental product that can be used to increase the formation of vasculature.
  • the present technology discloses a method of cryopreserving a placental product that preserves the viability of specific beneficial cells that are a primary source of factors for the promotion of wound healing while selectively depleting immunogenic cells (e.g. killing or rendering non-immunogenic).
  • the present technology discloses a bioassay to test immunogenicity of manufactured placental products.
  • the present technology discloses a placental product exhibiting a therapeutic ratio of MMP to TIMP comparable to that exhibited in vivo.
  • the present inventors have identified a need for the development of placental products exhibiting a ratio of MMP-9 and TIMP1 of about 7-10 to one.
  • the present technology discloses a placental product that comprises a2-macroglobulin.
  • a placental product that is capable of inactivating serine proteases, cysteine proteases, aspartic proteases, and metalloproteases.
  • a placental product that inactivates serine proteases there is now provided a placental product that inactivates cysteine proteases.
  • a placental product that inactivates aspartic proteases there is now provided a placental product that inactivates metalloproteases.
  • the present technology discloses a placental product that comprises bFGF.
  • the present technology discloses a placental product exhibiting a MMP to TIMP ratio favorable for wound healing.
  • the present technology discloses a cell migration assay capable of evaluating the wound-healing potential of a placental product.
  • IGFBP1 and adiponectin are among the factors contemplated herein that are important for wound healing. Evaluation of proteins derived from placental products prepared according to the presently disclosed technology reveal that EGF is one of the major factors secreted in higher quantities by these tissues. Additionally, the importance of EGF for wound healing together with high levels of EGF detected in the presently disclosed amniotic membranes support selection of EGF as a potency marker for evaluation of membrane products manufactured for clinical use pursuant to the present disclosure.
  • the present technology discloses a cryopreservation procedure for a placental product that selectively depletes immunogenic cells and preserves the viability of other beneficial cells (including at least one or more of mesenchymal stem cells, epithelial cells and fibroblasts).
  • the beneficial cells are the primary source of factors for the promotion of healing.
  • Placental products, their usefulness, and their immunocompatability are surprisingly enhanced by depletion of maternal trophoblast and selective elimination of CD14+ fetal macrophages.
  • Immunocompatability can be demonstrated by any means commonly known by the skilled artisan, such demonstration can be performed by the mixed Lymphocyte Reaction (MLR) and by lipopolysaccharide (LPS)-induced Tumor Necrosis Factor (TNF)-a secretion.
  • MLR mixed Lymphocyte Reaction
  • LPS lipopolysaccharide
  • TNF Tumor Necrosis Factor
  • the instant placental products contain basic Fibroblast Growth Factor (bFGF) optionally at a substantial concentration.
  • bFGF basic Fibroblast Growth Factor
  • the instant placental products provide and/or optionally secrete factors that stimulate cell migration and/or wound healing.
  • the presence of such factors can be demonstrated by any commonly recognized method.
  • conditioned medium from the present placental products enhance cell migration.
  • the placental products disclosed herein are useful in treating a number of wounds including: tendon repair, cartilage repair (e.g. femoral condyle, tibial plateau), ACL replacement at the tunnel/bone interface, dental tissue augmentation, fistulas (e.g. Crohn's disease, G-tube, tracheoesophogeal), missing tissue at adhesion barriers (e.g. nasal septum repair, vaginal wall repair, abdominal wall repair, tumor resection), dermal wounds (e.g. partial thickness burns, toxic epidermal necrolysis, epidermolysis bullosa, pyoderma gangrenosum, ulcers e.g. diabetic ulcers (e.g.
  • an epithelial cell layer wherein the approximate number of cells per cm2 of the amniotic membrane is about 500 to about 360,000; about 10,000 to about 360,000; about 10,000 to about 200,000; or about 20,000; about 25,000; about 30,000; about 35,000; about 40,000; about 45,000; about 50,000; about 55,000; about 60,000; about 65,000; about 70,000; about 75,000; about 80,000; about 85,000; about 90,000; about 95,000; about 100,000; about 105,000; about 1 10,000; about 1 15,000; about 120,000; about 125,000; about 130,000; about 135,000; about 140,000; about 145,000; about 150,000; about 155,000; about 160,000; about 165,000; about 170,000; about 175,000; about 180,000; about 185,000; about 190,000; or about 195,000.
  • a thick basement membrane comprising one or more of Collagen Type I, III, IV, laminin, and fibronectin
  • amniotic membrane with a thickness of about 20 to about 500 ⁇ , e. high thrombin activity
  • IL-1 a reduces pro-inflammatory proteins such as IL-1 a and TNF-a
  • antibacterial proteins such as defensins and allantoin (bacteriolytic proteins), o. angiogenic and mitogenic factors that promote blood vessel formation and re- epithelialization such as EGF, HGF, and VEGF,
  • hAECs Human Amniotic Epithelial Cells
  • t. cells with a capacity to differentiate to neural, hepatocyte, and pancreatic cells
  • hAMSCs Human Amniotic Mesenchymal Stem Cells with the capacity to differentiate to mesodermal lineages (osteogenic, chondrogenic, and adipogenic) and to all three germ layers-ectoderm (neural), mesoderm (skeletal muscle, cardiomyocytic, and endothelial), and ectoderm (pancreatic),
  • v. hAMSCs express CD49d and this distinguishes them from hAECs, w. hAMSCs that positive for the embryonic cytoplasmic marker Oct-4 that plays a role in maintaining sternness, or multipotency and self-renewal, x. hAECs that are positive for SSEA-3, SSEA-4, TRA-1 -60, TRA-1 -81 , and negative for SSEA-4 and non-tumorigenic.
  • the present inventors have now identified a need for the development of placental products exhibiting a ratio of MMP to TIMP comparable to that exhibited in vivo.
  • the present inventors have now identified a need for the development of placental products exhibiting a ratio of MMP-9 and TIMP1 of about 7-10 to one.
  • the present inventors have now identified a need for the development of placental products that inactivate serine proteases, cysteine proteases, aspartic proteases, and metalloproteases.
  • the present inventors have now identified a need for the development of placental products that inactivate serine proteases.
  • the present inventors have now identified a need for the development of placental products that inactivate cysteine proteases.
  • the present inventors have now identified a need for the development of placental products that inactivate aspartic proteases.
  • the present inventors have now identified a need for the development of placental products that inactivate metalloproteases.
  • 00141 The present inventors have now identified a need for the development of a placental product for wound healing that comprises mesenchymal stem cells, epithelial cells and fibroblasts.
  • the disclosure provides a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a membrane comprising cryopreserved amniotic membrane, wherein after cryopreservation and subsequent thawing the amniotic membrane comprises:
  • C) depleted amounts of one or more types of functional immunogenic cells, wherein greater than 40% of the combined cells in the stromal layer and epithelial layer are viable cells.
  • the disclosure provides a membrane comprising cryopreserved amniotic membrane having one or more tissue components, wherein after cryopreservation and subsequent thawing the one or more tissue components comprise:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • the disclosure provides a membrane comprising cryopreserved amniotic membrane having one or more tissue components, wherein after cryopreservation and subsequent thawing the one or more tissue components comprise:
  • tissue cells wherein said tissue cells are native to the amniotic membrane and greater than 40% of said tissue cells are viable;
  • amniotic membrane has depleted amounts of types of functional immunogenic cells, and wherein the one or more tissue components is present in an amount effective to:
  • the disclosure provides a membrane comprising cryopreserved placental membrane, wherein after cryopreservation and subsequent thawing the placental membrane comprises:
  • tissue cells wherein said tissue cells are native to the placental membrane and greater than 40% of said tissue cells are viable;
  • the placental membrane comprises amniotic membrane and chorionic membrane.
  • the depleted amounts of functional immunogenic cells produce immunogenic factors in amounts that are below levels sufficient to produce an immune response. In some embodiments, the depleted amounts of functional immunogenic cells produce immunogenic factors in amounts below detectable limits. As described herein, the depleted amounts of functional immunogenic cells may be selected from any one or more of maternal blood cells, neonatal blood cells, cord blood cells, tissue macrophages, and trophoblasts. In further embodiments, the depleted amounts of functional immunogenic cells comprise one or more tissue macrophage.
  • the tissue macrophage can be of any characterized type of macrophage such as, for example, tissue macrophages selected from the group consisting of CD1 1 b+, CD14+, CD18+, CD40+, and CD86+ and/or combinations thereof.
  • the depleted amounts of functional immunogenic cells produce one or more immunogenic factors (e.g., TNF-a, and/or other immunogenic factors described herein or otherwise known) in amounts that are below levels sufficient to produce an immune response.
  • the immunogenic factors may be produced in amounts that are negligible or below detectable limits.
  • less than 10% of viable cells are functional immunogenic cells (e.g., less that 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less that 1 % of viable cells).
  • the membrane comprises tissue cells wherein about 50% to about 100% of said tissue cells are viable. In some embodiments, about 60% to about 100% of said tissue cells are viable. In further embodiments, about 70% to about 100% of said tissue cells are viable. In further embodiments the viable cells may comprise mesenchymal stems cells (MSCs), fibroblasts, and/or epithelial cells.
  • MSCs mesenchymal stems cells
  • the membranes provide one or more tissue components (e.g., viable cells, one or more therapeutic factors, and/or extracellular matrix) in an amount that is effective to promote any of the activities of (i) - (viii) in vitro or in vivo.
  • tissue components e.g., viable cells, one or more therapeutic factors, and/or extracellular matrix
  • 00151 In vitro describes the experiments and/or procedures performed outside of the living organism (e.g. under tissue culture conditions using artificial culture medium). In vivo describes experiments and/or procedures performed within an organism, for example, an animal or human. 00152 Various embodiments of the above-described aspects may further comprise a delivery substrate, such that the membrane is fixed to the delivery substrate.
  • the membrane may be stored for an extended period of time prior to subsequent thawing.
  • the extended period of time is from about 6 months to at least about 36 months or more, alternatively from about 6 months to at least about 24 months or greater, alternatively from about 6 months to about 25 months or greater, alternatively from about 6 months to at least about at least about 12 months or greater, alternatively from about 6 months to about 10 months, alternatively from about 6 months, alternatively from about 3 months to about 6 months, alternatively from about 1 month to about 3 months, including other monthly and day derivations thereof for the various time periods described herein.
  • the viability of the tissue cells is substantially maintained upon thawing. In some embodiments, the viability of the tissue cells is substantially maintained for at least 24 months when stored frozen.
  • the membrane can be thawed and ready for use within 30 minutes of the start of a thawing method, such as described herein or as modified from generally known methods.
  • the membrane can be stored in saline up to an hour after thawing and still maintain about 70% viable cells.
  • One embodiment of the present technology provides a placental product comprising a cryopreservation solution and an amniotic membrane, wherein the amniotic membrane comprises viable native therapeutic cells and native therapeutic factors, and wherein the cryopreservation solution comprises an amount of a cryopreservative that is effective to provide for a cryopreserved product.
  • the amniotic membrane is substantially free of at least one at least one or both of the following immunogenic cell types: CD14+ macrophages and maternal blood cells.
  • the amniotic membrane comprises one or more layers which exhibit the architecture of the native amniotic membrane (e.g. has not been homogenized or treated with collagenase).
  • the placental membrane is suitable for dermal application to a wound.
  • the placental product is manufactured by steps taught herein.
  • the present placental products are not limited to products manufactured by the methods taught here.
  • products of the present technology could be produced through methods that rely on screening steps; e.g. steps to screen for preparations with the described technical features and superior properties.
  • the present placental product comprises one or more of the following technical features:
  • the viable therapeutic native cells are capable of differentiating into cells of more than one lineage (e.g. osteogenic, adipogenic and/or chonodrogenic lineages), b. the native therapeutic factors include IGFBP1 ,
  • the native therapeutic factors include adiponectin,
  • the native therapeutic factors include a2-macroglobulin,
  • the native therapeutic factors include bFGF,
  • the native therapeutic factors include EGF,
  • the native therapeutic factors include particular amounts of MMP-9 and TIMP1 , h. the native therapeutic factors include MMP-9 and TIMP1 in a ratio of about 7 to about 10,
  • the placental product does not comprise culture expanded cells
  • cryopreservative solution may be present in an amount of greater than about 15 ml_,
  • cryopreservative comprises DMSO
  • cryopreservation solution further comprises albumin, optionally wherein the albumin is Human Serum Albumin (HSA),
  • HSA Human Serum Albumin
  • cryopreservative comprises DMSO and albumin (e.g. HSA),
  • n. comprises about 5,000 to about 500,000 cells/cm 2 , about 5,000 to about 240,000 cells/cm 2 or about 20,000 to about 60,000 cells/cm 2 ,
  • the amniotic membrane comprises at least: about 2,000, or about 2,400, or about 4,000 or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000 stromal cells per unit cm 2 of the amniotic membrane,
  • the amniotic membrane comprises about 2,000 to about 15,000 of stromal cells per cm 2 of the amniotic membrane
  • q. comprises stromal cells wherein about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4% or about 90%, or about 92.5% or at least about 100% of the stromal cells are viable after a freeze-thaw cycle
  • r. comprises stromal cells wherein about 40% to about 92.5% of the stromal cells are viable after a freeze-thaw cycle
  • the amniotic membrane has a thickness of about 20 ⁇ to about 500 ⁇ ; about 20 ⁇ to about 200 ⁇ ; or about 20 ⁇ to about 100 ⁇ .
  • t. secretes less than about any of: 350 pg/cm 2 , 225 pg/cm 2 , 100 pg/cm 2 or 70 pg/cm 2 or less TNF-a into a tissue culture medium upon placing a 2 cm x 2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours,
  • TNF-a 350 pg/cm 2 , 225 pg/cm 2 , 100 pg/cm 2 or 70 pg/cm 2 or less TNF-a into a tissue culture medium upon placing a 2 cm x 2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours,
  • v. further comprises an chorionic membrane
  • w. amniotic membrane comprises a layer of epithelial cells
  • x. further comprises an chorionic membrane, wherein the amniotic membrane and the chorionic membrane are associated to one another in the native configuration
  • y. further comprises an chorionic membrane, wherein the amniotic membrane and the chorionic membrane are not attached to one another in the native configuration
  • z. further comprises a chorionic membrane wherein the chorionic membrane comprises about 2 to about 4 times more stromal cells relative to the amniotic membrane
  • aa. does not comprise a chorionic membrane
  • bb. comprises chorionic membrane, wherein the chorionic membrane comprises about 2 to about 4 times more stromal cells relative to the amniotic membrane, and cc. is suitable for dermal application to a wound.
  • a placental product comprises native therapeutic cells of the amniotic membrane.
  • the cells comprise one or more of MSCs, fibroblasts, and epithelial cells.
  • the native therapeutic cells comprise viable MSCs.
  • the native therapeutic cells comprise viable fibroblasts.
  • the native therapeutic cells comprise viable epithelial cells.
  • the native therapeutic cells comprise viable MSCs and viable fibroblasts.
  • the native therapeutic cells comprise viable MSCs, viable fibroblasts, and viable epithelial cells.
  • the therapeutic native cells are viable cells capable of differentiating into cells of more than one lineage (e.g. osteogenic, adipogenic and/or chonodrogenic lineages).
  • the placental product comprises about 500 to about 360,000 cells per cm 2 or about 40,000 to about 90,000 cells per cm 2 .
  • the placental product comprises at least: about 2,000, or about 2,400, or about 4,000, or about 6,000, or about 8,000, or about 10,000, or about 10,585, or about 15,000, or about 180,000 stromal cells per unit cm 2 of the placental product.
  • the placental product comprises about 2,000 to about 15,000 of stromal cells per cm 2 of the placental product.
  • the placental product comprises stromal cells wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4% or about 90%, or about 92.5%, or about 100% of the stromal cells are viable after a freeze-thaw cycle.
  • the placental product comprises stromal cells wherein about 40% to about 100% of the stromal cells are viable after a freeze-thaw cycle, alternatively from about 40% to about 99.9% of the stromal cells are viable, alternatively wherein about 70% to about 99% of the stromal cells are viable.
  • the placental product comprises less than about 1 % of CD14+ macrophages per total cells.
  • the amniotic membrane of the placental product comprises about 2 to about 4 times less stromal cells relative to a chorionic membrane derived from the same placenta.
  • the placental product further comprises chorionic membrane containing about 2 to about 4 times more stromal cells relative to the amniotic membrane.
  • the amniotic membrane of the placental product comprises MSCs in an amount of up to about 70% of the total number of cells in the amniotic membrane product.
  • the product comprises MSCs in an amount of: at least about 1 %, at least about 2%, at least about 3%, at least about 4%, at least about 5%, about 1 % to about 10%, or about 3% to about 100%, relative to the total number of cells in the amniotic membrane of the placental product.
  • of the total number of MSCs in the amniotic membrane product at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the MSCs are viable after a f reeze-thaw cycle.
  • the amniotic membrane of the placental product comprises fibroblasts in an amount of up to about 100% of the total number of cells in the amniotic membrane product.
  • the product comprises fibroblasts in an amount of: about 1 %, about 20%, about 5% to about 15%, at least about 1 %, at least about 2%, at least about 3%, or at least about 4% relative to the total number of cells in the amniotic membrane of the placental product.
  • of the total number of fibroblasts in the amniotic membrane product at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the fibroblasts are viable after a freeze-thaw cycle.
  • the amniotic membrane of the placental product comprises stromal cells in an amount of: about 5% to about 100%, about 5% to about 50%, about 5% to about 30%, about 10% to about 30%, about 15% to about 25%, at least about 5%, at least about 10%, or at least about 15%, relative to the total number of cells in the amniotic membrane of the placental product.
  • at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the stromal cells are viable after a freeze-thaw cycle.
  • the amniotic membrane of the placental product comprises epithelial cells in an amount of: about 60% to about 100%, about 60% to about 90%, about 70% to about 90%, about 40% to about 90%, about 50% to about 90%, at least about 40%, at least about 50%, least about 60%, or at least about 70%, relative to the total number of cells in the amniotic membrane of the placental product.
  • at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the epithelial cells are viable after a freeze-thaw cycle.
  • the total number of functional macrophages may comprise less than about 10% of viable cells (e.g., less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or less than about 1 %.
  • the amniotic membrane of the placental product comprises fibroblasts and MSCs in a ratio of: about 4:1 to about 1 :1 or about 3:1 to about 3:2, or about 2:1 .
  • the amniotic membrane of the placental product comprises MSCs in an amount of: at least about 1 ,000 cells/cm 2 at least about 2,000 cells/cm 2 , about 1 ,000 to about 5,000 cells/cm 2 , or about 2,000 to about 5,000 cells/cm 2 .
  • at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the MSCs are viable after a freeze-thaw cycle.
  • the amniotic membrane of the placental product comprises fibroblasts in an amount of: at least about 2,000 cells/cm 2 , at least about 4,000 cells/cm 2 , about 2,000 to about 9,000 cells/cm 2 , or about 2,000 to about 9,000 cells/cm 2 .
  • at least: about 40%, about 50%, about 60%,about 70%, or about 100% of the fibroblasts are viable after a freeze-thaw cycle.
  • the amniotic membrane of the placental product comprises stromal cells in an amount of: at least about 4,000, at least about 8,000 cells/cm 2 , about 4,000 to about 18,000 cells/cm 2 , or about 4,000 to about 18,000 cells/cm 2 .
  • at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the stromal cells are viable after a freeze-thaw cycle.
  • the amniotic membrane of the placental product comprises epithelial cells in an amount of: at least about 10,000 cells/cm 2 , at least about 20,000 cells/cm 2 , at least about 32,000 cells/cm 2 , about 10,000 to about 72,000 cells/cm 2 , about 20,000 to about 72,000 cells/cm 2 , or about 32,000 to about 72,000 cells/cm 2
  • at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the epithelial cells are viable after a freeze-thaw cycle.
  • the amniotic membrane of the placental product comprises functional macrophages in an amount of: less than about 3,000 cells/cm 2 , less than about 1 ,000 cells/cm 2 , or less than about 500 cells/cm 2 .
  • the placental product comprises a layer of epithelial cells.
  • the placental product comprises a chorionic membrane but is substantially free of trophoblasts. 00189 In one embodiment, the placental product is substantially free of functional CD14+ macrophages.
  • the placental product is substantially free of maternal blood cells.
  • the placental product is substantially free of trophoblasts, functional CD14+ macrophages, and maternal blood cells.
  • the placental product comprises viable stromal cells.
  • the placental product comprises viable MSCs.
  • the placental product comprises viable fibroblasts.
  • the placental product comprises viable epithelial cells.
  • the placental product comprises viable MSCs, fibroblasts, and epithelial cells.
  • the placental product comprises a chorionic membrane but is substantially free of maternal blood cells.
  • the placental product comprises a chorionic membrane but is substantially free of maternal blood cells and/or trophoblast cells.
  • the placental product is substantially free of culture expanded cells.
  • a placental product comprises native therapeutic factors of the amniotic membrane, such as defined above and otherwise described herein in various aspects and embodiments, including the Examples.
  • Table 10 provides a list of therapeutic factors tested and their functions.
  • the factors include one or more of: IGFBP1 , adiponectin, a2- macroglobulin, bFGF, EGF, MMP-9, and TIMP1 .
  • the factors are present in amounts/cm 2 that are substantially similar to that of a native amniotic membrane or layer thereof (e.g. ⁇ 10% or 20%).
  • the factors include VEGF, PDFG, HGF, SDF-1 , KGF, IGFBP1 , adiponectin, a2-macroglobulin, bFGF, EGF, MMP-9, and TIMP1 , and the like.
  • the factors are present in ratios that are substantially similar to that of a native amniotic membrane thereof.
  • the factors are present in amounts/cm 2 that are substantially similar to that of a native amniotic membrane or layer thereof (e.g. ⁇ 10% or 20%).
  • the factors include MMP-9 and TIMP1 in a ratio of about 7 to about 10 (e.g. about 7).
  • the factors are present in amounts/cm 2 that are substantially similar to that of a native amniotic membrane or layer thereof (e.g. ⁇ 10% or 20%).
  • the factors include one or more (e.g. a majority or all) of the factors listed in Table 10.
  • the factors are present in ratios that are substantially similar to that of a native amniotic membrane or layer thereof.
  • the factors are present in amounts/cm 2 that are substantially similar to that of a native amniotic membrane or layer thereof (e.g. ⁇ 10% or 20%).
  • the placental product thereof comprises substantially less TNF-a/ cm 2 than a native amniotic membrane or layer thereof, respectively.
  • the placental product thereof secretes substantially less TNF-a/ cm 2 than a native placental product or layer thereof, respectively.
  • the placental product secretes less than about any of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-a into a tissue culture medium upon placing a 2 cm x 2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours.
  • the placental product secretes less than about any of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-a into a tissue culture medium upon placing a 2 cm x 2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours.
  • the placental product further comprises an exogenously added inhibitor of TNF-a.
  • the inhibitor of TNF-a is PGE2.
  • the product may be treated with an antibiotic or an antibiotic cocktail.
  • an antibiotic cocktail includes gentamicin sulfate, vancomycin HCI, and amphotericin B.
  • a placental product of the present technology comprises one or more layers which exhibit the architecture of the native amniotic membrane.
  • placental layers that exhibit native architecture for example, layers that have not been homogenized or treated with collagenase or other enzyme that substantially disrupts the layer.
  • the placental product comprises a stromal layer with native architecture of the amniotic membrane.
  • the placental product comprises a basement membrane with native architecture of the amniotic membrane.
  • the placental product comprises an epithelial layer with native architecture of the amniotic membrane.
  • the placental product comprises a stromal layer and an epithelial cell layer with native architecture of the amniotic membrane.
  • the placental product or amniotic membrane thereof has a thickness of about 20 ⁇ to about 500 ⁇ ; about 20 ⁇ to about 200 ⁇ ; or about 20 ⁇ to about 100 ⁇ .
  • the placental product comprises a chorionic membrane but is substantially free of trophoblasts.
  • the placental product comprises a basement membrane with native architecture of the chorionic membrane and the chorionic membrane is substantially free of trophoblasts.
  • the maternal portion in contact with the chorionic membrane comprises fragments of extracellular matrix proteins.
  • the placental product has been treated with Dispase (e.g. Dispase II) and/or a substantial portion of the extracellular matrix protein fragments comprises terminal leucine or phenylalanine.
  • the placental product further comprises a chorionic membrane.
  • the amniotic membrane and the chorionic membrane in the placental product are associated to one another in the native configuration.
  • the amniotic membrane and the chorionic membrane are not attached to one another in the native configuration.
  • the placental product does not comprise a chorionic membrane.
  • the placental product can be formulated with a cryopreservation solution.
  • the cryopreservation solution comprising one or more cell- permeating cryopreservatives, one or more non cell-permeating cryopreservatives, or a combination thereof.
  • the cryopreservation solution comprises one or more cell-permeating cryopreservatives selected from DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, or a combination thereof.
  • the cryopreservation solution comprises one or more non cell- permeating cryopreservatives selected from polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof.
  • cryopreservation BioFiles Volume 5 Number 4 -Sigma-Aldrich® datasheet.
  • the cryopreservation solution comprises a cell-permeating cryopreservative, wherein the majority of the cell-permeating cryopreservative is DMSO.
  • the cryopreservation solution does not comprise a substantial amount of glycerol.
  • the cryopreservation solution comprises DMSO.
  • the cryopreservation solution does not comprise glycerol in a majority amount.
  • the cryopreservation solution does not comprise a substantial amount of glycerol.
  • the cryopreservation solution comprises additional components such as albumin (e.g. HSA or BSA), an electrolyte solution (e.g. Plasma-Lyte), or a combination thereof.
  • albumin e.g. HSA or BSA
  • electrolyte solution e.g. Plasma-Lyte
  • the cryopreservation solution comprises 1 % to about 15% albumin by volume and about 5% to about 20% cryopreservative by volume (e.g. about 10%).
  • the cryopreservative comprises DMSO.
  • the cryopreservation solution comprises about 5% to about 100% of the cryopreservative, alternatively about 5% to about 20%.
  • the placental product is formulated in greater than about 20 ml_ or about 50 ml_ of cryopreservation solution.
  • the cryopreservative solution comprises at least one cryopreservative (or cryopreservative agent).
  • the at least one cryopreservative comprises DMSO (e.g. if there is more than one cryopreservative, DMSO is found in a majority amount of total cryopreservative).
  • the cryopreservation solution does not comprise a substantial amount of glycerol.
  • the composition comprises cryopreservation solution.
  • the cryopreservation solution may be added to a container containing the placental product, optionally as membrane-mounted composition (e.g., on nitrocellulose).
  • a sufficient amount of cryopreservation solution is added to protect the membrane during the subsequent freezing steps.
  • the infusion of the membrane with the cryopreservation solution maintains viability of the cells contained within the membrane.
  • suitable cryopreservation solutions are known in the art, in one embodiment, the cryopreservation comprises storage in a cryopreservation solution comprising one or more cell-permeating cryopreservatives, one or more non-cell permeating cryopreservatives, or a combination thereof.
  • Suitable cryopreservatives include, but are not limited to, DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, propanediol, polyethylene glycol (PEG), 1 ,2- propanediol (PROH) or a combination thereof.
  • the cryopreservation solution may contain one or more non-cell permeating cryopreservative selected from polyvinyl pyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide, an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinyl pyrrolidone, hydroxyethyl strarch, autologous plasma or a combination thereof.
  • Other examples of useful cryopreservatives are described in Cryopreservation (BioFiles, Volume 5, Number 4 Sigma-Aldrich® Datasheet).
  • a suitable cryopreservation solution comprises a cryopreservative, in an amount of at least about 0.001 % to 100%, suitably in an amount from about 2% to about 20%, preferably about 5% to about 10% by volume, for example DMSO.
  • the cryopreservation solution comprises at least about 2 % cryopreservative (e.g. DMSO).
  • the cryopreservation solution may comprise serum albumin or other suitable proteins.
  • the cryopreservation solution comprises from about 1 % to about 20% serum albumin or other suitable proteins, alternatively from about 1 % to about 10%.
  • Serum albumin or other suitable proteins are present to help stabilize the membrane during the freeze-thaw process and to reduce the damage to cells, maintaining viability.
  • Serum albumin may be human serum albumin or bovine serum albumin.
  • the cryopreservation solution may further comprise a physiological buffer or saline, for example, phosphate buffer saline.
  • a container may be filled with a sufficient amount of the cryopreservation solution to cover the placental membrane.
  • the amount of the cryopreservation solution necessary can depend on a number of factors including, for example, the type of container and mounting used as well as the size of the membranes to be preserved. The lower the amount of cryopreservation solution necessary to top (or cover) the composition/device, the faster the composition is able to thaw. Thus, it is desirable to use the least amount of cryopreservation solution that allows for top coverage of the membrane without compromising viability of the cells during the freeze thaw. Further, the smaller the membrane and the smaller the container used, the less cryopreservation solution can be used.
  • a bag is used containing cryopreservation solution in an amount from about 7 ml_ to about 50 ml, alternatively from about 10 mL to about 50 ml, alternatively from about 15 mL to about 50 ml, alternatively from about 15 mL to about 25 ml.
  • about 15 mL of cryopreservation solution is added to the container or bag.
  • the amount of cryopreservation solution can be sufficient to fully submerge the membrane. The amount will depend on the size of the bag used and the size of the membrane being cryopreserved. If a small bag is being used with a small (e.g. smaller than 2 cm x 2 cm membrane), about 3 mL to about 10 ml, alternatively 3 mL to about 7 mL of cryopreservation solution may be used.
  • a container is used containing from about 7 mL to about 50 ml, alternatively from about 5 mL to about 20 ml, alternatively from about 7 mL to about 20 ml, alternatively from about 7 mL to about 15 ml.
  • the amount of cryopreservation solution can be sufficient to fully submerge the membrane within the container. The amount will depend on the size of the container used and the size of the membrane being cryopreserved.
  • the amount of cryopreservation solution is sufficient to protect cells during the freezing and subsequent thawing procedures.
  • at least 70% cell viability is maintained after a freeze-thaw.
  • at least 75% cell viability is maintained, alternatively about 80% cell viability is maintained, alternatively 85% cell viability is maintained, alternatively about 90% cell viability is maintained, alternatively about 95% cell viability is maintained, alternatively about 100% cell viability is maintained.
  • At viability of the membrane is at least 70%, at least 71 %, at least 72%, at least 73%, at least 74 %, at least 75%, at least 75 %, at least 78%, at least 80%, at least 82 %, at least 85%, at least 88%, at least 89%, at least 90%, at least 92%,and percentages in between.
  • the amount of cryopreservation solution is sufficient to protect the structural, architectural, and/or 3-D structure of the membrane,.
  • the cryopreservation solution contains a cell-permeating cryopreservative in an amount of 0.01 % to about 100%, alternatively from about 2 % to about 100 %.
  • the cryopreservation solution contains polysaccharides or monosaccharides.
  • the placental product is placed on nitrocellulose paper.
  • the placenta, amniotic, and/or chorionic membrane may be cut into a plurality of sections.
  • the membranes may be any suitable size and are customizable depending on the type of membrane and the particular end application or usage of that membrane. Suitable sizes of membrane include, but are not limited to, about 1 .5 cm x about 1 .5 cm, about 2 cm x about 2 cm, about 3 cm x about 3cm, about 4 cm x about 4 cm, about 5 cm x about 5 cm, about 6 cm x about 6 cm, about 7 cm x about 7 cm, about 8 cm x about 8 cm, about 7. 5 cm x about 15 cm, about 1 .
  • the cryopreserved membranes have a surprisingly long shelf-life or stability and retain viable cells when frozen for extended periods of time.
  • the cryopreserved products may be stored at about -20 °C to about -196 °C (e.g., about -45 to about -80 °C) for two years or more with retention of high cell viability (at least 40%, 50%, 60%, or 70% or more retention of viable cells) once thawed.
  • cryopreserved membranes can be stored at about -20 °C to about -196 °C (e.g., about -45 to about -80 °C) for at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 15 months, at least about 24 months, at least about 25 months, at least about 36 months, or more before thawing with a high retention of viable cells (e.g., at least 40% viable cells, alternatively at least 50% viable cells, alternatively at least about 70% viable cells, alternatively at least about 80%, about 85%, 90%, 95% or 100% viable cells.
  • viable cells e.g., at least 40% viable cells, alternatively at least 50% viable cells, alternatively at least about 70% viable cells, alternatively at least about 80%, about 85%, 90%, 95% or 100% viable cells.
  • kits for treating a wound or a tissue defect comprising:
  • the kit may comprise an additive that is selected from one or more antibiotics, emollients, keratolytic agents, humectants, antioxidants, preservatives, therapeutics, bandages, tools, cutting device, buffer, thawing medium, handling media, forceps, container and combinations thereof.
  • a placental product of the present technology can be manufactured from a placenta in any suitable manner that provides the technical features taught herein.
  • a placenta product comprises at least an immunocompatible amniotic membrane.
  • a placental product is manufactured by a method comprising: a. obtaining a placenta,
  • the method comprises a step of removing maternal blood cells from the placental membrane, for example, by lysing red blood cells, by removing blood clots, or a combination thereof.
  • the method comprises a step of treating the placental membranes with one or more antibiotics.
  • the method comprises a step of selectively depleting CD14+ macrophages , optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release.
  • the step of cryopreserving the placental membranes comprises freezing the membrane in a cryopreservation solution which comprises one or more cell- permeating cryopreservatives, one or more non cell-permeating cryopreservatives, or a combination thereof.
  • the step of cryopreserving the placental membranes comprises placing at about 2°C to about 8°C for a period of time and then freezing at -80 °C, thereby selectively depleting CD14+ macrophages optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release.
  • the method comprises retaining a layer of epithelial cells of the amniotic membrane.
  • the method comprises a step of removing the chorionic membrane or portion thereof.
  • the method comprises removing trophoblasts from the chorionic membrane while retaining the stromal layer of the chorionic membrane.
  • An exemplary placental membrane of the present technology can be manufactured or provided with a bandage or wound dressing.
  • the technology the placental product or membrane is immunocompatible. Immunocompatability can be accomplished by any selective depletion step that removes immunogenic cells or factors or immunogenicity from the placenta (or amniotic membrane thereof).
  • the placental product is made immunocompatible by selectively depleting it of functional immunogenic cells.
  • a placenta can be made immunocompatible by selectively removing immunogenic cells from the placental product (or amniotic membrane thereof) while retaining the therapeutic cells. For example, immunogenic cells can be removed by killing the immunogenic cells or by purification of the placenta there from.
  • the placental product is made immunocompatible by selectively depleting trophoblasts, for example, by removal of the trophoblast layer.
  • the placental product is made immunocompatible by selective depletion of functional CD14+ macrophages, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release or by MLR assay.
  • the placental product is made immunocompatible by selective depletion of maternal blood cells.
  • the placental product is made immunocompatible by selective depletion of functional CD14+ macrophages, trophoblasts, and maternal blood cells.
  • the placental product is made immunocompatible by selective depletion of trophoblasts and/or CD14+ macrophages, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release or by MLR assay.
  • immunocompatability is accomplished by removal or depletion of trophoblasts from the placental product. Removal of trophoblasts from the chorionic membrane is conducted while retaining the stromal layer of the chorionic membrane.
  • a placental product has one or more of the following superior features:
  • a. is substantially non-immunogenic
  • c. provides enhanced therapeutic efficacy.
  • trophoblasts are removed while retaining the stromal layer of the chorionic membrane.
  • Trophoblasts can be removed in any suitable manner which substantially diminishes the trophoblast content of the placental product.
  • the trophoblasts are selectively removed.
  • the trophoblasts are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the chorionic membrane (e.g. MSCs, therapeutic factors, extracellular matrix etc).
  • a majority (e.g. substantially all) of the trophoblasts are removed.
  • One method of removing trophoblasts comprises treating the placenta (e.g. chorion or amnio-chorion) with a digestive enzyme such as dispase (e.g. dispase II) and separating the trophoblasts from the placenta.
  • the step of separating comprises mechanical separation such as peeling or scraping.
  • scraping comprises scraping with a soft instrument such as a finger.
  • One method of removing trophoblasts comprises treating the chorionic membrane with dispase for about 30 to about 45 minutes separating the trophoblasts from the placenta.
  • the dispase is provided in a solution of about less than about 1 % (e.g. about 0.5%).
  • the step of separating comprises mechanical separation such as peeling or scraping.
  • scraping comprises scraping with a soft instrument such as a finger.
  • trophoblasts are removed before cryopreservation.
  • 00262 In one embodiment, functional macrophages are depleted or removed from the placental product. Surprisingly, such a placental product has one or more of the following superior features:
  • a. is substantially non-immunogenic
  • c. provides enhanced therapeutic efficacy.
  • Functional macrophages can be removed in any suitable manner which substantially diminishes the macrophage content of the placental product.
  • the macrophages are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the placenta (e.g. MSCs, therapeutic factors, etc).
  • a majority (e.g. substantially all) of the macrophages are removed.
  • One method of removing immune cells such as macrophages comprises killing the immune cells by rapid freezing rates such as 60-100°C/min.
  • Another method of removing immune cells comprises killing the immune cells by holding the cells at about 2°C to about 8°C for a period of time, and then freezing the immune cells (e.g., at about -20°C) at a rate of about 1 °C/min.
  • stromal cells e.g. MSCs
  • the present inventors have discovered a method of selectively killing CD14+ macrophages by placing the placental membrane at about 2°C to about 8°C for a period of time (e.g. for at least about 10 min such as for about 30-60 mins) at a temperature above freezing (e.g. incubating at 2-8°C) and then freezing the placenta (e.g. incubating at -80°C ⁇ 5°C).
  • the step of freezing comprises freezing at a rate of less than 10°/min (e.g. less than about 5°/min such as at about 1 min).
  • the step of placing the placental membrane at about 2°C to about 8°C comprises soaking the placental membrane in a cryopreservation solution (e.g. containing DMSO) for a period of time sufficient to allow the cryopreservation solution to penetrate (e.g. equilibrate with) the placental tissues.
  • a cryopreservation solution e.g. containing DMSO
  • the step of freezing comprises reducing the temperature at a rate of about 17min.
  • the step of freezing comprises freezing at a rate of less than 107min (e.g. less than about 57min such as at about 1 min).
  • the step of placing the placental membrane at about 2°C to about 8°C comprises soaking the placental membrane in a cryopreservation solution (e.g. containing DMSO) at a temperature of about -10-15°C (e.g. at 2-8°C) for at least about any of: 10 min, 20 min, 30 min, 40 min, or 50 min.
  • the step of placing the placental membrane at about 2°C to about 8°C comprises soaking the placental membrane in a cryopreservation solution (e.g. containing DMSO) at a temperature of about -10-15°C (e.g. at 2-8°C) for about any of: 10-120, 20-90 min, or 30-60 min.
  • the step of freezing comprises freezing at a rate of less than 107min (e.g. less than about 57min such as at about 1 min).
  • maternal blood cells are depleted or removed from the placental product.
  • a placental product has one or more of the following superior features:
  • a. is substantially non-immunogenic
  • c. provides enhanced therapeutic efficacy.
  • Maternal blood cells can be removed in any suitable manner which substantially diminishes such cell content of the placental product.
  • the maternal blood cells are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the placenta (e.g. therapeutic cells (e.g., MSCs), therapeutic factors, including angiogenic factors, antioxidant agents, anti-inflammatory agents, etc).
  • therapeutic cells e.g., MSCs
  • therapeutic factors including angiogenic factors, antioxidant agents, anti-inflammatory agents, etc.
  • removal of maternal blood cells comprises rinsing the amniotic membrane (e.g. with buffer such as PBS) to remove gross blood clots and any excess blood cells.
  • buffer such as PBS
  • removal of maternal blood cells comprises treating the amniotic membrane with an anticoagulant (e.g. citrate dextrose solution).
  • an anticoagulant e.g. citrate dextrose solution
  • removal of maternal blood cells comprises rinsing the amniotic membrane (e.g. with buffer such as PBS) to remove gross blood clots and any excess blood cells, and treating the amniotic membrane with an anticoagulant (e.g. citrate dextrose solution).
  • an anticoagulant e.g. citrate dextrose solution
  • the chorionic membrane is retained and removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord. The chorion on the umbilical side of the placenta is not removed due to the vascularization on this side.
  • the chorionic membrane is retained and removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord and rinsing the amniotic membrane and chorionic membrane (e.g. with buffer such as PBS) to remove gross blood clots and any excess blood cells.
  • buffer such as PBS
  • the chorionic membrane is retained and removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord and treating the amniotic membrane and chorionic membrane with an anticoagulant (e.g. citrate dextrose solution).
  • an anticoagulant e.g. citrate dextrose solution
  • the chorionic membrane is retained and removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord, rinsing the chorionic membrane amniotic membrane (e.g. with buffer such as PBS) to remove gross blood clots and any excess blood cells, and treating the amniotic membrane with an anticoagulant (e.g. citrate dextrose solution).
  • buffer such as PBS
  • an anticoagulant e.g. citrate dextrose solution
  • the placental product is selectively depleted of immunogenicity as demonstrated by a reduction in LPS stimulated TNF-a release. In one embodiment, the placental product is selectively depleted of macrophages.
  • TNF-a is depleted by killing or removal of macrophages.
  • TNF-a is functionally depleted by treatment with PGE2, which suppresses TNF-a secretion.
  • TNF- a is inhibited at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.
  • a placental product of the present technology may be used fresh or may be preserved for a period of time. Surprisingly, cryopreservation in the instant technology results in immunocompatible placental products.
  • a placental product is cryopreserved.
  • a placental product may be cryopreserved by incubation at freezing temperatures (from about -20 °C to about - 196°C, e.g. a -80 °C ⁇ 5°C) in a cryopreservative solution.
  • Cryopreservation can comprise, for example, incubating the placental product at about 2°C to about 8°C for 30-60 min, and then incubating at -80 °C until use. The placental product may then be thawed for use. Optionally, the placental product is cryopreserved in a manner such that cell viability is retained surprisingly well after a freeze-thaw cycle.
  • cryopreservation comprises storage in a cryopreservation solution comprising one or more cell-permeating cryopreservatives, one or more non cell- permeating cryopreservatives, or a combination thereof.
  • the cryopreservation solution comprises one or more cell-permeating cryopreservatives selected from DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, or a combination thereof.
  • the cryopreservation solution comprises one or more non cell-permeating cryopreservatives selected from polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof.
  • cryopreservatives selected from polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof.
  • Other examples of useful cryopreservatives are described in "Cryopreservation” (BioFiles Volume 5 Number 4 -Sigma-Aldrich® datasheet).
  • the cryopreservation solution comprises a cell-permeating cryopreservative, wherein the majority of the cell-permeating cryopreservative is DMSO.
  • the cryopreservation solution does not comprise a substantial amount of glycerol.
  • the cryopreservation solution comprises DMSO.
  • the cryopreservation solution does not comprise glycerol in a majority amount.
  • the cryopreservation solution does not comprise a substantial amount of glycerol.
  • the cryopreservation solution comprises additional components such as albumin (e.g. HSA or BSA), an electrolyte solution (e.g. Plasma-Lyte), saline solution, or any combination thereof.
  • albumin e.g. HSA or BSA
  • electrolyte solution e.g. Plasma-Lyte
  • saline solution e.g. saline solution, or any combination thereof.
  • the composition comprises cryopreservation solution.
  • the cryopreservation solution is added to the container containing the membrane-mounted device or composition.
  • a sufficient amount of cryopreservation solution is added to the container to protect the membrane during the subsequent freezing steps.
  • Suitable cryopreservation solutions are known in the art.
  • the cryopreservation comprises storage in a cryopreservation solution comprising one or more cell-permeating cryopreservatives, one or more non-cell permeating cryopreservatives, or a combination thereof.
  • Suitable cryopreservatives include, but are not limited to, DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, propanediol, polyethylene glycol (PEG), 1 ,2- propanediol (PROH) or a combination thereof.
  • the cryopreservation solution may contain one or more non-cell permeating cryopreservative selected from polyvinyl pyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide, an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinyl pyrrolidone, hydroxyethyl starch, autologous plasma or a combination thereof.
  • Other examples of useful cryopreservatives are described in Cryopreservation (BioFiles, Volume 5, Number 4 Sigma-Aldrich® Datasheet).
  • a suitable cryopreservation solution comprises a cell-permeating cryopreservative, in an amount of at least about 0.001% to 100%, suitably in an amount from about 2% to about 20%, preferably about 5% to about 10% by volume. In some instances, the cryopreservation solution comprises at least about 2 % cell-permeating cryopreservative. Further, the cryopreservation solution may comprise serum albumin or other suitable proteins. In some embodiments, the cryopreservation solution comprises from about 1 % to about 20% serum albumin or other suitable proteins, alternatively from about 1 % to about 10%.
  • the cryopreservation solution comprises 1 % to about 15% albumin by volume and about 5% to about 20% cryopreservative by volume (e.g. about 10%).
  • the cryopreservative comprises DMSO (e.g. in a majority amount).
  • Serum albumin or other suitable proteins are present to help stabilize the membrane during the freeze-thaw process and to reduce the damage to cells in order to maintain viability.
  • Serum albumin may be human serum albumin or bovine serum albumin.
  • the cryopreservation solution may further comprise a physiological buffer or saline, for example, phosphate buffer saline.
  • cryopreservation comprises placing the placental membrane on nitrocellulose paper.
  • the placental membrane is cut into a plurality of sections before cryopreservation.
  • the sections are placed on nitrocellulose paper before placing the placental membrane sections at about 2°C to about 8°C. .
  • the placental products, and particularly the cryopreserved membranes described herein provide an amount of viable cells, therapeutic factors, and extracellular matrix that are effective to promote a number of beneficial therapeutic activities and effects.
  • the membranes may be applied and provide an environment that can promote cells to produce any number of therapeutic factors, as well as provide amounts of therapeutic factors, viable cells, and extracellular matrix that provide the same or similar therapeutic benefit.
  • the disclosure provides a method of treating a wound on a subject comprising administering a membrane comprising a cryopreserved amniotic membrane as described herein, wherein upon administration the membrane provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to promote one or more of:
  • the disclosure provides a method for accelerating wound healing comprising administering a membrane according to any of the aspects and embodiments described herein.
  • the administering is effective to promote wound closure by 12 weeks after an initial administering step.
  • the administering is effective to promote wound closure by 5-6 weeks after an initial administering step.
  • the administering is effective to promote reduction in wound size by 50% or more 28 days after an initial administering step.
  • the administering is effective to improve wound closure rate by at least about 44% relative to standard wound treatment.
  • Standard wound treatment or standard of care for wound treatment may refer to and include any treatment that does not incorporate a membrane or product as described herein.
  • Suitable standard wound treatments are known in the art and include, but are not limited to, application of dressings (e.g., gauze, bandages, barriers, or bioengineered membranes that contain no detectable or low numbers of viable cells), debridement, antibiotics, salves, ointment, and the like and any combinations thereof.
  • Standard wound treatment may include debridement in conjunction with a wound dressing or bandage and an offloading device.
  • the disclosure provides a method of treating a subject for a wound that is refractory to a prior wound healing treatment, the method comprising administering at or near the site of the wound a membrane according to any of the aspects and embodiments described herein.
  • the administering is effective to promote wound closure by 12 weeks after an initial administering step.
  • the administering is effective to promote wound closure by 5-6 weeks after an initial administering step.
  • the administering is effective to promote reduction in wound size by 50% or more 28 days after an initial administering step.
  • the disclosure provides a method for treating a chronic wound comprising administering to the site of the wound a membrane as disclosed herein wherein the administering provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to promote healing of the chronic wound.
  • the wound is selected from the group consisting of lacerations, scrapes, burns, incisions, punctures, wound caused by a projectile, an epidermal wound, skin wound, chronic wound, acute wound, external wound, internal wound, congenital wound, ulcer, pressure ulcer, diabetic ulcer, tunnel wound, wound caused during or as an adjunct to a surgical procedure, venous skin ulcer, and avascular necrosis.
  • the membrane may either directly or may indirectly promote one or more of: (i) a reduction of the amount and/or activity of pro-inflammatory cytokines; (ii) an increase in the amount and/or activity of anti-inflammatory cytokines; (iii) a reduction of the amount and/or activity of reactive oxygen species; (iv) an increase in the amount and/or activity of antioxidant agents; (v) a reduction of the amount and/or activity of proteases; (vi) an increase in cell proliferation; (vii) an increase in angiogenesis; and/or (viii) an increase in cell migration.
  • the disclosure provides a method of treating an inflammatory ocular condition in a subject comprising administering to the subject a membrane as disclosed herein, wherein the administration provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to treat the inflammatory ocular condition.
  • the method can comprise administration of the membrane using any technique that may be directed to promote epithelialization, reduce pain, and/or to generally reduce inflammation of eye tissue.
  • the membrane can be administered via a surgical inlay or grafting technique, via an onlay or patching technique, or via a combination of inlay and onlay techniques.
  • the method may be associated with eye surgery (e.g., photorefractive keratectomy (PRK)), eye trauma (e.g., lacerations, burns, or scrapes), or an eye disease that is characterized by inflammation or the treatment of which may result in an amount of inflammation in ocular tissue.
  • eye surgery e.g., photorefractive keratectomy (PRK)
  • eye trauma e.g., lacerations, burns, or scrapes
  • an eye disease that is characterized by inflammation or the treatment of which may result in an amount of inflammation in ocular tissue.
  • Non-limiting examples of indications that include an "inflammatory ocular condition" encompassed by the method include general repair/reconstruction of the corneal or conjunctiva surface(s) such as, for example, persistent epithelial defects; corneal ulceration; corneal transplant; descemetocele; corneal perforations; defects following excision of epithelial or subepithelial lesions or tumors (conjunctival tumors, conjunctival intraeptithelial neoplasia, subepithelial lesions, band keratopathy, scars, conjunctival folds parallel to the edges of eyelids); acute chemical burns; acute keratitis; painful bullous keratopathy; partial or complete limbal stem cell deficiency (with stem cell grafting); acute Stevens-Johnson syndrome; symbelpharon; fornix reconstruction; anophthalmia; bleb revisions; scleral thinning; and pterygium (see, e.g
  • the disclosure provides a method of promoting tissue repair and/or tissue regeneration in a subject comprising administering to the subject a membrane as disclosed herein wherein the administration provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to promote tissue repair and/or tissue regeneration.
  • the method is used in combination with a surgical procedure selected from the group consisting of a tissue graft procedure, tendon surgery, ligament surgery, bone surgery, and spinal surgery.
  • the tissue is human tissue.
  • the human tissue is cartilage, skin, ligament, tendon, or bone.
  • the membrane may directly or indirectly stimulates tissue regeneration.
  • the disclosure provides a method of modulating inflammatory response comprising administering to the wound a membrane as disclosed herein wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to reduce the amount and/or activity of proinflammatory cytokines, and/or increase the amount and/or activity of anti-inflammatory cytokines.
  • the pro-inflammatory cytokine is selected from TNF-a and IL- 1 a, or a combination thereof.
  • the anti-inflammatory cytokine may be selected from IL-10 or PGE2 or a combination thereof.
  • the disclosure provides a method of modulating protease activity comprising administering to the wound a membrane as described herein wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to reduce protease activity, and/or increase the amount and/or activity of protease inhibitors.
  • the protease may be selected from the group consisting of a matrix metalloproteinase (MMP) and elastase, or any combinations thereof.
  • the one or more protease inhibitors comprise a tissue inhibitor of matrix metalloproteinase (TIMP).
  • the disclosure provides a method of reducing the amount and/or activity of reactive oxygen species (ROS) comprising administering to the wound a membrane as described herein, wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to reduce the amount and/or activity of ROS and/ or increase the amount and/or activity of antioxidant agents.
  • the antioxidant capacity provided by the membrane may be equivalent to up to about a 250 mM solution of ascorbic acid.
  • the membrane may rescue cells from oxidant-induced apoptosis.
  • the membrane may reduce or prevent cells from undergoing apoptosis after being exposed to an oxidant and/or undergoing oxidative injury.
  • the products are able to reduce the amount of cells that undergo oxidant-induced apoptosis by at least about 50%, alternatively at least about 60%, alternatively at least about 70%, alternatively at least about 80%, alternatively at least about 90%.
  • the disclosure provides a method of increasing angiogenesis comprising administering to wound a membrane as described herein, wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to increase angiogenesis.
  • the membrane provides an increase the amount and/or activity of a vascular endothelial growth factor (VEGF) or epidermal growth factor (EGF) or a combination thereof.
  • VEGF vascular endothelial growth factor
  • EGF epidermal growth factor
  • the membrane promotes vessel formation.
  • the membrane is able to promote and/or enhance the formation of closed tubes or vessels in a tissue.
  • membranes promote the formation of closed tubes by HUVECs.
  • the disclosure provides a method of increasing cell migration comprising administering to the wound a membrane as described herein, wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective increase cell migration.
  • the membrane induces the cell migration of cells selected from the group consisting of endothelial cells, fibroblasts, and epithelial cells, and combinations thereof.
  • the disclosure provides a method of increasing cell proliferation comprising administering to wound a membrane as described herein, wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective increase cell proliferation.
  • the disclosure provides a method of preventing or reducing scar or contracture formation in a subject comprising administering to the subject a membrane as described herein, wherein the administration provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to prevent or reduce scar or contracture formation.
  • the membrane may increase the amount and/or activity of IFN-2a and/or TGF-33 in an amount effective to prevent or reduce scar formation.
  • any of the above aspects relating to methods of modulating inflammatory response, modulating protease activity, reducing the amount of reactive oxygen species/increasing the amount of antioxidant agents, increasing/promoting angiogenesis, increasing cell migration, increasing cell proliferation, or preventing or reducing scar or contracture formation the administration of the membrane may directly or indirectly stimulate or induce the method.
  • the placental products of the present technology may be used to treat any tissue injury.
  • a method of treatment may be provided, for example, by administering to a subject in need thereof, a placental product of the present technology.
  • a typical administration method of the present technology is topical administration.
  • Administering the present technology can optionally involve administration to an internal tissue where access is gained by a surgical procedure.
  • Placental products can be administered autologously, allogeneically or xenogeneically.
  • a present placental product is administered to a subject to treat a wound.
  • the wound is a laceration, scrape, thermal or chemical burn, incision, puncture, or wound caused by a projectile.
  • the wound is an epidermal wound, skin wound, chronic wound, acute wound, external wound, internal wounds, congenital wound, ulcer, or pressure ulcer.
  • Such wounds may be accidental or deliberate, e.g., wounds caused during or as an adjunct to a surgical procedure.
  • the wound is closed surgically prior to administration.
  • a present placental product is administered to a subject to treat a burn.
  • the burn is a first-degree burn, second-degree burn (partial thickness burns), third degree burn (full thickness burns), infection of burn wound, infection of excised and unexcised burn wound, loss of epithelium from a previously grafted or healed burn, or burn wound impetigo.
  • a present placental product is administered to a subject to treat an ulcer, for example, a diabetic ulcer (e.g. foot ulcer).
  • an ulcer for example, a diabetic ulcer (e.g. foot ulcer).
  • a placental product is administered by placing the placental product directly over the skin of the subject, e.g., on the stratum corneum, on the site of the wound, so that the wound is covered, for example, using an adhesive tape. Additionally or alternatively, the placental product may be administered as an implant, e.g., as a subcutaneous implant.
  • a placental product is administered to the epidermis to reduce rhytids or other features of aging skin. Such treatment is also usefully combined with so-called cosmetic surgery (e.g. rhinoplasty, rhytidectomy, etc.).
  • a placental product is administered to the epidermis to accelerate healing associated with a dermal ablation procedure or a dermal abrasion procedure (e.g. including laser ablation, thermal ablation, electric ablation, deep dermal ablation, sub-dermal ablation, fractional ablation, and microdermal abrasion).
  • a dermal ablation procedure or a dermal abrasion procedure e.g. including laser ablation, thermal ablation, electric ablation, deep dermal ablation, sub-dermal ablation, fractional ablation, and microdermal abrasion.
  • traumatic wounds e.g. civilian and military wounds
  • surgical scars and wounds spinal fusions
  • spinal cord injury e.g., avascular necrosis, reconstructive surgeries, ablations, and ischemia.
  • a placental product of the present technology is used in a tissue graft procedure.
  • the placental product is applied to a portion of the graft which is then attached to a biological substrate (e.g. to promote healing and/or attachment to the substrate).
  • tissue such as skin, cartilage, ligament, tendon, periosteum, perichondrium, synovium, fascia, mesenter and sinew can be used as tissue graft.
  • a placental product is used in a tendon or ligament surgery to promote healing of a tendon or ligament.
  • the placental product is applied to portion of a tendon or ligament which is attached to a bone.
  • the surgery can be any tendon or ligament surgery, including, e.g. knee surgery, shoulder, leg surgery, arm surgery, elbow surgery, finger surgery, hand surgery, wrist surgery, toe surgery, foot surgery, ankle surgery, and the like.
  • the placental product can be applied to a tendon or ligament in a grafting or reconstruction procedure to promote fixation of the tendon or ligament to a bone.
  • placental products of the present technology provide superior treatment (e.g. healing, healing time and/or healing strength) for tendon and ligament surgeries.
  • Tendon and ligament surgeries can involve the fixation of the tendon or ligament to bone.
  • osteogenic and/or chondrogenic potential of MSCs in the present placental products promotes healing process and healing strength of tendons or ligaments.
  • the present placental products provide an alternative or adjunctive treatment to periosteum-based therapies. For example, useful periosteum based treatments are described in Chen et al.
  • a tendon is sutured to and/or wrapped or enveloped in a placental membrane and the tendon is attached to a bone.
  • the tendon is placed into a bone tunnel before attached to the bone.
  • the tendon or ligament surgery is a graft procedure, wherein the placental product is applied to the graft.
  • the graft is an allograft, xenograft, or an autologous graft.
  • the tendon or ligament surgery is repair of a torn ligament or tendon, wherein the placental product is applied to the torn ligament or tendon.
  • Non-limiting examples of tendons to which a placental product can be applied include a digitorum extensor tendon, a hamstring tendon, a bicep tendon, an Achilles Tendon, an extensor tendon, and a rotator cuff tendon.
  • a placental product of the present technology is used to reduce fibrosis by applying the placental product to a wound site.
  • a placental product of the present technology is used as an anti-adhesion wound barrier, wherein the placental product is applied to a wound site, for example, to reduce fibrosis (e.g. postoperative fibrosis).
  • fibrosis e.g. postoperative fibrosis
  • Non-limiting examples of wound sites to which the placental product can be applied include those that are surgically induced or associated with surgery involving the spine, laminectomy, knee, shoulder, or child birth, trauma related wounds or injuries, cardiovascular procedures, requiring angiogenesis stimulation, brain/neurological procedures, burn and wound care, and ophthalmic procedures.
  • the wound site is associated with surgery of the spine and the stromal side of the placental product is applied to the dura (e.g. the stromal side facing the dura).
  • the dura e.g. the stromal side facing the dura
  • a placental product of the present technology can optionally be used to reduce adhesion or fibrosis of a wound.
  • Postoperative fibrosis is a natural consequence of all surgical wound healing.
  • postoperative peridural adhesion results in tethering, traction, and compression of the thecal sac and nerve roots, which cause a recurrence of hyperesthesia that typically manifests a few months after laminectomy surgery.
  • Repeated surgery for removal of scar tissue is associated with poor outcome and increased risk of injury because of the difficulty of identifying neural structures that are surrounded by scar tissue. Therefore, experimental and clinical studies have primarily focused on preventing the adhesion of scar tissue to the dura matter and nerve roots.
  • Spinal adhesions have been implicated as a major contributing factor in failure of spine surgery. Fibrotic scar tissue can cause compression and tethering of nerve roots, which can be associated with recurrent pain and physical impairment.
  • placental products taught herein are useful to reduce adhesion or fibrosis of a wound, at least in part, because the placental products can function in-situ to provide an environment that includes reduced numbers of immune cells as well as an increased number of cellular factors (e.g., TGF-P3, HGF, VGF, EGF, HE, hyaluronic acid, etc.).
  • TGF-P3, HGF, VGF, EGF, HE, hyaluronic acid, etc. One advantage of the wound dressings and processes of the present technology is that an anti-adhesion barrier is provided which can be used to prevent adhesions following surgery, and in particular following back surgery.
  • the disclosure relates to a method of manufacturing a placental product (or alternatively, a "membrane” in the examples that follow) comprising an amniotic membrane from placenta post-partum.
  • a placental product or alternatively, a "membrane” in the examples that follow
  • One such method includes:
  • Example 2 Exemplary Manufacturing Process of a Placental Product Containing a Chorionic Membrane
  • One method of manufacturing a placental product comprising a chorionic membrane and optionally an amniotic membrane from placenta post-partum includes:
  • Example 3 Exemplary Manufacturing Process of a chorioamniotic membrane product
  • the disclosure provides a method of manufacturing a placental product comprising an amniotic membrane and a chorionic membrane from placenta post-partum that includes:
  • Example 4 Exemplary Placental Product Manufacturing Process 00342 Further details regarding one method for manufacturing a placental product comprising an amniotic membrane according to the presently disclosed manufacturing procedure are provided below.
  • the placenta was processed inside a biological safety cabinet.
  • the umbilical cord was first removed, and the amniotic membrane was peeled from the underlying chorionic membrane using blunt dissection.
  • the membrane was rinsed with phosphate buffered saline (PBS) (Gibco Invitrogen, Grand Island, NY) to remove gross blood clots and any excess blood cells.
  • PBS phosphate buffered saline
  • USP 1 1 % anticoagulant citrate dextrose solution
  • ACD-A anticoagulant citrate dextrose solution
  • 00345 The amnion was then each disinfected in vented flasks with 250 mL of antibiotic solution consisting of gentamicin sulfate (50 ⁇ g/mL) (Abraxis Pharmaceutical Products, Schaumburg, IL), vancomycin HCI (50 ⁇ g/mL) (Hospira Inc., Lake Forest, IL), and amphotericin B (2.5 ⁇ g/mL) (Sigma Aldrich, St. Louis, MO) in Dulbecco's Modified Eagle Medium (DMEM) at 37°C ⁇ 2°C for 24-28 hours. After the incubation period, the membrane was washed with PBS to remove any residual antibiotic solution.
  • DMEM Dulbecco's Modified Eagle Medium
  • Optitran BA-S 85 reinforced nitrocellulose paper (Whatman, Dassel, Germany) and cut to the appropriate size prior to packaging into an FP- 90 cryobag (Charter Medical Ltd., Winston-Salem, NC). The stromal side of the amnion was mounted towards the nitrocellulose paper.
  • HSA human serum albumin
  • the FP-90 cryobag was placed into a mangar bag (10 in. x 6 in.) (Mangar Industries, New England, PA), which was then heat sealed.
  • the mangar bag was placed into a packaging carton (10.5 in. x 6.5 in. x 0.6 in.) (Diamond Packaging, Rochester, NY). All cartons were refrigerated at 2-8°C for 30-60 minutes prior to freezing at -80°C ⁇ 5°C inside a Styrofoam container.
  • Example 4.1 Thawing time for membrane mounted on nitrocellulose paper. 00348
  • the cryopreserved membranes described herein can exhibit thawing properties that are more rapid than other cryopreserved products.
  • a rapid thaw profile allows for the membranes described herein to be used more efficiently and effectively for on-demand uses and application.
  • One thawing protocol is discussed below.
  • Table 1 Thawing time for placental membrane products cryopreserved with 50ml_ cryopreservation solution.
  • Example 5 Exemplary Manufacturing Process of a chorioamniotic membrane product
  • chorioamniotic membrane was then disinfected in vented flasks with 500 ml_ of antibiotic solution consisting of gentamicin sulfate (50 ⁇ g/mL) (Abraxis Pharmaceutical Products, Schaumburg, IL), vancomycin HCI (50 ⁇ g/mL) (Hospira Inc., Lake Forest, IL), and amphotericin B (2.5 ⁇ g/mL) (Sigma Aldrich, St. Louis, MO) in DMEM at 37°C ⁇ 2°C for 24-28 hours. After the incubation period, the membranes were washed with PBS to remove any residual antibiotic solution. The trophoblast layer of the chorion was gently removed using blunt dissection.
  • gentamicin sulfate 50 ⁇ g/mL
  • vancomycin HCI 50 ⁇ g/mL
  • amphotericin B 2.5 ⁇ g/mL
  • 00353 The membranes were mounted on Optitran BA-S 85 reinforced nitrocellulose paper (Whatman, Dassel, Germany) and cut to the appropriate size prior to packaging into an FP-90 cryobag (Charter Medical Ltd., Winston-Salem, NC. Once a membrane unit was placed into the FP-90 cryobag and the cryobag was heat sealed, 50 ml_ of a cryopreservation solution containing 10% dimethyl sulfoxide (DMSO) (Bioniche Teo. Inverin Co., Galway, Ireland) and 5% human serum albumin (HSA) (Baxter, West Lake Village, CA) in PlasmaLyte-A (Baxter Healthcare Corp., Deerfield, IL) were added through the center tubing line. Any excess air was removed, and the tubing line was subsequently sealed.
  • DMSO dimethyl sulfoxide
  • HSA human serum albumin
  • the FP-90 cryobag was placed into a mangar bag (10 in. x 6 in.) (Mangar Industries, New England, PA), which was then heat sealed.
  • the mangar bag was placed into a packaging carton (10.5 in. x 6.5 in. x 0.6 in.) (Diamond Packaging, Rochester, NY). All cartons were refrigerated at 2-8°C for 30-60 minutes prior to freezing at -80°C ⁇ 5°C inside a Styrofoam container.
  • the placental membranes were removed from the FP-90 cryobag and placed into a reservoir containing saline (Baxter Healthcare Corp., Deerfield, IL) for a minimum of 1 minute and a maximum of 60 minutes. Each membrane was detached from the reinforced nitrocellulose paper prior to digestion.
  • saline Boxter Healthcare Corp., Deerfield, IL
  • the suspension was filtered through a 100 ⁇ cell strainer nylon filter to remove any debris. Pass 15ml DMEM through the strainer into the same conical tube. Centrifugation at 2000 rpm for 10 minutes was performed, and supernatant was removed. Cell pellets were reconstituted with a volume of DMEM that was proportional to the pellet size, and 20 ⁇ _ of the resuspended cell suspension were mixed with 80 ⁇ _ of trypan blue (Sigma Aldrich, St. Louis, MO) for counting. The cell count sample was placed into a hemocytometer and evaluated using a microscope.
  • 00357 Chorionic membranes were digested with 25 ml_ of 0.75% collagenase solution at 37°C ⁇ 2°C for 20-40 minutes on a rocker. After collagenase digestion, the suspension was filtered through a 100 ⁇ cell strainer nylon filter to remove any debris. Centrifugation at 2000 rpm for 10 minutes was performed, and supernatant was removed. Cell pellets were reconstituted with a volume of DMEM that was proportional to the pellet size, and 20 ⁇ _ of the resuspended cell suspension were mixed with 80 ⁇ _ of trypan blue for counting. The cell count sample was placed into a hemocytometer and evaluated using a microscope.
  • Table 2 contains average cells per cm 2 and cell viability values for the amniotic and chorionic membranes from 32 placenta lots.
  • placental product comprising an amniotic membrane containing about 10,000 to about 360,000 cells/cm 2 . Since the amniotic membrane consists of epithelial cells and stromal cells, experiments were conducted to determine the ratio of epithelial cells to stromal cells. Amniotic membranes from 3 placenta lots were analyzed. First, a 5 cm x 5 cm piece of amniotic membrane was digested with approximately 25 ml_ of 0.05% trypsin-EDTA (Lonza, Walkersville, MD) at 37°C ⁇ 2°C in a water bath for 30 minutes.
  • epithelial cells were removed by gently scraping the cells from the membrane. After rinsing with PBS (Gibco Invitrogen, Grand Island, NY), the membrane was subsequently digested in the same manner as chorionic membrane (described above). In addition, another intact 5 cm x 5 cm piece of amniotic membrane was digested using the standard procedure (described above) to determine the total number of cells. The percentage of stromal cells was then determined by dividing the cell count from the amniotic membrane with the epithelial cells removed with the cell count from the intact membrane.
  • Cryopreservation is a method that provides a source of tissues and living cells.
  • a main objective of cryopreservation is to minimize damage to biological materials during low temperature freezing and storage.
  • general cryopreservation rules are applicable to all cells, tissues, and organs, a specific cryopreservation solution and procedure must be developed for each type of biological material.
  • the present application discloses a cryopreservation procedure for placental membrane products that can selectively deplete immunogenic cells from the placental membranes and preserve viability of other beneficial cells that are the primary source of factors for the promotion of healing.
  • cryopreservation method development for placental membranes the present inventors evaluated key parameters of cryopreservation including volume of cryopreservation solution, effect of tissue equilibration prior to freezing, and cooling rates for a freezing procedures.
  • Cryopreservation is an approach which can be utilized to reduce tissue immunogenicity. This approach is based on differential susceptibility of different cell types to freezing injury in the presence of DMSO; leukocytes are sensitive to fast cooling rates. The freezing rate of 1 °C/min is considered optimal for cells and tissues including immune cells. Rapid freezing rates such as 60- 100°C/min eliminate immune cells. However, this type of procedure is harmful to other tissue cells, which are desirable for preservation according to the present technology.
  • the developed cryopreservation procedure utilized a cryopreservation solution containing 10% DMSO, which is a key component protecting cells from destruction when water forms crystals at low temperatures.
  • the second step of cryopreservation was full equilibration of placental membrane in the cryopreservation solution, which was achieved by soaking membranes in the cryopreservation solution for 30-60 min at 4°C. This step allowed DMSO to penetrate the placental tissues.
  • condition 6 and condition 2 involved direct freezing (-80°C ⁇ 5°C) of the cryobags either inside a Styrofoam container or on the freezer shelf.
  • condition 6 and condition 2 exhibited the most gradual temperature decreases. Gradual temperature decreases are typically desired in order to preserve cell viability.
  • condition 2 included a 30-minute refrigeration step. Therefore, the decrease in temperature from the start of freezing to -4°C, where latent heat evolution upon freezing occurs, was examined further.
  • the rate of cooling was approximately -1 °C/minute during this period.
  • the rate of cooling for condition 2 was approximately -0.4°C/minute during the same timeframe. Therefore, condition 2 was selected for incorporation into a non-limiting cryopreservation process since slower rates of cooling are generally desired to maintain optimal cell viability.
  • Figure 2A depicts the effects of cryopreservation solution volume on process (cryopreservation) cell recovery for the amniotic membrane.
  • Figure 2B depicts the effects of cryopreservation solution volume on cell viability for the amniotic membrane.
  • cryopreservation cell recovery was calculated by comparing the number of viable cells before and after the cryopreservation process (as described previously herein). The intention was to identify cryopreservation conditions that provide maximum cell recovery. However, the utilized assay (trypan blue exclusion) requires taking tissue samples from different parts of the placenta, and since each tissue sample contains different cell counts, it is impossible to obtain an accurate cell recovery measurement. This explains the great range of process cell recovery (10% - 410%) we have encountered. On the other hand, cell viability was calculated by comparing the number of live cells with the total number of cells in the same piece of tissue. Therefore, cell viability was used to optimize the parameters of the cryopreservation procedure.
  • the 50ml_ volume of cryopreservation solution volume provided equivalent cell recovery compared to that of the 10 ml_ and 20ml_ for a 5x5 placental membrane.
  • 10 ml_ cryopreservation solution volume is sufficient to provide placental product with >70% cell viability according to the present technology.
  • the same results were obtained for chorionic membrane as seen by Figures 3A and 3B.
  • the mechanism of the assay is based on that fact that the metabolic reduction of the soluble tetrazolium salt to a blue formazan precipitate is dependent on the presence of viable cells with intact mitochondrial function.
  • Samples were incubated in the MTT assay medium for 3-4 hours. At the end of the conversion period, samples were extracted for 1 hour at 37°C in DMSO. At the end of the extraction period, 0.2 ml_ of each extract was transferred to a well of a 96-well plate.
  • the absorbencies, which are proportional to cell viability, were read on a plate reader (Spectramax 340PC, Molecular Devices) at 570 nm with the DMSO extraction buffer as a blank.
  • Figure 4 shows the MTT values obtained from fresh and thawed cryopreserved samples. As indicated from the data, cell viability did not show significant difference between fresh and post-thaw cryopreserved chorioamniotic samples.
  • FIG. 5A amniotic membrane
  • Figure 5B chorionic membranes
  • the first condition involved directly freezing the product unit on a shelf within the freezer (-80°C ⁇ 5°C).
  • the second condition also contained a direct freeze, but the product unit was placed into a Styrofoam container within the freezer.
  • the third condition included a refrigeration (about 2°C to about78°C) period of 30 minutes prior to the freezing step.
  • 3 placenta lots were evaluated.
  • Two (2) placenta lots were analyzed for the chorionic membrane. Results indicated that the third condition was optimal for both membrane types.
  • Membranes can be 66,565 6.2% 14.3% held in saline for up to 1 hr.
  • cm centimeter
  • min minutes
  • temp temperature
  • hr hour
  • SD standard deviation
  • cryopreservation method is able to preserve cell viability for prolonged periods of time.
  • three lots of amniotic membranes were manufacturing using the final manufacturing and cryopreservation process.
  • Cell viability was determined using the quantitative method described in example 6 at Time 0 (1 week in storage), 12 months, 24 months and 25 months after storage. Table 6 demonstrates that after prolonged storage and subsequent thawing, cell viability remained above 70% for all lots at all time points.
  • amniotic and chorionic membranes were stained using a LIVE/DEAD ® Viability/Cytotoxicity kit (Molecular Probes Inc., Eugene, OR) to qualitatively assess cell viability. Staining was performed as per the manufacturer's protocol. Membrane segments of approximately 0.5 cm x 0.5 cm were used. Evaluation of stained membranes was performed using a fluorescent microscope. An intense uniform green fluorescence indicated the presence of live cells, and a bright red fluorescence indicated the presence of dead cells. Images of fresh amniotic and chorionic membranes as well as cryopreserved amniotic and chorionic membranes demonstrated that the manufacturing process did not alter the phenotypic characteristics of the membranes post thaw.
  • FIG. 7 contains representative images of the epithelial layer of fresh amniotic membrane (A), epithelial layer of cryopreserved amniotic membrane (B), stromal layer of fresh amniotic membrane (C), stromal layer of cryopreserved amniotic membrane (D), fresh chorionic membrane (E), and cryopreserved chorionic membrane (F). Live cells are green, and dead cells are red. These images demonstrated that the manufacturing process did not alter the phenotypic characteristics of the membranes and the proportion of viable cell types (epithelial and stromal cells) in the membranes post thaw.
  • amnion and chorion One unique feature of the human amnion and chorion is the absence of fetal blood vessels that prevent mobilization of leukocytes from fetal circulation. On the fetal side, macrophages resident in the chorioamniotic mesodermal layer represent the only population of immune cells. Thus, fetal macrophages present in the amnion and chorion are the major source of tissue immunogenicity.
  • the present inventors believe that removal of the maternal trophoblasts and CD14+ fetal macrophages, among other immunogenic cell types, from placental membranes prevents activation of lymphocytes in vitro. Removal of the maternal trophoblasts can be achieved by direct cleaning, whereas cryopreservation process described in this technology eliminates CD14+ cells.
  • MLR ixed Lymphocyte Reaction
  • LPS Lipopolysaccharide
  • TNF Tumor Necrosis Factor
  • An MLR is a widely used type of in vitro assay to test cell and tissue immunogenicity.
  • the assay is based on the ability of immune cells (responders) derived from one individual to recognize allogeneic Human Leukocyte Antigen (HLA) and other antigenic molecules expressed on the surface of allogeneic cells and tissues (stimulators) derived from another individual when mixed together in a well of an experimental tissue culture plate.
  • the response of immune cells to stimulation by allogeneic cells and tissues can be measured using a variety of methods such as secretion of particular cytokines (e.g., Interleukin (IL-2), expression of certain receptors (e.g., IL-2R), or cell proliferation, all of which are characteristics of activated immune cells.
  • cytokines e.g., Interleukin (IL-2)
  • IL-2R Interleukin
  • cell proliferation all of which are characteristics of activated immune cells.
  • Placental tissue samples representing different steps of the presently disclosed manufacturing process were used for immunogenicity testing. These samples included amnion with choriotrophoblast (ACT) as a starting material, separated choriotrophoblast (CT), chorion (CM), trophoblast (T), amnion (AM), and amnion and chorion (A/C) Both freshly purified and cryopreserved (final products) tissues were tested.
  • ACT amnion with choriotrophoblast
  • CT choriotrophoblast
  • CM chorion
  • T trophoblast
  • AM amnion
  • A/C amnion and chorion
  • MLR assay cells from placental tissues were isolated using 280 U/mL of collagenase type II (Worthington, Cat No. 4202). Tissues were treated with enzyme for 60- 90 min at 37°C ⁇ 2°C, and the resulting cell suspension was filtered through a 100 ⁇ filter to remove tissue debris. Single cell suspensions were then centrifuged using a Beckman, TJ-6 at 2000 rpm for 10 min and washed twice with DPBS. Supernatant was discarded after each wash, and cells were resuspended in 2 mL of DMEM (Invitrogen, Cat No.
  • hPBMCs Human Peripheral Blood Mononuclear Cells alone were used as a negative control, and a mixture of two sets of hPBMCs derived from two different donors was used as a positive MLR control.
  • IL-2Ra was measured in cell lysates using the IL-2Ra ELISA kit (R&D Systems, Cat No. SR2A00) according to the manufacturer's protocol.
  • IL-2Ra The level of IL-2Ra is a measure of activation of T-cells in response to immunogenic molecules expressed by allogeneic cells. Results presented in Figures 8 and 9 demonstrate a method of manufacture of placental membranes, resulting in low immunogenicity of the final products.
  • FIG. 8 demonstrates the manufacturing process serially reduces immunogenicity of the placental product.
  • Samples representing different steps of the manufacturing process were co-cultured with hPBMCs for 4 days.
  • IL-2Ra was measured in cell lysates as a marker of T-cell activation.
  • Negative control shows a basal level of immune cell activation: PBMCs derived from one donor were cultured alone.
  • Positive control a mixture of PBMCs derived from 2 different donors.
  • Figure 9 demonstrates selective depletion of immunogenicity results from the present cryopreservation process of producing the present placental products, as evidenced by the significant decrease in immunogenicity upon cryopreservation.
  • fetal macrophages present in the amnion and chorion are a major source of tissue immunogenicity.
  • tissue immunogenicity Macrophages in fetal placental membranes respond to bacterial LPS by secretion of inflammatory cytokines such as TNF-a. Therefore, secretion of TNF- ⁇ in response to LPS is used here to characterize tissue immunogenicity of placental membranes at each critical manufacturing step. Samples from each manufacturing step included trophoblast (T), amnion with chorio trophoblast (ACT), choriotrophoblast (CT), chorion (CM), and amnion (AM).
  • T trophoblast
  • ACT amnion with chorio trophoblast
  • CT choriotrophoblast
  • CM chorion
  • AM amnion
  • TNF-a detected in the culture medium from greater than about 70 pg/cm 2 (corresponding to 280 pg/mL) for both spontaneous and LPS-induced TNF-a secretion was considered immunogenic (Fortunato, et al.1996).
  • CT Choriotrophoblast membranes
  • Figure 1 1 CT cells were cultured with PBMCs for 4 days.
  • IL-2Ra was measured in cell lysates as a marker of T-cell activation.
  • Positive control a mixture of PBMCs derived from 2 different donors. Results of this assay, as seen in Figure 1 1 , showed a correlation with the MLR data: tissues that produce high levels of TNF-a in response to LPS are immunogenic in the MLR assay
  • the low levels of TNF-a and the absence of the response to LPS by AM and CM indicates the exemplary cryopreservation method described in the current technology eliminates viable functional macrophages from the amniotic and chorionic membranes, which ensures the safety of such an allogeneic product.
  • amnion and chorionic membranes are important for developing a thorough understanding of potential functional roles in wound healing and immunogenicity.
  • amnion also contains epithelial cells.
  • both membranes comprise resident fetal macrophages.
  • the close proximity to maternal blood circulation and decidua provide a potential source of immunogenic cells (maternal leukocytes and trophoblast cells) and therefore are a potential source of immunogenicity.
  • FACS Fluorescence-activated cell sorting
  • CD45-PE BD 555483 Cell surface Pan Hematopoetic cell marker
  • CD34 - APC BD 340667 Cell surface Hematopoetic marker
  • Table 8 shows the characterization of the cellular composition of placental membranes based on selective CD markers.
  • Table 9 provides a FACS analysis of cells isolated from the amniotic and chorionic membranes that were cultured in 10% FBS in DMEM at 37°C ⁇ 2°C until confluency (passage 0 cells).
  • Example 13 Adherence of cells derived from Placental Products.
  • a cell in addition to the presence of specific cellular markers, a cell can be classified as an MSC if it shows plastic adherence properties under normal culture conditions and has a fibroblast-like morphology.
  • Cells were isolated from the amniotic and chorionic membrane products as described in this technology, plated into MSC media and cultured at 37 °C ⁇ 2°C until they reach confluency. Their ability to adhere to the plastic culture dishes was then evaluated.
  • Figure 12 demonstrates plastic adherence of cells isolated and cultured from amniotic (Figure 12A) and chorionic (Figure 12B), which is similar to MSCs isolated and expanded from human bone marrow aspirate ( Figure 12C). These data show that cells derived from amniotic and chorionic membranes retain a phenotype similar to MSCs.
  • MSCs are also defined by their ability to differentiate into different connective tissue types.
  • placental-derived cells to undergo osteogenic differentiation was tested.
  • Placental cells were isolated and cultured at 37 °C ⁇ 2 °C until they reach confluency.
  • osteogenic medium was added to the culture and expression of alkaline phosphatase was measured.
  • Alkaline phosphatase is an enzyme involved in the mineralization of bone and is a well known osteogenic marker.
  • Figure 12D shows that several cells are stained with a purple dye which represents alkaline phosphatase expression. This demonstrates that MSCs present in placental membranes retain their differentiation potential.
  • VEGF Vascular endothelial growth factor
  • GuHCI 8M guanidine hydrochloride
  • ELISA Enzyme-Linked Immunosorbant Assay
  • Angiogenesis Angiotensin-2 (Ang-2), Fibroblast Growth
  • bFGF Factor basic (bFGF), heparin-bound Function Therapeutic Factors
  • HB-EGF Epidermal Growth Factor
  • EGF Keratinocyte Growth Factor
  • KGF-7 Keratinocyte Growth Factor
  • PDGF Platelet derived Growth Factors
  • VEGF Vascular Endothelial Growth Factor
  • HGF Hepatocyte Growth Factor
  • PIGF Placental Growth Factor
  • PEDF Pigment Epithelium Derived Factor
  • TSP-2 Trombospondin-1
  • EGF Epidermal Growth Factor
  • KGF Keratinocyte Growth Factor
  • Adiponectin Acrp-30
  • IGF Insulin Growth Factor 1
  • IGFBP 1 Insulin-like growth factor binding protein
  • TGFa Transforming Growth Factor a
  • Chemoattractant Stromal Cell Derived Factor 1 Beta (SDF- 1 b), bFGF, EGF, KGF
  • MMP1 Matrix Metalloproteinase 1
  • MMP2 Protease Inhibitors
  • MMPs (TIMP1 and 2), Alpha-2- macroglobulin, Fibronectin Function Therapeutic Factors
  • G- CSF Immunoregulatory Granulocyte Colony-Stimulating Factor
  • IL- 1 RA Interlaukinl receptor antagonist
  • LIF Leukemia Inhibitory Factor
  • IFN-2a Interferon 2a
  • PLAB Placental Bone Morphogenetic Protein
  • Amniotic and chorionic membranes were isolated and packaged at -80°C ⁇ 5°C according to the manufacturing protocols disclosed herein in Examples 1 and 2. Packaged membranes were then thawed in a 37°C ⁇ 2°C water bath and washed 3 times with DPBS. Membranes were cut into 8 cm 2 pieces. For tissue lysate samples, one 8 cm 2 piece of membrane was snap frozen in liquid nitrogen followed by pulverization using a mortar and pestle. Crushed tissue was transferred to a 1 .5 ml_ microcentrifuge tube and 500 ⁇ _ of Lysis buffer (Cell Signaling Technologies, Cat No. 9803) with protease inhibitor (Roche, Cat No.
  • Placental product lysates were analyzed for the presence of proteins that are important in tissue repair.
  • Table 1 1 depicts the biochemical profile of the lysates of exemplary placental tissue products of the present technology.
  • Placental products of the present technology also demonstrate a durable effect, which is desirable for wound healing treatments.
  • the extracellular matrix and presence of viable cells within the amniotic membrane described herein allow for a cocktail of proteins that are known to be important for wound healing to be present for at least 14 days.
  • Amniotic membranes were thawed and plated onto tissue culture wells and incubated at 37 °C ⁇ 2°C for 3, 7, and 14 days. At each time point, a sample of the culture supernatant was collected and measured through protein array analysis as described in Example 15.1 .
  • Table 12 illustrates the level of various secreted factors in tissue culture supernatants of amniotic membrane lots at 3, 7 and 14 days as measured through protein array analysis. Table 12. Levels of proteins secreted in amnion tissue culture supernatants at different time points (pg/ml).
  • IFN-2a and TGF-33 are cytokine/growth factor known to reduce fibrosis in various tissues.
  • IFN-2a and TGF-33 have been suggested to modulate wound healing by the prevention of scar and contracture formation (Ishida, Kondo et al. 2004; Ferguson, Duncan et al. 2009).
  • IFN-2a may serve a role to inhibit fibroblast proliferation, decrease collagen and fibronectin synthesis and fibroblast-mediated wound contracture (Wang, Crowston et al. 2007).
  • IFN-2a has been administered subcutaneously and shown to improve scar quality (Nedelec et al, Lab Clin Med 1995, 126:474).
  • TGF- ⁇ 3 regulates the deposition of extracellular matrix and has been shown to decrease scar formation when injected in rodent cutaneous wound models. Clinically, TGF- ⁇ 3 has been shown to improve scar appearance when injected at the wound site (Occleston et al., J Biomater Sci Polym Ed 2008, 19:1047). TGF-33 works as a TGF- ⁇ antagonist, modulating fibroblast-myofibroblast differentiation, and restricting profibrotic gene transcription (Chang, Kishimoto et al. 2014).
  • Placental products described in the present technology have been analyzed for the presence of IFN-2a and TGF-33. Briefly, after thawing, the membranes were homogenized and centrifuged at 16,000g to collect the resulting supernatants. Supernatants were analyzed on a commercially available ELISA kit from MabTech (IFN-2a) and R&D Systems (TGF-33). Figure14 shows significant expression of IFN-2a (A) and TGF-33 (B) in placental product homogenates.
  • bFGF modulates a variety of cellular processes including angiogenesis, tissue repair, and wound healing (Presta et al., 2005, Reuss et al., 2003, and Su et al., 2008). In wound healing models, bFGF has been shown to increase wound closure and enhance vessel formation at the site of the wound (Greenhalgh et al., 1990). Evaluation of proteins derived from amniotic and chorionic membranes prepared pursuant to the presently disclosed manufacturing process revealed that bFGF is one of the major factors in placental tissue protein extracts.
  • Figure 14 B depicts expression of bFGF by amniotic membranes (AM) and chorionic membranes (CM) from two donors detected during the protein profile evaluation of placental membranes.
  • AM amniotic membranes
  • CM chorionic membranes
  • BMP-2 and BMP-4 may stimulate differentiation of MSCs to osteoblasts in addition to promote cell growth; placental BMP or PLAB is a novel member of the BMP family that is suggested to mediate embryonic development.
  • IGF-1 insulin-like growth factor 1
  • PIGF Placental derived growth factor
  • a2-macroglobulin is known as a plasma protein that inactivates proteinases from all 4 mechanistic classes, serine proteinases, cysteine proteinases, aspartic proteinases, and metalloproteinases. Another important function of this protein is to serve as a reservoir for cytokines and growth factors, examples of which include TGF, PDGF, and FGF. In the chronic wounds like diabetic ulcers or venous ulcers, the presence of high amount of proteases leads to rapid degradation of growth factors and delays in wound healing. Thus, the presence of o2-macroglobulin in products designed for chronic wound healing will be beneficial.
  • the membrane was thawed and washed according to the manufacturer's instructions. Dermagraft membrane was cut into 7.5 cm 2 pieces. For tissue lysates, one 7.5 cm 2 piece of membrane was snap frozen in liquid nitrogen followed by pulverization using a mortar and pestle. Crushed tissue was transferred to a 1 .5 mL microcentrifuge tube and 500 ⁇ _ of Lysis buffer (Cell Signaling Technologies, Cat No. 9803) with protease inhibitor (Roche, Cat No. 1 1836153001 ) was added and incubated on ice for 30 min with frequent vortexing. The sample was then centrifuged at 16000 g for 10 min.
  • Lysis buffer Cell Signaling Technologies, Cat No. 9803
  • protease inhibitor Roche, Cat No. 1 1836153001
  • the supernatant was collected and sent for protein array analysis by Aushon Biosystems.
  • tissue culture one 7.5 cm 2 piece of membrane was plated onto a well of a 12-well dish and 2 ml_ of DMEM +1 % HSA+ antibiotic/antimycotic were added and incubated at 37°C ⁇ 2°C for 3, 7, or 14 days. After incubation, tissue and culture media were transferred to a 15 mL conical tube and centrifuged at 2000 rpm for 5 min. Culture supernatant was collected and sent for protein array analysis by Aushon Biosystems.
  • the membrane was cut into 7.3 cm 2 pieces.
  • tissue lysates one 7.3 cm 2 piece of membrane was snap frozen in liquid nitrogen followed by pulverization using a mortar and pestle. Crushed tissue was transferred to a 1 .5 mL microcentrifuge tube and 500 ⁇ of Lysis buffer (Cell Signaling Technologies, Cat No. 9803) with protease inhibitor (Roche, Cat No. 1 1836153001 ) was added and incubated on ice for 30 min with frequent vortexing. The sample was then centrifuged at 16000 g for 10 min. The supernatant was collected and sent for protein array analysis by Aushon Biosystems.
  • tissue culture For tissue culture, one 7.3 cm 2 piece of membrane was plated onto a well of a 12-well dish and 2 mL of DMEM +1 % HSA+ antibiotic/antimycotic were added and incubated at 37°C ⁇ 2°C for 3, 7, or 14 days. After incubation, tissue and culture media were transferred to a 15 mL conical tube and centrifuged at 2000 rpm for 5 min. Culture supernatant was collected and sent for protein array analysis by Aushon Biosystems. Example 16.2 Therapeutic Factors present in Day 3 Supernatants of Exemplary Placental Tissues and Commercially Available Products
  • Protein array data analyses showed that the majority of selected testing factors (refer to Table 1 1 ) were expressed in amniotic membrane, chorionic membrane, Apligraf, and Dermagraft. Three proteins were identified as unique for the amniotic membrane and/or the chorionic membrane which are undetectable in Apligraf and Dermagraft. These proteins are EGF, IGFBP1 , and Adiponectin. All three proteins are important for wound healing.
  • Figure 16 depicts expression of EGF (A), IGFBP1 (B), and Adiponectin (C) in amniotic (AM), chorionic membrane (CM) and commercially available products.
  • AM75 and AM 78 are cryopreserved placental products of the present technology (e.g. cryopreserved), while CM75 and CM78 are cryopreserved chorionic membrane products. These proteins are believed by the inventors to facilitate the therapeutic efficacy of the present placental products for wound healing.
  • Table 14 depicts the biochemical profile of the supernatants of exemplary placental products of the present technology and two commercially available products (results adjusted per 8 cm 2 after subtraction of the negative background).
  • AM75 and AM 78 are placental products of the present technology (e.g. cryopreserved) and CM75 and CM78 are cryopreserved chorionic membrane products.
  • MMPs and TIMPs are among the factors that are important for wound healing. However, expression of these proteins must be highly regulated and coordinated. Excess of MMPs versus TIMPs is a marker of poor chronic wound healing. We investigated expression of MMPs and TIMPs and its ratio in amniotic membrane and chorionic membrane and compared it to the expression profile in Apligraf and Dermagraft.
  • MMP to TIMP ratio is higher in cases of nonhealing wounds.
  • the ratio between MMP-9 and TIMP1 is approximately 7 - 10 to one for good healing and 18-20 or higher for poor healing.
  • Analysis of the ratio between MMPs and TIMPs secreted by placental tissues, Apligraf, and Dermagraft showed that the amniotic and chorionic membrane products contain MMPs and TIMPs at an approximate ratio of 7, which is favorable for wound healing. In contrast, Dermagraft had a ratio > 20, and Apligraf had a ratio >200.
  • EGF is among the factors that are important for wound healing (Schultz et al., 1991 , Komarcevic, 2000, and Hong et al., 2006). The absence or decreased amount of EGF is one characteristic of chronic wounds (Harding et al., 2002). Evaluation of proteins derived from amniotic membrane samples prepared according to the developed manufacturing process disclosed by the present application reveal that EGF is one of the major factors secreted in higher quantities by these tissues. The importance of EGF for wound healing together with high levels of EGF detected in the presently disclosed amniotic membranes support selection of EGF as a potency marker for evaluation of membrane products manufactured for clinical use pursuant to the present disclosure.
  • ELISA qualification meets the standards established by the FDA and ICH guidances for bioanalytical assay validation (Validation of Analytical Procedures: Text and Methodology Q2(R1 ), 1994; ICH Harmonized Tripartite Guideline and Guidance for Industry Bioanalytical Method Validation, 2001 ).
  • Amniotic membranes evaluated for expression of EGF by this method confirmed protein array data and further demonstrated that EGF was a unique factor expressed at clinically significant levels in these tissues.
  • Chronic wounds fail to progress through the normal stages of healing, often stalling in the inflammatory stage (Maxson, Lopez et al. 2012).
  • the characteristics of the chronic wounds environment include 1 ) high levels of proinflammatory cytokines such as TNF-a and IL-1 a, 2) low levels of anti-inflammatory cytokines, 3) high levels of proteases and low levels of their inhibitors, as well as 4) high levels of oxidants and low levels of antioxidant to counter balance. Therefore inflammation control at the site of injury is the key to restart/progress the healing process.
  • the effect of placental membranes on antiinflammatory activity was investigated for wound healing.
  • Tissue samples were cultured with and without 1 ng/ml of TNF-a (proinflammatory factor) for 18 hours. Supernatants were collected and the concentration of prostaglandin E2 (PGE2) was then measured using PGE2 monoclonal enzyme immunoassay assay (EIA) kit (Cayman).
  • PGE2 monoclonal enzyme immunoassay assay (EIA) kit (Cayman). Figure 19 showed that our placental membrane products (amniotic membrane product) produce high levels of anti-inflammatory cytokine PGE2 when exposed to TNF-a.
  • PBMC peripheral blood mononuclear cells
  • T cell mitogens - anti-CD3 monoclonal antibodies (CD3) and anti-CD28 monoclonal antibodies (CD28) were added at ⁇ g per mL to activate immune cells.
  • Tissue samples were then incubated with activated PBMCs for 48 hours at 37°C and 5% C0 2 in a humidified atmosphere.
  • TNF-a, and IL-1 a production were measured in supernatants using ELISA Duoset kits (R&D systems).
  • MMPs and neutrophil elastase activity are involved in normal physiological and pathologic processes, such as degradation of basement membrane, remodeling of ECM, connective tissue turnover, angiogenesis, reproduction and wound repair.
  • MMPs and neutrophil elastase destroy components of ECM and damage growth factors and their receptors that are essential for healing.
  • placental membrane products mediate the inhibition of MMPs and elastase.
  • Azocoll is an insoluble, ground collagen to which a purple azodye is impregnated. Upon proteolysis, soluble azodye is released and can be detected by absorbance at 550nm. Therefore, azocoll is often used as a chromogenic non-specific substrate to examine the protease activity in the environment.
  • the assay was performed using a modified method developed by Jiang et al (Jiang, Tan et al. 2007). Azocoll was washed and suspended in 10mM PBS, pH 7.4, at a final concentration of 1 .5 mg/mL.
  • Collagenase IV was used as the positive control because its two active forms, MMP-2 and MMP-9 (72kD and 92kD, respectively), have been shown to have elevated expression in wound fluid from chronic leg ulcer, which is correlated with poor healing (Trengove, Stacey et al. 1999).
  • Amniotic membrane product was incubated with 0.1 % (w/v) collagenase IV (Life Technologies) and azocoll suspension under gentle end-to-end rotation at 37°C incubator for 5 hours. The reaction was stopped by centrifuging the samples at 10,000 xg for 8 minutes.
  • ROS reactive oxygen species
  • NHDFs normal human dermal fibroblasts
  • tbOOH tert-butyl hydroperoxide
  • Neovascularization is a crucial step in the wound healing process.
  • the formation of new blood vessels is necessary to provide the fibroblasts with sufficient nutrient supply for the production of a provisional granulation matrix and the survival of keratinyocytes.
  • HUVECs (Lonza) were seeded with conditioned medium derived from amniotic membrane product, at a concentration of 10 4 cells/well on Matrigel (BD)-coated culture wells.
  • Conditioned medium was obtained by culture amniotic membrane products in endothelial cell growth medium EBM (Lonza) supplemented with 2% FBS for three days.
  • EBM supplemented with all necessary cocktail of growth factors were used positive controls. After 8 hours of incubation, fields from each sample were randomly photographed by inverted microscopy, and the number of closed tubes were counted and plotted by ImageJ (NIH).
  • Figure 25 demonstrates that conditioned medium from amniotic membrane products enhances tube formation, equivalent to positive control.
  • Example 20 Placental tissues enhance cell migration and wound healing
  • the process of wound healing is highly complex and involves a series of structured events controlled by growth factors (Goldman, 2004). These events include increased vascularization, infiltration by inflammatory immune cells, and increases in cell proliferation.
  • the beginning stages of wound healing revolve around the ability of individual cells to polarize towards the wound and migrate into the wounded area In order to close the wound area and rebuild the surrounding tissue.
  • Three types of cells are mainly involved in the cell migration process. They are vascular endothelial cells, fibroblasts and keratinocytes. Vascular endothelial cells migrate to the area of the wound and form new blood vessels, which provides the necessary oxygen and nutrients for proper wound healing.
  • Fibroblasts need to migrate into the wound site to form granulation tissue, in order to reconstitute the various connective tissue components.
  • Re-epithelialization requires the directional migration of keratinocytes. Therefore, we sought to determine if factors secreted from amniotic and chorionic membranes produced at Osiris promote these three types of cell migration and wound field closure. To accomplish this, we utilized a commercially available wound healing assay as seen in Figure 26 (Cell Biolabs) and a transwell cell migration assay.
  • Cell lines include human microvascular endothelial cells (HMVEC, Lonza Inc.), human umbilical endothelial cells (HUVEC, Lonza Inc.), human dermal fibroblasts (HDF, Lonza Inc.) and diseased human keratinocytes (D-HEK Lonza Inc.)- Results indicate that cell migration of all three types of cells is enhanced by treatment with conditioned media from placental membranes.
  • HMVEC human microvascular endothelial cells
  • HMVEC human umbilical endothelial cells
  • HEVEC human umbilical endothelial cells
  • HDF human dermal fibroblasts
  • D-HEK Lonza Inc. diseased human keratinocytes
  • 00444 Cells are collected via trypsinization, pelleted, and counted before being resuspended in complete keratinocyte media at a density of 2X10 5 cells/mL. 250ul (5X10 4 cells) of cell suspension is then pipetted into each side of a well containing a wound healing insert (Cytoselect 24 well Wound Healing Assay Plate, Cell Biolabs). The cells are grown for 24 hours in complete media. After 24 hours, the wound inserts are removed. At the same time, complete keratinocyte media is removed and replaced with experimental media. Complete keratinocyte media and basal keratinocyte media were used as positive and negative controls, respectively.
  • placental membranes are incubated for three days in DMEM with 1 % human serum albumin (HSA) in a tissue culture incubator. The resulting tissue and media are then placed in eppendorf tubes and spun at high speed in a microcentrifuge. The supernatants are collected and stored at -80 C until use.
  • conditioned media from placental membranes is diluted 1 :20 in basal keratinocyte media before being added to experimental wells. After 18 hours, the media is removed and the cells are fixed for 20 min in 4% parafolmaldehyde and stained with crystal violet. The wound field in each well is then photographed. Wound healing in determined by the amount of wound field still visible at the end of the experiment when compared to control pictures taken before conditioned media is added to the wells.
  • Conditioned media from amniotic and chorionic membranes was used to assess the potential for these membrane to promote endothelial cell migration and wound field closure.
  • Conditioned media from placental amniotic, chorionic, and a combination of amniotic/chorionic membranes supported migration of endothelial cells into the experimental wound field (Figure 27)
  • Example 20.2 Placental membrane conditioned media supports endothelial cells, fibroblasts, and keratinocytes migration.
  • 00446 Migration assay was performed on human umbilical vein endothelial cells, human normal dermal fibroblasts, and human diseased keratinocytes (type II diabetes) (Lonza) using a FluroBlok transwell system with 8 ⁇ pores (BD). 100,000 cells were suspended in DMEM with 0.1 % FBS and added to the upper chamber of the transwell to arrest mitosis. The next day, conditioned medium derived from tissue samples were added to lower chambers and incubated overnight. After incubation, cells that had migrated through the filter were fixed and stained for calcein (Molecular Probe). The field was visualized by a fluorescence inverted microscope and pictures were taken from four randomly chosen fields under 10x magnification. Our results demonstrated that amniotic products promote the migration of endothelial cells ( Figure 27 and 28), fibroblasts ( Figure 29), and keratinocytes ( Figure 30).
  • cryopreserved placental membrane product which provides necessary angiogenic and anti-inflammatory growth factors was introduced in an effort to improve outcomes of patients with chronic skin ulceration at-risk for amputation.
  • a chronic, cutaneous ulcer is defined as a wound that has failed to proceed through an orderly and timely series of events to produce a durable structural, functional and cosmetic closure(2).
  • the most common chronic wounds include pressure ulcers and leg ulcers.
  • the triad of peripheral neuropathy, deformity, and minor trauma has emerged as the most frequent causes of insult that lead to foot ulcerations.
  • an appropriate benchmark for a chronic wound is a decrease of 10% to 15% in size every week, or 50% decrease in size over a one-month period.
  • a three-year retrospective cohort study performed by Ramsey et al. of 8,905 patients in a large health maintenance organization who have diabetes reported a 5.8% cumulative incidence of ulceration. At the time of diagnosis, 15% of these patients developed osteomyelitis and 16% required partial amputation of a lower limb.
  • a cryopreserved placental membrane was made as described herein, comprising an allograft derived from the amnion including a bilayer of native epithelial cells and a stromal layer consisting of neonatal fibroblasts, extracellular matrix (ECM) and mesenchymal stem cells (MSC).
  • ECM extracellular matrix
  • MSC mesenchymal stem cells
  • the vascular exam revealed non-palpable pulses in the dorsalis pedis and posterior tibial arteries.
  • Doppler exam revealed a monophasic dorsalis pedis pulse with a biphasic posterior tibial artery pulse.
  • the fifth digit had gangrenous changes and was cold on palpation. There were ischemic changes of the fourth digit.
  • Radiographic evaluation revealed scattered air densities indicative of soft tissue gas in the fourth interspace as well as the tip of the fifth digit.
  • Panel A First application of a cryopreserved placental membrane product; B: 8 weeks post first cryopreserved placental membrane an instant membrane product application; C: 10 1/2 weeks post first cryopreserved placental membrane an instant membrane product application; D: 12 weeks post first cryopreserved placental membrane an instant membrane product application; E: 19 weeks post first cryopreserved placental membrane an instant membrane product application.
  • the patient was instructed to remain strictly non-weight bearing to the affected limb, and returned for follow-up on post-operative day 6.
  • the dressing was clean, dry and intact. There were no post-operative complications such as abscess, cellulitis, discomfort, or drainage and no clinical signs of infection.
  • the patient received a total of 7 applications of cryopreserved placental membrane product over the course of the next 8 weeks.
  • the wound was inspected for clinical signs of infection. Evaluation at each visit revealed marked development in granulation tissue to the wound base and significant decrease in size. Eight weeks after the initial application of the allograft tissue the wound was closed.
  • FIG. 32 Photographs of the remarkable wound healing mediated by a placental product of the present technology as shown in Figure 32.
  • Panel A Osteomyelitis, tendon exposed, probed to bone. First cryopreserved placental membrane graft was applied after surgical debridement; B: Status post 1 application of cryopreserved placental membrane graft, wound is granular in nature and no signs of infection; C: 3 weeks post-surgical intervention; 2 applications on the cryopreserved placental membrane graft, the wound is considerably smaller in circumference and depth; D: 6 weeks post-surgical intervention the wound is almost closed; At 8 weeks and 7 applications of the cryopreserved placental membrane graft, the wound is closed.
  • Key inclusion criteria included confirmed Type I or Type II diabetes, patient age between 18 years and 80 years, index wound present between 4 weeks and 52 weeks, wound located below the malleoli on plantar or dorsal surface of the foot, and ulcer between 1 cm 2 and 15cm 2 .
  • Main exclusion criteria included hemoglobin A1 c above 12%, evidence of active infection including osteomyelitis or cellulitis, inadequate circulation to the affected foot defined by an ankle brachial index ⁇ 0.70 or > 1 .30, or toe brachial index ⁇ 0.50 or Doppler study with inadequate arterial pulsation, exposed muscle, tendon, bone or joint capsule, and reduction of wound area by > 30% during the screening period.
  • 00469 Following a one (1 ) week screening period, patients were randomized to the cryopreserved placental product or control treatment arm in a 1 :1 ratio. Patients randomized to placental product treatment received an application of placental product once a week ( ⁇ 3 days) for up to 84 days (Blinded Treatment Phase). Patients in the control group received standard wound therapy once a week ( ⁇ 3 days) for up to 84 days. All wounds were appropriately cleaned and surgically debrided to remove all nonviable soft tissue from the wound by scalpel, tissue nippers and/or curettes at each weekly visit. For patients randomized to the placental product group, the cryopreserved placental membrane was placed to come in full contact with the wound and edges.
  • the study sample size was based on an assumed closure rate of 30% in the control arm and 50% in the placental product group with a 30% drop out rate. Under these assumptions, 94 completed patients in each treatment arm were required to meet the 2-sided Type 1 error rate of 0.05 with 80% power.
  • a pre-specified interim analysis was planned at 50% enrollment. The interim analysis utilized a one-sided superiority design based on an Emerson-Fleming symmetric group sequential design using an O'Brien-Fleming boundary shape (Emerson and Fleming, 1989). The analysis was performed by an unblinded statistician and reported to the blinded review committee. Following the interim analysis, the blinded review committee recommended to terminate study enrollment due to overwhelming superiority of the placental product arm versus the control arm.
  • the probability of closure for the placental product group was 67.1 % compared to 27.1 % for the standard care group (Log- Rank, p ⁇ 0.0001 ).
  • Placental product patients also required fewer study visits (i.e. applications) to achieve closure compared to patients in the control arm (6 vs. 12, p ⁇ 0.001 ) as depicted in Figure 35.
  • Cox proportional hazard regression analysis was performed with treatment group, duration of ulcer, baseline ulcer area, glucose control (glycated hemoglobin), ulcer location, and BMI as covariates. Following adjustment for these variables, placental product was found to have a significant effect on time to closure (p ⁇ 0.0001 ). The hazard ratio was 4.77 (95% CI 2.279, 9.971 ), indicating superior odds of closure with placental product relative to standard wound therapy.

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Abstract

La présente invention concerne un produit dérivé de la membrane placentaire comprenant une membrane amniotique immunocompatible. Lesdits produits dérivés de la membrane placentaire peuvent être cryoconservés et contenir des facteurs thérapeutiques et des cellules viables après décongélation. Les produits dérivés de la membrane placentaire sont utilisés pour cicatriser les plaies et régénérer/réparer les tissus car ils sont capables de favoriser l'angiogenèse, réduire l'inflammation, inhiber les protéases et l'oxydation provoquée par les radicaux libres, réduire la formation des cicatrices, et d'autres actions favorisant la cicatrisation. La présente invention concerne des produits visant à protéger le tissu lésé ou endommagé, ou destinés à être utilisés comme revêtement pour prévenir les adhérences, protéger des bactéries, inhiber l'activité bactérienne et/ou favoriser la cicatrisation ou la croissance tissulaire. L'invention porte également sur des méthodes de fabrication et des méthodes d'utilisation de ce types de produits dérivés de membranes.
PCT/US2014/037208 2014-05-07 2014-05-07 Produits dérivés d'une membrane amniotique immunocompatible WO2015171144A1 (fr)

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EP14891554.9A EP3139936A4 (fr) 2014-05-07 2014-05-07 Produits dérivés d'une membrane amniotique immunocompatible
KR1020167034202A KR20170002572A (ko) 2014-05-07 2014-05-07 면역적합성 양막 생성물
AU2014393404A AU2014393404A1 (en) 2014-05-07 2014-05-07 Immunocompatible amniotic membrane products
PCT/US2014/037208 WO2015171144A1 (fr) 2014-05-07 2014-05-07 Produits dérivés d'une membrane amniotique immunocompatible
JP2016567041A JP2017514879A (ja) 2014-05-07 2014-05-07 免疫適合性のある羊膜生成物

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3203835A4 (fr) * 2014-10-07 2018-03-07 Nutech Medical, Inc. Procédé et composition pour conservation hypothermique de tissu placentaire
US10314688B2 (en) 2016-08-24 2019-06-11 Arthrex, Inc. Tissue use for repair of injury
CN112584878A (zh) * 2018-08-30 2021-03-30 樱花精机株式会社 腱和腱鞘的再生、修复材料及修复材料的应用
US11510947B2 (en) 2010-02-18 2022-11-29 Osiris Therapeutics, Inc. Methods of manufacture of immunocompatible amniotic membrane products
US11511017B2 (en) 2019-03-12 2022-11-29 Arthrex, Inc. Ligament reconstruction

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2948129A1 (fr) * 2014-05-07 2015-11-12 Osiris Therapeutics, Inc. Produits de membranes choriales immunocompatibles

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110206776A1 (en) * 2010-02-18 2011-08-25 Samson Tom Methods of manufacture of immunocompatible amniotic membrane products
US20110251566A1 (en) * 2010-04-09 2011-10-13 Dmitry Zimnitsky Apparatuses, methods, and compositions for the treatment and prophylaxis of chronic wounds
US20120095455A1 (en) * 2000-02-11 2012-04-19 The General Hospital Corporation Photochemical tissue bonding

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6152142A (en) * 1997-02-28 2000-11-28 Tseng; Scheffer C. G. Grafts made from amniotic membrane; methods of separating, preserving, and using such grafts in surgeries
US20020039788A1 (en) * 2000-02-29 2002-04-04 Isseroff Roslyn R. Corneal epithelial graft composites
US20030187515A1 (en) * 2002-03-26 2003-10-02 Hariri Robert J. Collagen biofabric and methods of preparing and using the collagen biofabric
WO2005021014A1 (fr) * 2003-09-03 2005-03-10 Jiuchang Miao Preparation d'un amnios biologique conservable a temperature ambiante
WO2007023750A1 (fr) * 2005-08-26 2007-03-01 National University Corporation University Of Toyama Amnion seche et procede de sechage de l’amnion
US8372437B2 (en) * 2006-08-17 2013-02-12 Mimedx Group, Inc. Placental tissue grafts
WO2011074208A1 (fr) * 2009-12-18 2011-06-23 国立大学法人東北大学 Procédé pour la préparation d'un matériau de transplantation cornéenne par mise en transparence d'un derme cutané ou d'un amnios
CA2948129A1 (fr) * 2014-05-07 2015-11-12 Osiris Therapeutics, Inc. Produits de membranes choriales immunocompatibles

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120095455A1 (en) * 2000-02-11 2012-04-19 The General Hospital Corporation Photochemical tissue bonding
US20110206776A1 (en) * 2010-02-18 2011-08-25 Samson Tom Methods of manufacture of immunocompatible amniotic membrane products
US20140037598A1 (en) * 2010-02-18 2014-02-06 Osiris Therapeutics, Inc. Therapeutic products comprising vitalized placental dispersions
US20110251566A1 (en) * 2010-04-09 2011-10-13 Dmitry Zimnitsky Apparatuses, methods, and compositions for the treatment and prophylaxis of chronic wounds

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP3139936A4 *
SEN ET AL.: "Oxygen, Oxidants, and Antioxidants in Wound Healing An Emerging Paradigm", ANN. N.Y. ACAD. SCI., vol. 957, 1 May 2002 (2002-05-01), pages 239 - 249, XP055234848 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11590173B2 (en) 2010-02-18 2023-02-28 Osiris Therapeutics, Inc. Methods of manufacture of therapeutic products comprising vitalized placental dispersions
US12109241B2 (en) 2010-02-18 2024-10-08 Osiris Therapeutics, Inc. Methods of manufacture of immunocompatible chorionic membrane products
US11986498B2 (en) 2010-02-18 2024-05-21 Osiris Therapeutics, Inc. Therapeutic products comprising vitalized placental dispersions
US11510947B2 (en) 2010-02-18 2022-11-29 Osiris Therapeutics, Inc. Methods of manufacture of immunocompatible amniotic membrane products
US11638725B2 (en) 2010-02-18 2023-05-02 Osiris Therapeutics, Inc. Methods of manufacture of immunocompatible chorionic membrane products
US11590172B2 (en) 2010-02-18 2023-02-28 Osiris Therapeutics, Inc. Immunocompatible chorionic membrane products
EP3203835A4 (fr) * 2014-10-07 2018-03-07 Nutech Medical, Inc. Procédé et composition pour conservation hypothermique de tissu placentaire
EP4035530A1 (fr) * 2014-10-07 2022-08-03 Prime Merger Sub, LLC Stockage hypothermique de tissu placentaire
US10987209B2 (en) 2016-08-24 2021-04-27 Arthrex, Inc. Tissue use for repair of injury
US11918453B2 (en) 2016-08-24 2024-03-05 Arthrex, Inc. Tissue use for repair of injury
US10314688B2 (en) 2016-08-24 2019-06-11 Arthrex, Inc. Tissue use for repair of injury
CN112584878A (zh) * 2018-08-30 2021-03-30 樱花精机株式会社 腱和腱鞘的再生、修复材料及修复材料的应用
US11511017B2 (en) 2019-03-12 2022-11-29 Arthrex, Inc. Ligament reconstruction

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