WO2015053739A1 - Procédés de combinaison de cellules souches mésenchymateuses et d'allogreffes cartilagineuses, et produits obtenus après combinaison desdites cellules souches mésenchymateuses et allogreffes cartilagineuses - Google Patents

Procédés de combinaison de cellules souches mésenchymateuses et d'allogreffes cartilagineuses, et produits obtenus après combinaison desdites cellules souches mésenchymateuses et allogreffes cartilagineuses Download PDF

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WO2015053739A1
WO2015053739A1 PCT/US2013/063674 US2013063674W WO2015053739A1 WO 2015053739 A1 WO2015053739 A1 WO 2015053739A1 US 2013063674 W US2013063674 W US 2013063674W WO 2015053739 A1 WO2015053739 A1 WO 2015053739A1
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Prior art keywords
stem cells
mesenchymal stem
cartilage
adipose tissue
allograft
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PCT/US2013/063674
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English (en)
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Simon Bogdansky
Yaling Shi
Brent Atkinson
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Allosource
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Priority to KR1020157026775A priority Critical patent/KR20160058721A/ko
Priority to CA2904989A priority patent/CA2904989A1/fr
Priority to EP13895259.3A priority patent/EP2954044A4/fr
Priority to PCT/US2013/063674 priority patent/WO2015053739A1/fr
Publication of WO2015053739A1 publication Critical patent/WO2015053739A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • 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
    • A61L27/3612—Cartilage, synovial fluid
    • 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
    • A61L27/3641—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 site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/3654—Cartilage, e.g. meniscus
    • 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
    • A61L27/38—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 containing added animal cells
    • A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • 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
    • A61L27/38—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 containing added animal cells
    • A61L27/3839—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 containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3852—Cartilage, e.g. meniscus
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1311—Osteocytes, osteoblasts, odontoblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1317—Chondrocytes

Definitions

  • a method of combining mesenchymal stem cells with an osteochondral allograft comprising providing adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells, e.g., a stromal vascular fraction comprising a heterogenous population of mesenchymal stem cells and unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the osteochondral allograft so as to form a seeded osteochondral allograft; and allowing the cell suspension to adhere to the osteochondral allograft by incubating the seeded osteochondral allograft for a period of time to allow the mesenchymal stem cells to attach.
  • an allograft product including a combination of mesenchymal stem cells with an osteochondral allograft, and the combination manufactured by obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the
  • mesenchymal stem cells and the unwanted cells adding the cell suspension with the mesenchymal stem cells to seed the osteochondral allograft so as to form a seeded osteochondral allograft; and allowing the cell suspension to adhere to the seeded
  • mesenchymal stem cells with an osteochondral allograft comprising providing adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells to acquire a stromal vascular fraction comprising a heterogenous population of mesenchymal stem cells and unwanted cells, wherein the digesting includes making a collagenase I solution, and filtering the solution through a 0.2 ⁇ m filter unit, mixing the adipose solution with the collagenase I solution, and adding the adipose solution mixed with the collagenase I solution to a shaker flask; placing the shaker with continuous agitation at about 75 RPM for about 45 to 60 minutes so as to provide the adipose tissue with a visually smooth appearance; aspirating a supernatant containing mature adipocytes so as to provide a pellet; adding the cell suspension with the mesenchy
  • an allograft product including a combination of mesenchymal stem cells with an osteochondral allograft, and the combination manufactured by obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the
  • the digesting includes making a collagenase I solution, and filtering the solution through a 0.2 ⁇ m filter unit, mixing the adipose solution with the collagenase I solution, and adding the adipose solution mixed with the collagenase I solution to a shaker flask; placing the shaker with continuous agitation at about 75 RPM for about 45 to 60 minutes so as to provide the adipose tissue with a visually smooth appearance; aspirating a supernatant containing mature adipocytes so as to provide a pellet; adding the cell suspension with the mesenchymal stem cells to seed the osteochondral allograft so as to form a seeded osteochondral allograft; and allowing the cell suspension to adhere to the osteochondral allograft for a period of time to allow the mesenchymal stem cells to attach.
  • a method of combining mesenchymal stem cells with decellularized, morselized cartilage comprising providing adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells, e.g., a stromal vascular fraction comprising a heterogenous population of mesenchymal stem cells and unwanted cells; adding the cell suspension with the mesenchymal stem cells (e.g., stromal vascular fraction) to seed the morselized cartilage so as to form seeded morselized cartilage; and allowing the cell suspension to adhere to the decellularized, morselized cartilage by incubating the seeded decellularized, morseling cartilage for a period of time to allow the mesenchymal stem cells to attach.
  • an allograft product including a combination of mesenchymal stem cells with decellularized, morselized cartilage, and the combination manufactured by obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells; adding the cell suspension with the mesenchymal stem cells (e.g., stromal vascular fraction) to seed the morselized cartilage so as to form seeded morselized cartilage; and allowing the cell suspension to adhere to the decellularized, morselized cartilage by incubating the seeded decellularized, morselized cartilage for a period of time to allow the mesenchymal stem cells to attach.
  • mesenchymal stem cells e.g., stromal vascular fraction
  • a method of combining mesenchymal stem cells with decellularized, morselized cartilage comprising providing adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells to acquire a stromal vascular fraction, wherein the stromal vascular fraction comprises a heterogenous population of mesenchymal stem cells and unwanted cells, and wherein the digesting includes making a collagenase I solution, and filtering the solution through a 0.2 ⁇ m filter unit, mixing the adipose solution with the collagenase I solution, and adding the adipose solution mixed with the collagenase I solution to a shaker flask; placing the shaker with continuous agitation at about 75 RPM for about 45 to 60 minutes so as to provide the adipose tissue with a visually smooth appearance; aspirating a
  • an allograft product including a combination of mesenchymal stem cells with decellularized, morselized cartilage, and the combination manufactured by obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells; digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells to acquire a stromal vascular fraction, and the digesting includes making a collagenase I solution, and filtering the solution through a 0.2 ⁇ m filter unit, mixing the adipose solution with the collagenase I solution, and adding the adipose solution mixed with the collagenase I solution to a shaker flask; placing the shaker with continuous agitation at about 75 RPM for about 45 to 60 minutes so as to provide the adipose tissue with a visually smooth appearance; aspirating a supernatant containing mature adipocytes so as to provide a pellet; adding the cell
  • a method of combining mesenchymal stem cells with an osteochondral allograft comprising obtaining the mesenchymal stem cells from adipose tissue of a cadaveric donor; obtaining the osteochondral allograft from the same cadaveric donor; adding the mesenchymal stem cells to seed the osteochondral allograft so as to form a seeded osteochondral allograft; and allowing the cell suspension to adhere to the osteochondral allograft for a period of time to allow the mesenchymal stem cells to attach.
  • an allograft product including a combination of mesenchymal stem cells with an osteochondral allograft, and the combination manufactured by combining mesenchymal stem cells with an osteochondral allograft, the method comprising providing the mesenchymal stem cells from adipose tissue of a cadaveric donor; providing the osteochondral allograft from the same cadaveric donor; adding the mesenchymal stem cells to seed the osteochondral allograft so as to form a seeded osteochondral allograft; and allowing the cell suspension to adhere to the seeded
  • a method of combining mesenchymal stem cells with decellularized, morselized cartilage comprising obtaining the mesenchymal stem cells from adipose tissue of a cadaveric donor; obtaining the morselized cartilage from the same cadaveric donor; adding the mesenchymal stem cells to seed the morselized cartilage so as to form a seeded osteochondral allograft; and allowing the cell suspension to adhere to the decellularized, morselized cartilage for a period of time to allow the mesenchymal stem cells to attach.
  • an allograft product including a combination of mesenchymal stem cells with decellularized, morselized cartilage, and the combination manufactured by providing the mesenchymal stem cells from adipose tissue of a cadaveric donor; providing the morselized cartilage from the same cadaveric donor; adding the mesenchymal stem cells to seed the morselized cartilage so as to form seeded morselized cartilage; and allowing the cell suspension to adhere to the decellularized, morselized cartilage for a period of time to allow the mesenchymal stem cells to attach.
  • a method of combining mesenchymal stem cells with cartilage comprising providing the mesenchymal stem cells from adipose tissue of a cadaveric donor; providing the cartilage from the same cadaveric donor; adding the mesenchymal stem cells to seed the cartilage so as to form a seeded cartilage; and allowing the cell suspension to adhere to the mesenchymal stem cells and the cartilage by incubating the seeded cartilage for a period of time to allow the mesenchymal stem cells to attach.
  • cartilage e.g., decellularized cartilage
  • an allograft product including a combination of mesenchymal stem cells with cartilage (e.g., decellularized cartilage), and the combination manufactured by obtaining the mesenchymal stem cells from adipose tissue of a cadaveric donor; obtaining the cartilage from the same cadaveric donor; adding the mesenchymal stem cells to seed the cartilage so as to form a seeded cartilage; and allowing the cell suspension to adhere to the mesenchymal stem cells and the cartilage for a period of time to allow the mesenchymal stem cells to attach.
  • cartilage e.g., decellularized cartilage
  • FIGURE 1 illustrates a flow chart of an exemplary method of combining mesenchymal stem cells with an osteochondral allograft
  • FIGURE 2 illustrates a flow chart of an exemplary method of combining mesenchymal stem cells with decellularized, morselized cartilage
  • FIGURE 3 illustrates an exemplary osteochondral allograft
  • FIGURE 4 illustrates H&E staining of a cartilage control sample
  • FIGURE 5 illustrates H&E staining of adiposed-derived stem cells seeded cartilage.
  • MSCs mesenchymal stem cells
  • ASCs Adipose- derived stem cells or ASCs are stem cells that are derived from adipose tissue.
  • Stromal Vascular Fraction or SVF generally refers to the centrifuged cell pellet obtained after digestion of tissue containing MSCs.
  • the stromal vascular fraction comprises a heterogenous population of cells, including MSCs and unwanted cells (non- mesenchymal stem cells).
  • the SVF pellet may include multiple types of stem cells.
  • stem cells may include, for example, one or more of hematopoietic stem cells, epithelial stem cells, and mesenchymal stem cells.
  • mesenchymal stem cells are filtered from other stem cells by their adherence to an osteochondral graft (or cartilage or morselized cartilage), while the other stem cells (i.e., unwanted cells) do not adhere to the osteochondral graft (or cartilage or morselized cartilage).
  • Other cells that do not adhere to the osteochondral graft (or cartilage or morselized cartilage) may also be included in these unwanted cells.
  • a stromal vascular fraction that is seeded onto an allograft as described herein (e.g., an osteochondral allograft, a cartilage allograft, a morselized cartilage allograft, a decellularized cartilage allograft, or a decellularized morselized cartilage allograft) is directly seeded onto the allograft without an intermediate step of proliferating the mesenchymal stem cells.
  • Methods of obtaining a stromal vascular fraction are described, e.g., in WO 2010/059565, incorporated by reference herein.
  • adipose derived stem cells are isolated from cadavers and characterized using flow cytometry and tri-lineage differentiation (osteogenesis,
  • tissue engineering and regenerative medicine approaches offer great promise to regenerate bodily tissues.
  • the most widely studied tissue engineering approaches which are based on seeding and in vitro culturing of cells within the scaffold before implantation, is the cell source and the ability to control cell proliferation and differentiation.
  • Many researchers have demonstrated that adipose tissue-derived stem cells (ASCs) possess multiple differentiation capacities. See, for example, the following, which are incorporated by reference: Rada, T., R.L.
  • Adipose-derived stem cells characterization and current application in orthopaedic tissue repair. Exp Bioi Med (Maywood), 2009. 234(1): p. 1-9. van Dijk, A., et al., Differentiation of human adipose-derived stem cells towards
  • ASCs and bone marrow-derived stem cells are remarkably similar with respect to growth and morphology, displaying fibroblastic characteristics, with abundant endoplasmic reticulum and large nucleus relative to the cytoplasmic volume.
  • Gimble, J. and F. Guilak Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy, 2003. 5(5): p. 362-9. Gimble, J.M. and F. Guilak, Differentiation potential of adipose derived adult stem (ADAS) cells. Curr Top Dev Bioi, 2003. 58: p. 137-60.
  • Kim, Y., et al. Direct comparison of human mesenchymal stem cells derived from adipose tissues and bone marrow in mediating neovascu/arization in response to vascular ischemia. Cell Physiol Biochem, 2007. 20(6): p. 867-76. Lin, L., et al., Comparison of osteogenic potentials of BMP4 transduced stem cells from autologous bone marrow and fat tissue in a rabbit model of calvarial defects. Calcif Tissue lnt, 2009. 85(1): p. 55-65.
  • FIGURE 1 is a flow chart of a process for combining an osteochondral allograft with stem cells.
  • a stromal vascular fraction e.g., a heterogenous population of cells comprising mesenchymal stem cells and unwanted cells
  • a method of combining mesenchymal stem cells with an osteochondral allograft is provided.
  • the method is an ex vivo method.
  • the method may include obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells.
  • Unwanted cells may include hematopoietic stem cells and other stromal cells.
  • the method may further include digesting the adipose tissue to form a cell suspension having the mesenchymal stem cells and at least some or all of the unwanted cells (e.g., a stromal vascular fraction). In another embodiment, this digestion step may be followed by negatively depleting some of the unwanted cells and other constituents to concentrate mesenchymal stem cells.
  • the method includes adding the cell suspension with the mesenchymal stem cells to seed the osteochondral allograft. This may be followed by allowing the cell suspension to adhere to the osteochondral allograft (e.g., by incubating the seeded allograft under appropriate incubation conditions) for a period of time to allow the mesenchymal stem cells to attach.
  • the method may include rinsing the seeded osteochondral allograft to remove the unwanted cells from the seeded ostechondral allograft.
  • an allograft product may include a combination of
  • the adipose tissue may be obtained or recovered from a cadaveric donor.
  • a typical donor yields 2 liters of adipose containing 18 million MSCs.
  • an osteochondral allograft may be from the same cadaveric donor as the adipose tissue.
  • the adipose tissue may be obtained from a patient that will be undergoing the cartilage or osteochondral replacement/regeneration surgery.
  • both the osteochondral graft (or cartilage or morselized cartilage) and the adipose tissue may be obtained from the same cadaveric donor.
  • Adipose cells may be removed using liposuction.
  • Other sources, and combination of sources, of adipose tissue, other tissues, and osteochondral allografts may be utilized.
  • the adipose tissue may be washed prior to or during digestion. Washing may include using a thermal shaker at 75 RPM at 37°C for at least 10 minutes. Washing the adipose tissue may include washing with a volume of PBS substantially equal to the adipose tissue.
  • washing the adipose tissue includes washing with the PBS with 1% penicillin and streptomycin at about 37°C.
  • washing the adipose tissue may include agitating the tissue and allowing phase separation for about 3 to 5 minutes. This may be followed by aspirating off a supernatant solution. The washing may include repeating washing the adipose tissue multiple times until a clear infranatant solution is obtained.
  • washing the adipose tissue may include washing with a volume of growth media substantially equal to the adipose tissue.
  • FIGURE 2 is a flow chart of a process for combining morselized cartilage (e.g., decellularized morselized cartilage) with stem cells.
  • a stromal vascular fraction e.g., a heterogenous population of cells comprising mesenchymal stem cells and unwanted cells
  • a method of combining mesenchymal stem cells with decellularized, morselized cartilage is provided.
  • the method is an ex vivo method. The method may include obtaining adipose tissue having the mesenchymal stem cells together with unwanted cells.
  • Unwanted cells may include hematopoietic stem cells and other stromal cells.
  • the method may further include digesting the adipose-derived tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells (e.g., stromal vascular fraction). In another embodiment, this digesting step may be followed by naturally selecting MSCs and depleting some of the unwanted cells and other constituents to concentrate mesenchymal stem cells.
  • the method includes adding the cell suspension with the mesenchymal stem cells (e.g., the stromal vascular fraction) to the morselized cartilage.
  • an allograft product may include a combination of
  • the adipose tissue may be obtained from a cadaveric donor.
  • a typical donor yields 2 liters of adipose containing 18 million MSCs.
  • morselized cartilage may be from the same cadaveric donor as the adipose tissue.
  • the adipose tissue may be obtained from a patient.
  • both the osteochondral graft ⁇ or cartilage or morselized cartilage) and the adipose tissue may be obtained from the same cadaveric donor.
  • Adipose cells may be removed using liposuction.
  • Other sources, and combination of sources, of adipose tissue, other tissues, and morselized cartilage may be utilized.
  • the adipose tissue may be washed prior to or during digestion. Washing may include using a thermal shaker at 75 RPM at 37°C for at least 10 minutes. Washing the adipose tissue may include washing with a volume of PBS substantially equal to the adipose tissue. In an embodiment, washing the adipose tissue includes washing with the PBS with 1% penicillin and streptomycin at about 37°C.
  • washing the adipose tissue may include agitating the tissue and allowing phase separation for about 3 to 5 minutes. This may be followed by aspirating off a supernatant solution. The washing may include repeating washing the adipose tissue multiple times until a clear infranatant solution is obtained. In one embodiment, washing the adipose tissue may include washing with a volume of growth media substantially equal to the adipose tissue.
  • Digesting the cell suspension may include making a collagenase I solution, and filtering the solution through a 0.2 ⁇ m filter unit, mixing the adipose tissue with the collagenase I solution, and adding the cell suspension mixed with the collagenase 1 solution to a shaker flask. Digesting the cell suspension may further include placing the shaker with continuous agitation at about 75 RPM for about 45 to 60 minutes so as to provide the adipose tissue with a visually smooth appearance. [0045] Digesting the cell suspension may further include aspirating supernatant containing mature adipocytes so as to provide a pellet, which may be referred to as a stromal vascular fraction.
  • adding the cell suspension with the mesenchymal stem cells to the osteochondral allograft or the morselized cartilage may include using a cell pellet for seeding onto the osteochondral graft (or cartilage or morselized cartilage, e.g., decellularized cartilage or decellularized morselized cartilage).
  • the cell suspension is allowed to adhere to seeded allografts for a period of time to allow the mesenchymal stem cells to attach to the osteochondral allograft or the morselized cartilage.
  • the seeded osteochondral graft (or seeded cartilage or seeded morselized cartilage, e.g., seeded decellularized cartilage or seeded decellularized morselized cartilage) is incubated for a period of time under appropriate incubation conditions.
  • the seeded osteochondral graft (or seeded cartilage or seeded morselized cartilage, e.g., seeded decellularized cartilage or seeded decellularized morselized cartilage) is incubated in a humidified incubator. In some embodiments, the seeded osteochondral graft (or seeded cartilage or seeded morselized cartilage, e.g., seeded decellularized cartilage or seeded decellularized morselized cartilage) is incubated in the presence of culture medium and/or one or more growth factors.
  • the seeded osteochondral graft (or seeded cartilage or seeded morselized cartilage, e.g., seeded decellularized cartilage or seeded decellularized morselized cartilage) is rinsed to remove unwanted cells from the allograft.
  • a lab sponge or other mechanism may be used to pat dry the cells.
  • the method may include placing the osteochondral graft (or cartilage or morselized cartilage, e.g., decellularized cartilage or decellularized morselized cartilage) into a cryopreservation media after rinsing the osteochondral allograft or the morselized cartilage.
  • This cryopreservation media may be provided to store the final products.
  • the method may include maintaining the osteochondral allograft or the morselized cartilage into a frozen state after rinsing the osteochondral allograft or the morselized cartilage to store the final products.
  • the frozen state may be at about negative 80° C.
  • Ficoll density solution may be utilized.
  • negatively depleting the concentration of the mesenchymal stem cells may include adding a volume of PBS and a volume of Ficoll density solution to the adipose solution.
  • the volume of PBS may be 5 ml and the volume of Ficoll density solution may be 25 ml with a density of 1.073 g/ml.
  • Negatively depleting the concentration of the mesenchymal stem cells may also include centrifuging the adipose solution at about 1160 g for about 30 minutes at about room temperature.
  • the method may include stopping the centrifuging the adipose solution without using a brake.
  • Negatively depleting the concentration of the mesenchymal stem cells is optional and may next include collecting an upper layer and an interface containing nucleated cells, and discarding a lower layer of red cells and cell debris. Negatively depleting the concentration of the mesenchymal stem cells may also include adding a volume of D-PBS of about twice an amount of the upper layer of nucleated cells, and inverting a container containing the cells to wash the collected cells. Negatively depleting the concentration of the mesenchymal stem cells may include centrifuging the collected cells to pellet the collected cells using the break during deceleration.
  • negatively depleting the concentration of the mesenchymal stem cells may further include centrifuging the collected cells at about 900 g for about 5 minutes at about room temperature. Negatively depleting some of the unwanted cells may include discarding a supernatant after centrifuging the collected cells, and resuspending the collected cells in a growth medium.
  • Previous methods used autogenous osteochondral grafts, wherein a graft from one area of a donor knee was transplanted to same donor knee, but to an area that was damaged. However, this method causes trauma to the patient and creates a new area that is damaged. Allografts are currently used that prevent the trauma caused by autografts.
  • Non-processed osteochondral allografts suffer from being immune reactive. Processed osteochondral allografts suffer from either having no viable cells, reduced viability, or fully differentiated cells that are not capable of undergoing regeneration. Thus, there is a need to provide a cartilage graft that contains viable MSCs to recapitulate the regenerative cascade. [0052] The surface of cartilage, by its very nature, is not adherent to cells. The
  • mesenchymal stem cells are anchorage dependent, but this has been defined in the art as being adherent to tissue culture plastic, not to a biological tissue like cartilage. Surprisingly, the methods provided herein permit viable MSCs that bind to cartilage. [0053]
  • the methods provided herein describe the allograft processing that allows MSCs to adhere to the non-synthetic osteochondral or cartilage (e.g., decellularized cartilage) scaffolds described herein.
  • the method in the example demonstrates a blending and processing method that removes cells from the cartilage graft such that viable MSCs can adhere.
  • the mesenchymal stem cells are non-immunogenic and regenerate cartilage of the osteochondral allograft or the morselized cartilage.
  • the unwanted cells are generally anchorage independent. This means that the unwanted cells generally do not adhere to the osteochondral allograft or the morselized cartilage.
  • the unwanted cells may be
  • mesenchymal stem cells adhere to the osteochondral allograft or the morselized cartilage while unwanted cells, such as hematopoietic stem cells, are rinsed away leaving a substantially uniform population of mesenchymal stem cells on the osteochondral graft (or cartilage or morselized cartilage).
  • unwanted cells such as hematopoietic stem cells
  • the ability to mineralize the extracellular matrix and to generate cartilage is not unique to MSCs.
  • ASCs possess a similar ability to differentiate into chondrocytes under similar conditions. Human ASCs offer a unique advantage in contrast to other cell sources.
  • this method and combination product involve processing that does not alter the relevant biological characteristics of the tissue. Processing of the adipose/stem cells may involve the use of antibiotics, cell media, collagenase. None of these affects the relevant biological characteristics of the stem cells. The relevant biological characteristics of these mesenchymal stem cells are centered on renewal and repair. The processing of the stem cells does not alter the cell's ability to continue to differentiate and repair.
  • mesenchymal stem cells In the absence of stimulation or environmental cues, mesenchymal stem cells (MSCs) remain undifferentiated and maintain their potential to form tissue such as bone, cartilage, fat, and muscle. Upon attachment to an osteoconductive matrix, MSCs have been shown to differentiate along the osteoblastic lineage in vivo. See, for example, the following, which are incorporated by reference: Arinzeh T.L., Peter S.J., Archambault M.P., van den Bos C., Gordon S., Kraus K., Smith A., Kadiyala S. Allogeneic mesenchymal stem cells regenerate bone in a critical sized canine segmental defect. J Bone Joint Surg Am. 2003; 85-A:1927-35.
  • an osteochondral allograft 10 which may include cartilage 15 and bone 20 from a cadaver. Osteochondral allograft may be placed in the area of a knee 25 or other joint where cartilage is missing. This technique may be used where there is a large area of cartilage that is missing or if there both bone and cartilage are missing.
  • the donor allograft must be tested for contamination, which may include bacteria, hepatitis, and HIV. Having a single donor for both the osteochondral allograft and adipose-derived mesenchymal stem cells may reduce testing burdens and minimize other potential issues.
  • EXAMPLE [0059] The following example is offered to illustrate, but not to limit, the claimed invention. Cartilage Combined with Adipose-derived Stem Cells
  • the amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells.
  • FIGURE 4 illustrates H&E staining of cartilage control (10X magnification). Note that there were no live cells in the voids of the ground cartilage matrix.
  • FIGURE 5 illustrates H&E staining of ASCs seeded cartilage (10X magnification). Note the live cell nuclei in the voids.
  • the cartilage only control there were no live cells, only the dead cell debris was discovered. The cells seemed to be all dead and left the voids behind.
  • the ASCs seeded cartilage it seemed that all the seeded cells repopulated the voids left by pre-existing cells from the cartilage. There were no live cells on the cartilage surface that lacked decellularized zones.

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Abstract

Cette invention concerne des procédés de combinaison de cellules souches mésenchymateuses (MSC) avec une allogreffe ostéo-cartilagineuse, une allogreffe cartilagineuse morcellisée, ou une allogreffe cartilagineuse décellularisée, morcellisée. Dans certains modes de réalisation, le procédé comprend l'ensemencement d'une fraction vasculaire stromale comprenant des MSC et des cellules indésirables sur l'allogreffe.
PCT/US2013/063674 2013-10-07 2013-10-07 Procédés de combinaison de cellules souches mésenchymateuses et d'allogreffes cartilagineuses, et produits obtenus après combinaison desdites cellules souches mésenchymateuses et allogreffes cartilagineuses WO2015053739A1 (fr)

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KR1020157026775A KR20160058721A (ko) 2013-10-07 2013-10-07 중간엽 줄기세포 및 연골 함유 동종이식편의 조합 방법, 및 조합된 중간엽 줄기세포 및 연골 함유 동종이식편 제품
CA2904989A CA2904989A1 (fr) 2013-10-07 2013-10-07 Procedes de combinaison de cellules souches mesenchymateuses et d'allogreffes cartilagineuses, et produits obtenus apres combinaison desdites cellules souches mesenchymateuses et allogreffes cartilagineuses
EP13895259.3A EP2954044A4 (fr) 2013-10-07 2013-10-07 Procédés de combinaison de cellules souches mésenchymateuses et d'allogreffes cartilagineuses, et produits obtenus après combinaison desdites cellules souches mésenchymateuses et allogreffes cartilagineuses
PCT/US2013/063674 WO2015053739A1 (fr) 2013-10-07 2013-10-07 Procédés de combinaison de cellules souches mésenchymateuses et d'allogreffes cartilagineuses, et produits obtenus après combinaison desdites cellules souches mésenchymateuses et allogreffes cartilagineuses

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9808558B2 (en) 2008-11-20 2017-11-07 Allosource Allografts combined with tissue derived stem cells for bone healing
US10568990B2 (en) 2013-03-15 2020-02-25 Allosource Cell repopulated collagen matrix for soft tissue repair and regeneration
EP3479834A4 (fr) * 2016-07-04 2020-02-26 Kangstem Biotech Co., Ltd. Complexe comprenant un lysat exempt de cellules de cartilage et des cellules souches pour la stimulation de la différenciation du cartilage et son utilisation

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Publication number Priority date Publication date Assignee Title
WO2008036374A2 (fr) * 2006-09-21 2008-03-27 Medistem Laboratories, Inc. Allogreffes de cellules souches chez des receveurs non conditionnes
US20100124776A1 (en) * 2008-11-20 2010-05-20 Allosource Allografts combined with tissue derived stem cells for bone healing
US8221500B2 (en) * 2003-05-16 2012-07-17 Musculoskeletal Transplant Foundation Cartilage allograft plug

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US8221500B2 (en) * 2003-05-16 2012-07-17 Musculoskeletal Transplant Foundation Cartilage allograft plug
WO2008036374A2 (fr) * 2006-09-21 2008-03-27 Medistem Laboratories, Inc. Allogreffes de cellules souches chez des receveurs non conditionnes
US20100124776A1 (en) * 2008-11-20 2010-05-20 Allosource Allografts combined with tissue derived stem cells for bone healing

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9808558B2 (en) 2008-11-20 2017-11-07 Allosource Allografts combined with tissue derived stem cells for bone healing
US9814803B2 (en) 2008-11-20 2017-11-14 Allosource Allografts combined with tissue derived stem cells for bone healing
US10568990B2 (en) 2013-03-15 2020-02-25 Allosource Cell repopulated collagen matrix for soft tissue repair and regeneration
US11229725B2 (en) 2013-03-15 2022-01-25 Allosource Cell repopulated collagen matrix for soft tissue repair and regeneration
EP3479834A4 (fr) * 2016-07-04 2020-02-26 Kangstem Biotech Co., Ltd. Complexe comprenant un lysat exempt de cellules de cartilage et des cellules souches pour la stimulation de la différenciation du cartilage et son utilisation

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