WO2010059565A2 - Allogreffes combinées avec des cellules souches dérivées de tissu pour la cicatrisation osseuse - Google Patents

Allogreffes combinées avec des cellules souches dérivées de tissu pour la cicatrisation osseuse Download PDF

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WO2010059565A2
WO2010059565A2 PCT/US2009/064611 US2009064611W WO2010059565A2 WO 2010059565 A2 WO2010059565 A2 WO 2010059565A2 US 2009064611 W US2009064611 W US 2009064611W WO 2010059565 A2 WO2010059565 A2 WO 2010059565A2
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bone
bone substrate
accordance
substrate
mesenchymal stem
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PCT/US2009/064611
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WO2010059565A3 (fr
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Yaling Shi
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Allosource
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Priority to EP09828080.3A priority Critical patent/EP2358404B1/fr
Priority to KR1020117014011A priority patent/KR101704072B1/ko
Priority to CA2743869A priority patent/CA2743869C/fr
Publication of WO2010059565A2 publication Critical patent/WO2010059565A2/fr
Publication of WO2010059565A3 publication Critical patent/WO2010059565A3/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • 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
    • A61K35/37—Digestive system
    • A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
    • 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
    • A61L27/3604—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
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • 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
    • A—HUMAN NECESSITIES
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    • 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
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    • A—HUMAN NECESSITIES
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    • 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
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    • 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
    • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
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    • 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
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    • 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
    • A61L27/56—Porous materials, e.g. foams or sponges
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N5/0653—Adipocytes; Adipose tissue
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
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    • 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
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • A—HUMAN NECESSITIES
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    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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    • C12N2533/18—Calcium salts, e.g. apatite, Mineral components from bones, teeth, shells
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    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • Adipose-derived stem ce ⁇ is (ASCs) 1 which can be obtained in large quantities, have been utilized as c ⁇ lluiar therapy for the induction of bone formation in tissue engineering strategies.
  • Allografts may be combined with stem cells. This requires a significant amount of tissue processing and vacuular processing prior to seeding the ailograft substrate,
  • AiSografts seeded with living cells generally provide better surgical results.
  • a method of combining mesenchymal stem ceils with a bone substrate comprising obtaining adipose tissue having the mesenchymal stem celis together with unwanted DCis; digesting the adipose tissue to form a ceil suspension having the mesenchyme! stem cris and the unwanted celis; adding the DC! suspension with the mesenchymal stem ceils to seed the bone substrate so as to form a seeded bone substrate; c ⁇ ituring the mesenchymal stem celis on the seeded bone substrate for a period of time to allow the mesenchymal stem celis to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate,
  • an aiiograft product including a combination of mesenchyme! stem ceils with a bone substrate, and the combination manufactured by obtaining adipose tissue having the mesenchymal stem cris together with unwanted DCis; digesting the adipose tissue to form a celi suspension having the mesenchymal stem celis and the unwanted DCis; adding the cell suspension with the mesenchymal stem DCis to seed the bone substrate so as to form a seeded bone substrate; cuituring the mesenchymal stem cells on the seeded bone substrate for a period of time to aiiow the mesenchymal stem cris to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted celis from the bone substrate.
  • a method of combining mesenchymal stem ceils vvtth a bone substrate comprising obtaining adipose tissue having the mesenchymal stem cells together with unwanted DCis: digesting the adipose tissue to form a cell suspension having the mesenchymal stem cris and the unwanted celis to acquire a stromal vascular fraction, and the digesting includes making a coilagenase I solution, and filtering the solution through a 0.2 ⁇ m ftiter unit, mixing the adipose solution with the coilagenase I solution, and adding the adipose solution mixed with the coilagenase 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
  • suspension with the mesenchyme! stem ceils to seed the bone substrate so as to form a seeded bone substrate; cuituring the mesenchymal stem cells on the seeded bone substrate for a period of time to allow the mesenchyma! stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted celis from the bone substrate.
  • an ai ⁇ ograft product including a combination of mesenchymal stem cells wrth a bone substrate, and the combination manufactured by obtaining adipose tissue having the mesenchymal stem DCis together with unwanted celis; digesting the adipose tissue to form a celi suspension having the mesenchymal stem cells and the unwanted ceils to acquire a stroma! vascuiar fraction, and the digesting includes making a collagenase ! solution, and filtering the solution through a 0.2 ⁇ m filter unit, mixing the adipose solution with the coilagenase !
  • a method of combining mesenchymal stem cells with a bone substrate comprising obtaining tissue having the mesenchymai stem ceils together with unwanted DCis; digesting the tissue to form a ceil suspension having the mesenchymai stem ceils and the unwanted DCis; adding the DCi suspension with the mesenchymai stem cells to seed the bone substrate so as to form a seeded bone substrate; culturi ⁇ g the mesenchymai stem cells on the seeded bone substrate for a period of time to ailow the mesenchymal stem cells to adhere to the bone substrate: and rinsing the bone substrate to remove the unwanted cells from the bone substrate.
  • an ailograft product inciuding a combination of mesenchymai stem ceils with a bone substrate, and the combination manufactured by obtaining tissue having the mesenchymal stem celis together with unwanted DCis; digesting the tissue to form a eel! suspension having the mesenchymal stem cells and the unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; culturing the mesenchymal stem cells on the seeded bone substrate for a period of time to aiiow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate,
  • a method of combining mesenchymal stem cells with a bone substrate comprising obtaining bone marrow tissue having the mesenchymal stem cells together with unwanted cells; digesting the bone marrow tissue to form a cell suspension having the mesenchymal stem DCis and the unwanted cells; adding the cell suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate; cuituring the mesenchymal stem cells on the seeded bone substrate for a period of time to aiiow the mesenchymai stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.
  • an allograft product including a combination of mesenchymai stem cells with a bone substrate, and the combination manufactured by obtaining bone marrow tissue having the mesenchymal stem ceils together with unwanted cells; digesting the bone marrow tissue to form a cell suspension having the mesenchymal stem cells and the unwanted ceils; adding the ceil suspension with the mesenchymal stem cells to seed the bone substrate so as to form a seeded bone substrate: culturing the mesenchymal stem cells and the bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate; and rinsing the bone substrate to remove the unwanted cells from the bone substrate.
  • a method of combining mesenchymal stem cells with a bone substrate comprising obtaining muscle tissue having the mesenchymal stem celis together with unwanted ceils; digesting the muscle tissue to form a eel! suspension having the mesenchyme!
  • an aiiograft product including a combination of mesenchyme! stem ceils with a bone substrate, and the combination manufactured by obtaining muscle tissue having the mesenchymal stem ce ⁇ s together with unwanted DCis; digesting the muscie tissue to form a DC!
  • FIGURE 1 illustrates a flow chart of the combination of mesenchymal stem cells with a bone substrate
  • FIGURE 2 illustrates a prior art example of a pellet of a stromal vascular fraction containing the desired stem cells and unwanted ceils;
  • FIGURE 3 illustrates various examples of strips ( Figures 3A and 38 ⁇ and dowels ⁇ FIGURES 3C and 3D) which have a 3-D cancellous matrix structure and mesenchymal stem cells ⁇ MSCs ⁇ may adhere to;
  • FIGURE 4 illustrates a standard curve of total live ASCs using the CCK-8 assay
  • FIGURE 5 illustrates mineral deposition by ASCs cultured in osteogenic medium
  • FIGURE 6 illustrates H&E staining showed that cells adhered to the bone surface.
  • human aduit stem cells are generally referred to as mesenchymal stem ceils or MSCs
  • MSCs are pl ⁇ ripotent cells that have the capacity to differentiate in accordance with at least two discrete development pathways.
  • Adipose-derived stem celis 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. In one embodiment, the pellet may include m ⁇ Stipie types of stem cells.
  • These stem cells may include, for example, one or more of hematopoietic stem cells, epithelial progenitor celis, and mesenchymal stem cells, in an embodiment, mesenchymal stem ceils are filtered from other stem cells by their adherence to a bone substrate, while the other stem cells ⁇ i.e., unwanted ceils) do not adhere to the bone substrate. Other barrets that do not adhere to the bone substrate may also be included in these unwanted celis.
  • Adipose derived stem cells may be isolated from cadavers and characterized using flow cytometry and tri-lineage differentiation (osteogenesis, chondrogenesis and adipogenesis) may be performed in vitro. The final product may be characterized using histology for microstr ⁇ cture and biochemical assays for cell count. This consistent ceil-based product may be usefui for bone regeneration.
  • 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 impiantation, is the cell source and the abiiity to control cell proliferation and differentiation. Many researchers have demonstrated that adipose tissue-derived stem cells (ASCs) possess muitipie differentiation capacities. See, for example, the following, which are incorporated by reference;
  • AnghtSeri E., et al., Neuronal differentiation potential of human adipose-derived mesenchymal stem cells. Stern Cells Dev, 2008. 17 ⁇ 5): p.
  • Adipose-derived stem cells are a source for cell therapy of the corneal stroma. Stem Cells, 2008. 26(2): p. 570-9.
  • Ki ⁇ gham, PJ. , et al, Adipose-derived stem cells differentiate into a
  • adipose tissue is probably the most abundant and accessible source of adult stem cells. Adipose tissue derived stem cells have great potential for tissue regeneration. Nevertheless, ASCs and bone marrow-derived stem cells (BMSCs) are remarkably similar with respect to growth and morphology, displaying fibroblastic characteristics, with abundant endoplasmic reticulum and large nucleus relative to the cytoplasmic volume. See, for example, the following, which are incorporated by reference:
  • ADAS adipose derived adult stem
  • Hayashi, O., et aL Comparison of osteogenic ability of rat mesenchymal stem cells from hone marrow,, periosteum, and adipose tissue.
  • Adipose-derived stem cells characterization and current application in orthopaedic tissue repair. Exp Biol Med (Maywood), 2009.
  • Hpoaspirate transplant a healing process mediated by adipose-derived adult stem cells, Pl ast Reconstr Surg , 2007. 119(5); p. 1409-22; discussion
  • Demineralized bone substrate as an allogeneic material, is a promising bone tissue-engineering scaffold clue to its close relation to autoiogous bone in terms of structure and function. Combined with MSCs 1 these scaffolds have been demonstrated to accelerate and enhance bone formation within osseous defects when compared with the matrix aione. See, for example, the foilowing, which are incorporated by reference:
  • human ASCs seeded bone substrates may be characterized in terms of microstructure, cell number and ceil identity using histology, biochemical assay and flow cytometry.
  • these substrates may include bone material which has been previously subjected to a demineralization process.
  • FIGURE 1 is a flow chart of a process for making an allograft with stem cells product, in an embodiment, a stroma! vascular fraction may be used to seed the allograft It should be apparent from the present disclosure that the term "seed” relates to addition and placement of the stem cells within, or at least in attachment to, the allograft, but is not limited to a specific process.
  • FIGURE 2 illustrates a pellet of the stromal vascular fraction containing the desired stem ceils.
  • a method of combining mesenchymal stem ceils with a bone substrate is provided. The method may include obtaining adipose tissue having the mesenchymal stem celis together with unwanted ceils.
  • Unwanted ceils may inciude hematopoietic stem seils an ⁇ other stromal cells.
  • the method may further include digesting the adipose tissue to form a cell suspension having the mesenchymal stem ceils and at least some or all of the unwanted cells. In another embodiment, this may be followed by negatively depleting some of the unwanted celis and other constituents to concentrate mesenchymal stem celis.
  • the method includes adding the cell suspension with the mesenchymal stem cells to the bone substrate. This may be followed by culturing the mesenchymal stem cells and the bone substrate for a period of time to allow the mesenchymal stem celis to adhere to the bone substrate. In order to provide a desired product, the method includes rinsing the bone substrate to remove the unwanted cells from the bone substrate,
  • an allograft product may include a combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.
  • the adipose tissue may be obtained from a cadaveric donor.
  • a typical donor yields 2 liters of adipose containing 18 million MSCs.
  • a bone substrate may be from the same cadaveric donor as the adipose tissue,
  • the adipose tissue may be obtained from a patient
  • both the bone substrate and the adipose tissue may be obtained from the same patient. This may include, but is not limited to, removal of a portion of the ilium (e.g., the iliac crest) may be removed from the patient by a surgical procedure and adipose cells may be removed using liposuction.
  • Other sources, and combination of sources, of adipose tissue, other tissues, and bone substrates may be utilized.
  • the adipose tissue may be washed prior to or during digestion.
  • 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 infranatant solution.
  • the washing may include repeating washing the adipose tissue multiple times until a clear infra ⁇ atant 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.
  • a method of combining mesenchymal stem cells with a bone substrate is provided. The method may include obtaining bone marrow tissue having the mesenchymal stem cells together with unwanted cells.
  • Unwanted cells may include hematopoietic stem sells and other stromal cells.
  • the method may further include digesting the bone marrow tissue to form a cell suspension having the mesenchymal stem cells and the unwanted cells. In another embodiment, this may be followed by naturally selecting IvISCs 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 to the bone substrate. This may be followed by cuituring the mesenchymal stem ceils and the bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate. In order to provide a desired product, the method includes rinsing the bone substrate to remove the unwanted cells from the bone substrate,
  • an allograft product may include a combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.
  • a method of combining mesenchymal stem ceils with a bone substrate may include obtaining muscle tissue having the mesenchyma! stem cells together with unwanted cells. Unwanted ceils may inciude hematopoietic stem seiis and other stromal cells. The method may further include digesting the muscle tissue to form a cell suspension having the mesenchyma! stem ceils and the unwanted cells, in another embodiment, this may be followed by naturally selecting ySCs to concentrate mesenchymal stem cells,
  • ⁇ ⁇ t > tn ⁇ method includes adding the cell suspension with the mesenchyma! stem ceils to the bone substrate. This may be followed by cuituring the mesenchymal stem ceils and the bone substrate for a period of time to allow the mesenchymal stem cells to adhere to the bone substrate. In order to provide a desired product, the method includes rinsing the bone substrate to remove the unwanted ceils from the bone substrate,
  • an allograft product may inciude a combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment.
  • a method of combining mesenchymal stem cells with a bone substrate may include obtaining tissue having the mesenchymal stem cells together with unwanted cells. Unwanted cells may include hematopoietic stem sells and other stromal cells. The method may further include digesting the tissue to form a cell suspension having the mesenchymal stem cells and at least some of the unwanted cells. In another embodiment, this may be followed by negatively depleting some of the unwanted cells and other constituents to concentrate mesenchyma! stem ceils. [0044] Next, the method includes adding the cell suspension with the mesenchymal stem ceils to the bone substrate. In an embodiment, this substrate may include a bone materia!
  • this substrate may be a non-bone materia!, which may include (but is not limited to) a collagen based material. This may be followed by culturing the mesenchymal stem ceils and the bone substrate fora period of time to allow the mesenchymal stem ceils to adhere to the bone substrate.
  • the method includes rinsing the bone substrate to remove the unwanted cells from the bone substrate,
  • an allograft product may include a combination of mesenchymal stem cells with a bone substrate such that the combination is manufactured by the above exemplary embodiment
  • 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 I 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.
  • 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. (See, for example, FIGURE 2.)
  • a lab sponge or other mechanism Prior to seeding, a lab sponge or other mechanism may be used to pat dry bone substrate.
  • adding the eel! suspension with the mesenchymal stem ceils to the bone substrate may include using a eel! pellet for seeding onto the bone substrate, In an embodiment, adding the eel! suspension with the mesenchymal stem cells to the bone substrate may include using a cell pellet for seeding onto the bone substrate.
  • adding the DCi suspension with the mesenchymal stem ceils to the bone substrate may include using a cell pellet for seeding onto the bone substrate of cortical bone.
  • adding the eel! suspension with the mesenchymal stem cel!s to the bone substrate may include adding the cell pellet onto the bone substrate of canceilous bone.
  • adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the cell pellet onto the bone substrate of ground bone.
  • adding the eel! suspension with the mesenchymal stem ceils to the bone substrate may include adding the cell pellet onto the bone substrate of cortical/cancellous bone.
  • adding the cell suspension with the mesenchymal stem cells to the bone substrate may include adding the DCi pellet onto the bone substrate of demineralized cancellous bone.
  • the method may include placing the bone substrate into a cryopreservation media after rinsing the bone substrate.
  • This cryopres ⁇ rvatson media may be provided to store the final products.
  • the method may include maintaining the bone substrate into a frozen state after rinsing the bone substrate to store the final products.
  • the frozen state may be at about negative 80° C.
  • negatively depleting the concentration of the mesenchymal stem cells may include adding a volume of PBS an ⁇ a volume of Ficoll density solution to the adipose solution.
  • the volume of PBS may be 5 ml and the volume of Fiooll density solution may be 25 ml with a density of 1 .073 g/ml.
  • Negatively depleting the concentration of the mesenchymal stem cells may aiso include centrifuging the adipose solution at about 116O 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 ceils 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 ceils, 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 mesenchymai 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 ceils, and resuspending the collected cells in a growth medium.
  • adding the cell suspension with the mesenchymai stem ceils to the bone substrate may include adding the DCS peliet onto the bone substrate.
  • Adding the solution with the mesenchymal stem cells to the bone substrate may include adding cell pellet onto the bone substrate which was subjected to a demineralization process.
  • adding the ceil suspension with the mesenchymal stem ceils to the bone substrate may include adding the cell pellet onto the bone substrate of cortical bone.
  • adding the eel! suspension with the mesenchymal stem cells to the bone substrate includes adding the ceil peilet onto the bone substrate of cancellous bone.
  • adding the cell suspension with the mesenchymal stem cells to the bone substrate may inciude adding the ceil peliet onto the bone substrate of ground bone.
  • adding the cell suspension with the mesenchymal stern ceils to the bone substrate may include adding the cell pellet onto the bone substrate of cortical/cancel Sous bone, in another embodiment, adding the cell suspension with the mesenchymal stem DCis to the bone substrate may include adding the celi pellet onto the bone substrate of demineralized cancellous bone,
  • the method may further include placing the bone substrate into a cryopreservation media after rtnsing the bone substrate.
  • This cryopreservation media may be provided to store the final products.
  • the method may include maintaining the bone substrate into a frozen state after rinsing the bone substrate to store the final products.
  • the frozen state may be at about negative 80° C.
  • the seeded allografts are cultured for a period of time to allow the mesenchymal stem commiss to adhere to the bone substrate. The unwanted cells were rinsed and removed from the bone substrate. After culturing, a iab sponge or other mechanism may be used to pat dry the bone substrate, [OOS63 The mesenchymal stem ceils are anchorage dependent.
  • the mesenchymal stem cells naturally adhere to the bone substrate.
  • the mesenchymal stem ceils are non-immunogenic and regenerate bone.
  • the unwanted celis are generally anchorage independent. This means that the unwanted cells generally do not adhere to the bone substrate.
  • the unwanted cells may be immunogenic and may create blood and immune system ceils.
  • mesenchymal stem celis adhere to the bone while unwanted cells, such as hematopoietic stem sells, are rinsed away leaving a substantially uniform population of mesenchymal stem cells on the bone substrate.
  • bone substrates ⁇ e.g., cortical cancellous dowels, strips, cubes, blocks, discs, and granules, as well as other substrates formed in dowels, strips, cubes, blocks, discs, and granules
  • a demineralizaiion process to remove blood, lipids and other cells so as to leave a matrix.
  • FIGURE 3 illustrates various examples of strips (FIGURES 3A and 3B) and dowels (FIGURES 3C and 3D).
  • these substrates may have a 3-D cancellous matrix structure, which MSCs may adhere to.
  • 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, coilagenase. 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 ceils 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 osteoinductive matrix, MSCs have been shown to differentiate along the osteoblastic iineage in vivo. See, for example, the following, which are incorporated by reference;
  • Adipose was recovered from cadaveric donors. Adipose aspirate may be collected using liposuction machine and shipped on wet ice.
  • Adipose was washed with equal volume of pre ⁇ warmed phosphate buffered saline (PBS) at 37 0 C, 1 % penicillin/streptomycin. Next, the adipose was agitated to wash the tissue. Phase separation was allowed for about 3 to 5 minutes. The infranatant solution was aspirated. The wash was repeated 3 to 4 times until a clear infranatant solution was obtained.
  • PBS phosphate buffered saline
  • the digestate was transferred to centrifuge tubes and centrifuged for 5 minutes at about 300-50Og at room temperature.
  • the supernatant, containing mature adipocytes, was then aspirated.
  • the pellet was identified as the stromal vascuiar fraction (SVF).
  • the allografts may include cortical/cancellous or both which was subjected to a demin ⁇ ralization process.
  • a dynamic “seeding” process can be used for particular bone substrate. 10m! of a cell suspension and bone substrate were placed in a 50ml centrifuge tube on an orbital shaker and agitated at 100 to 300 rpm for 6 hours. [0076] After a few days (about 1 to 3 days), the allograft was taken out and rinsed thoroughly in PBS and sonicated to remove unwanted cells. The allograft was put into cryopres ⁇ rvation media (10% DMSO, 90% serum) and kept frozen at -80 0 C. The frozen allograft combined with the mesenchymal stem cells is a final product.
  • Adipose was recovered from cadaveric donors. Adipose aspirate may be collected using liposuction machine and shipped on wet ice
  • Adipose tissue was processed in a thermal shaker at RPy ⁇ 75. 37 0 C for 10 min. Adipose was washed with equal volume of pre-warmed phosphate buffered saline (PBS) at 37 0 C, 1 % penicillin/streptomycin. Next, the adipose was agitated to wash the tissue. Phase separation was allowed for about 3 to 5 minutes. The supernatant solution was sucked off. The wash was repeated 3 to 4 times until a clear infranatant solution was obtained
  • PBS pre-warmed phosphate buffered saline
  • Ficoii density solution was added to the bottom of the tube with a pipet.
  • the tubes were subjected to centrifugatio ⁇ at 1 160g for 30 min at room temperature and stopped with the brake off.
  • the upper iayer and interface, approximately 15 to 17 mi containing the nucleated celis were coilected with a pipet and transferred to a new 50ml disposable centrifuge tube.
  • the iower iayer contained red ceils and cell debris and was discarded.
  • a cell peiiet was used for "seeding" onto allografts. Allografts may include deminarealized bone, cortical/canceiious bone, or both. A very smal! volume of medium was added into the DCi peiiet and shaken. 100 ⁇ ! of cell mixtures were added onto a 15mm disc within a 24-weii culture piate.
  • 00893 After culturing the allograft in a CO2 incubator at about 37 0 C, 1 ml growth medium (DMEM/F12, 10% FBS with antibiotics) was added. This was a static “seeding" process. A dynamic "seeding" process can be used for a particular bone substrate,
  • the allograft was taken out and rinsed thoroughly in PBS to remove unwanted ceils.
  • the allograft was put into cryopreservation media (10% DMSO, 90% serum) and kept frozen at -80 0 C.
  • the frozen allograft combined with the stem ceils is a final product.
  • Adipose was recovered from cadaveric donors. Adipose aspirate may be collected using liposuction machine and shipped on wet ice. [00933 WASHiNG
  • the bone marrow sample is washed by adding 6 to 8 volumes of Dulbecco's phosphate buffered saline (D-PBS) in a 50ml disposable centrifuge, inverting gently and subjecting to centrifugation (80Og for 10 min) to pellet ceils to the bottom of the tube.
  • D-PBS Dulbecco's phosphate buffered saline
  • Skeletal muscle may be recovered from cadaveric donors. [00983 WASHiNG
  • J00100J Minced skeietai muscle (1 ⁇ 3mm cube) is digested in a 3 mg/mi collagenase D solution in ⁇ -MEM at 37 0 C for 3 hours.
  • the solution is filtered with "IGOum nylon mesh.
  • the solution is ceotrifuged at 500 g for 5 min.
  • 1001013 ACQUIRE STEM CELLS AND COMBINE ONTO ALLOGRAFT [00102J
  • the supernatant ts discarded and the cell pellets from all lubes are resuspended in 1-2 ml of growth medium (DMEM, low glucose, with 10% FBS and1 % pen/strap).
  • the cell mixtures are seeded onto allografts.
  • Adipose was recovered from a cadaveric donor within 24 hours of death and shipped in equal volume of DMEM in wet ice.
  • I0010S3 WASHiNG was recovered from a cadaveric donor within 24 hours of death and shipped in equal volume of DMEM in wet ice.
  • Adipose were washed 3 times with PBS and suspended in an equal volume of PBS supplemented with Coliagenase Type i prewarmed to 37 0 C.
  • the tissue was placed in a shaking water bath at 37 0 C with continuous agitation for 45 to 60 minutes and centrif ⁇ ged for 5 minutes at room temperature.
  • the supernatant, containing mature adipocytes, was aspirated.
  • the pe ⁇ let was identified as the SVF (stromal vascular fraction).
  • ASCs adipose-derived stem ceils
  • PE anti-CD73 (clone AD2) Becton Dickinson, PE an!i-CD90 (clone F15-42-1 ) AbD SeroTec, PE anti-CD 105 (clone SN6) AbD SeroTec, PE anti-Fibroblasts/Epitheiial Cells (clone D7-FIB) AbD SeroTec, FITC anti-CD34 (clone 8G12) Becton Dickinson, FITC anti-CD45 (clone 2D1) Becfo ⁇ Dickinson, and PE anti-CD271 (clone ME20.4-1.H4) Miltenyi BioTec.
  • the lsotype controls were FITC Mouse IgGI Kappa (clone MOPC-21) Becton Dickinson, PE Mouse IgGi Kappa (clone MOPC-21 ) Becton Dickinson, and PE Mouse lgG2a Kappa ⁇ clone G155-178) Becton Dickinson. [00114J A small aliquot of the cells were stained with a propidium iodide/detergent solution and fluorescent nuclei were counted using a hemocytometer on a fluorescent microscope.
  • This total ceil count was used to adjust the number of cells per staining tube to no more than 5.0 x 105 cells
  • the celts were washed with flow cytometric wash buffer (PBS supplemented with 2% FBS and 0.1% NaN3), stained with the indicated antibodies and washed again before acquisition Staining was for 15 minutes at room temperature (15-30 C). At least 20 : 000 cells were acquired for each sample on a FACScan flow cytometer equipped with a 15-mW, 488-nm, argon-ion laser (BD Smmunocytometry Systems, San Jose, CA). The cytometer QC and setup included running SpheroTech rainbow (3 ⁇ m, 6 peaks) calibration beads (SpheroTech Inc.) to confirm instrument functionality and linearity.
  • Flow cytometric data were collected and analyzed using CellQuest software (BD immunocytometry Systems).
  • the small and large ceils were identified by forward (FSC) and side-angle light scatter (SSC) characteristics. Autofluorescence was assessed by acquiring ceils on the flow cyiometer without incubating with fiuorochrome labeled antibodies. Surface antigen expression was determined with a variety of directly labeled antibodies according to the supplier's recommendations. Antibodies staining fewer than 20% of the cells relative to the isotype-matched negative control were considered negative (this is standard-of-practice for immunophenofyping leukocytes for leukemia lymphoma testing). The viability of the small and large cells was determined using the Becton Dickinson Via-Probe (7- AAD).
  • Osteogenesis - Confluent cultures of primary ASCs were induced to undergo osteogenesis by replacing the stromal medium with osteogenic induction medium (Stempro® osteogenesis differentiation kit, Snvitrogen). Cultures were fed with fresh osteogenic induction medium every 3 to 4 days for a period of up to 3 weeks. Cells were then fixed in 10% neutral buffered formalin and rinsed with D! water. Osteogenic differentiation was determined by staining for calcium phosphate with Alizarin red (Sigma).
  • Adipogenesis - Confluent cultures of primary ASCs were induced to undergo adipogenesis by replacing the stromal medium with adipogenic induction medium (Stempro® adipogenesis differentiation kit, Snvitrogen). Cultures were fed with fresh adipogenic induction medium every 3 to 4 days for a period of up to 2 weeks. Cells were then fixed in 10% neutral buffered formalin and rinsed with PBS, Adipogenic differentiation was determined by staining for fat globules with oil red O (Sigma).
  • Cell count may be preformed with a CCK-8 Assay.
  • Cell Counting Kit 8 (CCK-8 r Dojindo Molecular Technologies, Maryland) allows sensitive colorimetric assays for the determination of the number of viable calls in celi proliferation assays.
  • FIGURE 4 there is illustrated a standard curve of total live ASCs using the CCK-8 assay.
  • WST-8 [2- ⁇ 2 ⁇ methoxy ⁇ 4 ⁇ nitropheny! ⁇ 3 ⁇ (4 ⁇ nitropheny!) ⁇ 5 ⁇ 2 !
  • FIGURE 3 illustrates an appearance of strips, dowels and disks.
  • ali have a corticai bottom and cancellous top.
  • Other embodiments may be used,
  • the small cells (mean 97%) contain only a small percentage of the markers tested and therefore could not be immunophenotyped with this method: D7-FIB (5%), CD105 (6%), CD90 (15%), CD73 (6%) and CD34 (10%).
  • the SVF contained a significant population of CD34+ cells (Large CD34+ 62% and small GD34+ 10%).
  • the paucity of CD45+ cells (Large 15% and small 3%) would suggest that the SVF does not contain significant numbers of WBC (CO45+ : low FSC, low SSC) or hematopoietic stem cells (CD34+, low CD45+, medium FSC, Sow SSC).
  • the anti-Fibroblasts/Epithelial Cells (clone D7-FSB) antibody has been reported to be a good marker for MSC.
  • the large cells were D7-FIB+ 36% and the small cells were D7-FIB+ 5%.
  • CD271 should be negative on SVF cells and the large cells were CD271+ 10% and the small cells were CD271+ 0%.
  • the immunophenotype became more homogenous for both the large and small cells.
  • the large cells (53%) have the following immunophenotype and percentage; D7-FIB+ (93%), CD105+ (98%), CD90+ (96%) and CD73+ (99%).
  • the small cells (47%) have the following immunophenotype and percentage; D7-FIB+ (77%), CD105+ (75%), CD90+ (58%) and CD73+ (83%).
  • the ASCs has lost CD34 marker expression (P3; large 4% and small 1%) (P 1 : large 8% and small 6%) and the CD45+ cells remained low (P3: large 2% and small 2%) (P1 : large 3% and small 1% ⁇ . This would suggest thai there are few WBC (CD45+, low FSC, Sow SSC) or hematopoietic stem cells (CD34+, tow CD45+, medium FSC, low SSC) present.
  • the anti-Fibrabiasts/Epitheiial Ceil ⁇ clone D7-FIB) antibody for the adherent and cultured cells showed an increased expression.
  • the large ceils were D7-FI8+ 93% and the small creis were D7-FI8+ 77%.
  • CD271 should become positive following adherence and culture of the SVF.
  • the iarge cells were CD271 + 4% and the smali cells were CD271+ 1 %.
  • the large ceils were CD271+ 27% and the smaii cells were CD271+ 3%.
  • CD271 does not seem to be a useful marker for cuitured MSC but more data is required.
  • CD 105 was chosen to estimate the mean tota! percentage of MSC; although there is no single surface marker that can discern MSC in a mixed population.
  • Figure 5 illustrates mineral deposition by ASCs cultured in osteogenic medium (A) indicating eariy stages of bone formation.
  • the samples were stained with alizarin red S.
  • Negative controls (D) showed no sign of bone formation.
  • the samples were stained with Oi! red O.
  • the picture E is negative control.
  • the samples were stained with alcian blue.
  • the negative control (F) showed no sign of chondrogenesis, [00135]
  • morphoSogical changes appeared during the second week of the culture. At the end of the 21 -day induction period, some calcium crystals were clearly visible. Cell differentiation was confirmed by alizarin red staining (Fig. 3A).
  • adipogenic potential was assessed by induction of confluent ASCs, At the end of the induction cycles (7 to 14 days), a consistent cell vacuolation was evident in the induced cells. Vacuoles brightly stained for fatty acid with oil red O staining (Fig, 38). Chondrogenic potential was assessed by induction of confluent ASCs. At the end of the induction cycles (14 to 21 days), the induced ceils were clearly different from non-induced control cells. Cell differentiation was confirmed with Alcian blue staining (Fig. 3C).
  • FIGURE 6 is an illustration of H&E staining that showed that stem cells adhered to the bone surface.
  • ASCs possess a simiiar abiiity to differentiate into osteoblasts under similar conditions.
  • Human ASCs offer a unique advantage in contrast to other eel! sources.
  • the muitipotent characteristics of ASCs, as wells as their abundance in the human body, make these cells a popular source in tissue engineering applications. This consistent cell-based new product has the potential to be effective for bone regeneration.

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Abstract

La présente invention concerne un procédé de combinaison de cellules souches mésenchymateuses (MSC) avec un substrat osseux. Dans un mode de réalisation, le procédé comprend l’obtention de tissu ayant des MSC conjointement avec des cellules indésirables. Le tissu est digéré pour former une suspension de cellules comprenant des MSC et des cellules indésirables. La suspension de cellules est ajoutée au substrat. Le substrat est cultivé de manière à permettre aux MSC d’adhérer. Le substrat est rincé afin d’éliminer les cellules indésirables. Dans différents modes de réalisation, le tissu est un tissu adipeux, un tissu musculaire, ou un tissu de moelle osseuse. Dans un mode de réalisation, il est décrit un produit d’allogreffe comprenant une combinaison de MSC avec un substrat osseux où la combinaison est produite par culture de MSC disposées sur le substrat pendant un certain temps pour permettre aux MSC d’adhérer au substrat, puis rinçage du substrat pour enlever les cellules indésirables du substrat. D’autres modes de réalisation sont également décrits.
PCT/US2009/064611 2008-11-20 2009-11-16 Allogreffes combinées avec des cellules souches dérivées de tissu pour la cicatrisation osseuse WO2010059565A2 (fr)

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ITGE20120034A1 (it) * 2012-03-28 2013-09-29 Carlo Tremolada Preparato e metodo per la produzione di un preparato comprendente cellule staminali mesenchimali
US9186380B2 (en) 2012-11-15 2015-11-17 Allosource Minced cartilage systems and methods
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AU2014225458A1 (en) 2013-03-07 2015-07-09 Allosource Consistent calcium content bone allograft systems and methods
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US10857264B2 (en) 2009-06-04 2020-12-08 Universite Catholique De Louvain Multi-dimensional biomaterial and method for producing the same
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US9808558B2 (en) 2017-11-07
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US20160030639A1 (en) 2016-02-04
US9192695B2 (en) 2015-11-24
US9814803B2 (en) 2017-11-14
EP2358404A4 (fr) 2012-08-15
KR20110106855A (ko) 2011-09-29
CA2743869C (fr) 2017-03-14
EP2358404A2 (fr) 2011-08-24
US20160045640A1 (en) 2016-02-18
WO2010059565A3 (fr) 2010-08-19
CA2743869A1 (fr) 2010-05-27
EP3111965A1 (fr) 2017-01-04

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