WO2022102739A1 - 脱細胞化組織組成物 - Google Patents

脱細胞化組織組成物 Download PDF

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WO2022102739A1
WO2022102739A1 PCT/JP2021/041694 JP2021041694W WO2022102739A1 WO 2022102739 A1 WO2022102739 A1 WO 2022102739A1 JP 2021041694 W JP2021041694 W JP 2021041694W WO 2022102739 A1 WO2022102739 A1 WO 2022102739A1
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Prior art keywords
decellularized tissue
decellularized
protein
tissue composition
mass
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PCT/JP2021/041694
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English (en)
French (fr)
Japanese (ja)
Inventor
光将 本間
謙一郎 日渡
春樹 小原
拓矢 木村
敬太 木下
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Adeka Corp
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Adeka Corp
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Priority to EP21891984.3A priority Critical patent/EP4245845A4/en
Priority to CA3204817A priority patent/CA3204817A1/en
Priority to CN202180076247.XA priority patent/CN116615528A/zh
Priority to KR1020237018831A priority patent/KR20230107827A/ko
Priority to JP2022562197A priority patent/JPWO2022102739A1/ja
Priority to US18/036,218 priority patent/US20250108147A1/en
Priority to AU2021379450A priority patent/AU2021379450A1/en
Publication of WO2022102739A1 publication Critical patent/WO2022102739A1/ja
Anticipated expiration legal-status Critical
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    • 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
    • A61L27/3633—Extracellular matrix [ECM]
    • 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
    • A61L27/3625—Vascular tissue, e.g. heart valves
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • 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
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention relates to a decellularized tissue composition.
  • Decellularization means removing cellular components such as nucleic acids that are antigenic to the transplantee, thereby avoiding immune rejection.
  • the decellularized tissue can be produced by decellularizing the living tissue using, for example, a treatment liquid containing a surfactant (Patent Documents 1 to 3).
  • an object of the present invention is to provide a decellularized tissue exhibiting appropriate strength and good tissue regeneration.
  • the decellularized tissue composition according to [1] which further contains protein B having (a) a molecular weight of 3,000 to 15,000 and (b) an isoelectric point: pI3.00 to 5.50.
  • the decellularized tissue composition of the present invention since it exhibits appropriate strength and excellent cell adhesion, it exhibits good tissue regeneration ability (for example, cell-inducing effect and differentiation-inducing effect) in vivo. Can be done. In addition, by having excellent strength, it exhibits excellent handling performance at the time of treatment.
  • the decellularized tissue composition of the present invention comprises a decellularized tissue and a protein having (a) a molecular weight of 30,000 to 70,000 and (b) an isoelectric point: pI6.00 to 9.00 (hereinafter, protein). (Sometimes referred to as A) and.
  • the decellularized tissue composition of the present invention is a protein having (a) a molecular weight of 3,000 to 15,000 and (b) an isoelectric point: pI3.00 to 5.50 (hereinafter, may be referred to as protein B). ) May be included.
  • the decellularized tissue is a tissue from which cell components have been removed from animal-derived tissue, and is a tissue whose main component is extracellular matrix components such as elastin, collagen (type I, type IV, etc.) and laminin. It has a low rejection reaction at the time of transplantation, can be expected to establish and reconstruct the cells at the transplant destination, and functions as a scaffold for the cells.
  • a method for obtaining the decellularized tissue contained in the composition of the present invention a conventionally known method can be used.
  • the method for decellularization is not particularly limited as long as the effect of the present invention can be obtained, but for example, a method by high hydrostatic pressure treatment, a method by freeze-thaw treatment, and a surfactant are used.
  • Methods, treatment with an alcohol solvent, etc. may be mentioned, and two or more of these may be combined, and high hydrostatic pressure treatment is performed in order to efficiently obtain a decellularized tissue composition and to exert the effect of the present invention.
  • the method according to is preferable.
  • the decellularized tissue used in the decellularized tissue composition of the present invention is not particularly limited as long as it is a vertebrate-derived biological tissue, but since there are few rejection reactions, a mammalian or bird-derived biological tissue is preferable.
  • Mammalian livestock, bird livestock or human-derived biological tissues are more preferred because they are readily available.
  • Mammal livestock include cows, horses, camels, raccoons, donkeys, yaks, sheep, pigs, goats, deer, alpaca, dogs, raccoon dogs, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, mice, squirrels, Examples include raccoons.
  • livestock of birds examples include inco, parrot, chicken, duck, turkey, guinea fowl, guinea fowl, pheasant, ostrich, quail, and emu.
  • bovine, pig, rabbit, and human biological tissues are preferable because of the stability of availability.
  • a site having an extracellular matrix structure can be used, and such sites include, for example, liver, kidney, urinary tract, bladder, urethra, tongue, tongue, esophagus, stomach, and small intestine.
  • examples include the large intestine, anus, pancreas, heart, blood vessels, spleen, lungs, brain, bones, spinal cord, cartilage, testis, uterus, oviduct, ovary, placenta, cornea, skeletal muscle, tendon, nerve, and skin.
  • a site of living tissue it is preferably in cartilage, bone, liver, kidney, heart, heart membrane, aorta, skin, small intestinal submucosal tissue, lung, brain, internal thoracic artery, or spinal cord because of its high effect of tissue regeneration.
  • the heart membrane, internal thoracic artery, liver, cartilage, skin, small intestinal submucosal tissue, or spinal cord is preferably in cartilage, bone, liver, kidney, heart, heart membrane, aorta, skin, small intestinal submucosal tissue, lung, brain, internal thoracic artery, or spinal cord.
  • a hydrostatic pressure of 50 to 1500 MPa is applied to the tissue derived from a living body in a medium.
  • the hydrostatic pressure to be applied is preferably 50 MPa or more, a pressure vessel that can withstand the application is not required, a large amount of energy is not required, and the medium used for the application is an aqueous medium. Is preferably 1500 MPa or less from the viewpoint of producing ice and preventing the tissue from being damaged by the generated ice.
  • the hydrostatic pressure to be applied is more preferably 80 to 1300 MPa, further preferably 90 to 1200 MPa, further preferably 95 to 1100 MPa, further preferably 95 to 700 MPa, and 400 to 700 MPa are decellularization effect, sterilization effect and virus-free. It is most preferable from the viewpoint of activating effect and ease of application.
  • Examples of the medium used for applying the hydrostatic pressure include water, physiological saline, water for injection, propylene glycol or an aqueous solution thereof, glycerin or an aqueous solution thereof, and a saccharide aqueous solution.
  • Examples of the buffer solution include acetate buffer solution, phosphate buffer solution, citric acid buffer solution, borate buffer solution, tartrate buffer solution, Tris buffer solution, HEEPS buffer solution, MES buffer solution and the like. These media may contain a surfactant.
  • the temperature of the high hydrostatic pressure treatment is not particularly limited as long as it does not generate ice and does not damage the tissue due to heat, but it is 0 to 0 because the decellularization treatment is smoothly performed and the effect on the tissue is small. 45 ° C. is preferable, 4 to 37 ° C. is more preferable, and 15 to 35 ° C. is most preferable. If the time of high hydrostatic pressure treatment is too short, cells will not be sufficiently destroyed, and if it is long, it will lead to waste of energy. Therefore, in high hydrostatic pressure treatment, the time to maintain the target applied pressure is 1 to 1 to 1. 120 minutes is preferable, 5 to 60 minutes is more preferable, and 7 to 30 minutes is even more preferable.
  • the tissue treated with high hydrostatic pressure is preferably treated with a nucleolytic enzyme.
  • Nucleic acid-degrading enzymes remove nucleic acid components from living tissues to which hydrostatic pressure is applied, and are not particularly limited, but are, for example, pancreas-derived, spleen-derived, or Escherichia coli-derived DNase (for example, DNase I, DNase II) can be mentioned.
  • the nucleic acid-degrading enzyme can be added to the medium used for the high hydrostatic pressure treatment (for example, water, physiological saline, injection solution, buffer solution, etc.) and allowed to act.
  • the amount of enzyme to be added varies depending on the type of enzyme and the definition of the number of units (U), but can be appropriately set by those skilled in the art.
  • DNase I may be used at 50 to 200 U / mL.
  • the treatment temperature also varies depending on the nucleolytic enzyme used, but may be set to, for example, 1 ° C to 40 ° C.
  • the treatment time is also not particularly limited, but may be, for example, 1 to 120 hours (preferably 1 to 96 hours, more preferably 1 to 48 hours). Time processing is sufficient.
  • the tissue treated with high hydrostatic pressure is washed with a washing liquid.
  • the cleaning solution may be the same as or different from the medium for high hydrostatic pressure treatment.
  • the cleaning liquid preferably contains an organic solvent or a chelating agent.
  • the organic solvent can improve the efficiency of removing lipids, and the chelating agent inactivates calcium ions and magnesium ions in the decellularized tissue to make the particulate decellularized tissue of the present invention into a diseased part. It can prevent calcification when applied.
  • the organic solvent a water-soluble organic solvent is preferable, and ethanol, isopropanol, acetone, and dimethyl sulfoxide are preferable because the effect of removing lipids is high.
  • Chelating agents include ethylenediamine tetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriminpentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexacetic acid (TTHA), 1,3-propane.
  • EDTA ethylenediamine tetraacetic acid
  • NTA nitrilotriacetic acid
  • DTPA diethylenetriminpentaacetic acid
  • HEDTA hydroxyethylethylenediaminetriacetic acid
  • TTHA triethylenetetraminehexacetic acid
  • Diaminetetraacetic acid PDTA
  • 1,3-diamino-2-hydroxypropanetetraacetic acid DPTA-OH
  • HIDA hydroxyethyliminodiacetic acid
  • DHEG dihydroxyethylglycine
  • GEDTA glycol etherdiaminetetraacetic acid
  • Iminocarboxylic acid-based chelating agents such as dicarboxymethyl glutamic acid (CMGA), 3-hydroxy-2,2'-iminodysuccinic acid (HIDA), dicarboxymethyl aspartic acid (ASDA) or salts thereof; citric acid, tartrate acid, malic acid.
  • a hydroxycarboxylic acid-based chelating agent such as lactic acid or a salt thereof, and examples of the salt of these chelating agents include a sodium salt or a potassium salt.
  • the cleaning temperature is not particularly limited as long as it does not damage the tissue due to heat, but is preferably 0 to 45 ° C., more preferably 1 to 40 ° C., because the cleaning property is good and the effect on the tissue is small. Most preferably 2 to 35 ° C. In the case of washing, the washing liquid may be shaken or stirred as necessary.
  • the biological tissue is subjected to a temperature of -80 to -20 ° C (preferably -80 to -40 ° C) for 1 to 48 hours (preferably 10 to 10 to 40 ° C). After holding and freezing for 30 hours, the step of thawing at a temperature of 20 to 37 ° C. is repeated once or twice or more (preferably 2 to 5 times). After that, it is preferable to carry out the nucleolytic enzyme treatment, and the nucleolytic enzyme treatment may be carried out by the same method as the treatment in the high hydrostatic pressure treatment. In the freeze-thawed living tissue, the cells in the tissue are destroyed, and these cells are removed by the washing solution. The cleaning method may be the same as that used in the high hydrostatic pressure treatment.
  • the biological tissue is treated with a detergent solution (for example, 0.25 mass% sodium dodecyl sulfate (SDS) solution) from 1 to 48. Shake at 2-10 ° C (preferably 4 ° C) for an hour (preferably 12-36 hours).
  • a detergent solution for example, 0.25 mass% sodium dodecyl sulfate (SDS) solution
  • SDS sodium dodecyl sulfate
  • different surfactant solutions eg, 0.5% mass% Triton X (polyoxyethylene octylphenyl ether) solution
  • 1 to 48 hours preferably 12 to 36 hours
  • 2 to 10 ° C. preferably
  • It may be shaken at 4 ° C.
  • Surfactants include, but are not limited to, sodium dodecyl sulphate, alkyl sulfonates, alkyl sulphates, polyoxyethylene alkyl sulfates, ⁇ -sulfofatty acid ester salts, polyoxyethylene alkyl ethers, etc.
  • Examples thereof include polyoxyethylene alkyl phenyl ether (for example, polyoxyethylene octyl phenyl ether) and alkyl (poly) glycosides.
  • the tissue derived from the living body is sonicated (for example, intensity: 10 W / cm 2 , frequency: 10 kHz, action time: in physiological saline, for example). 2 minutes). Then, preferably in a surfactant solution (eg, 1% mass% Triton X (polyoxyethylene octylphenyl ether) solution) for 1 to 120 hours (preferably 12 to 120 hours), 2 to 10 ° C. (preferably). Shake at 4 ° C). Further, it is preferable to carry out the nucleolytic enzyme treatment, and the nucleolytic enzyme treatment may be carried out by the same method as the treatment in the high hydrostatic pressure treatment. After that, it is preferable to wash by the same method as the washing in the high hydrostatic pressure treatment.
  • a surfactant solution eg, 1% mass% Triton X (polyoxyethylene octylphenyl ether) solution
  • the obtained decellularized tissue is not limited, but is preferably freeze-dried. Freeze-drying can be omitted depending on the site of the living tissue. Further, the obtained decellularized tissue may be sterilized by gamma ray irradiation, UV irradiation or the like, and it is preferable to sterilize by gamma ray irradiation.
  • the content of the decellularized tissue is preferably 90.0 to 100.0% by mass, preferably 95.0 to 100.0% by mass, based on the decellularized tissue composition, from the viewpoint of exerting the effect of the present invention. % Is more preferable, 96.0 to 100.0% by mass is further preferable, and 97.0 to 100.0% by mass is most preferable.
  • the DNA content of the decellularized tissue composition is low, but in the decellularized tissue composition of the present invention, the DNA content per dry mass is higher than that of the decellularized tissue composition. It is preferably 0.0300% by mass or less. This can prevent rejection from occurring when used in regenerative medicine.
  • the decellularized DNA ratio of the decellularized tissue composition of the present invention is more preferably 0.0250% by mass or less, further preferably 0.0200% by mass or less, and 0. It is more preferably 0.0150% by mass or less, and most preferably 0.0120% by mass or less.
  • the decellularized DNA ratio is 0.0150% by mass or less, it is preferable in that the cell adhesion to the decellularized tissue composition is particularly easy to be enhanced.
  • the lower limit of the decellularized DNA ratio is not particularly limited, but is preferably as low as possible, preferably 0.0001% by mass or more, and further preferably 0.0002% by mass or more from the viewpoint of ease of realization.
  • the DNA content can be measured by the picogreen method.
  • a dry test piece of the decellularized tissue composition (hereinafter, may be referred to as a sample) is immersed in a proteolytic enzyme solution to dissolve it, and then treated with phenol / chloroform to remove the protein, and then subjected to an ethanol precipitation method. Collect DNA.
  • the recovered DNA is fluorescently stained with picogreen (Life Technologies) to quantify the DNA by measuring the fluorescence intensity, and the DNA content (mass) of the sample is calculated.
  • a calibration curve prepared using the standard DNA attached to Pico Green is used. From the dry mass and DNA content of the sample, the DNA ratio is calculated according to the following formula.
  • the decellularized tissue composition of the present invention comprises protein A having (a) a molecular weight of 30,000 to 70,000 and (b) an isoelectric point: pI 6.00 to 9.00.
  • the decellularized tissue composition of the present invention may further contain a protein having (a) a molecular weight of 3,000 to 15,000 and (b) an isoelectric point: pI3.00 to 5.50. Thereby, the effect of the present invention can be more exerted.
  • the molecular weight and isoelectric point (pI) of the protein can be determined, for example, by two-dimensional electrophoresis of the protein, and identification of the protein can be performed by mass spectrometry.
  • either isoelectric point separation or molecular weight separation may be performed first (as the first dimension), but from the viewpoint of improving the accuracy of measurement, the isoelectric point separation is performed in the first dimension. It is preferable to perform molecular weight separation in the second dimension.
  • Two-dimensional electrophoresis can be performed according to a conventional method, and commercially available kits and devices can be used. For example, isoelectric focusing is performed using a capillary gel or strip gel as a separation medium, and the gel after completion of the electrophoresis is used as a planar gel (for example, SDS-polyacrylamide gel) to develop the isoelectric focusing.
  • the molecular weight can be separated by performing electrophoresis in the direction perpendicular to the relative weight.
  • electrophoresis By staining the gel subjected to two-dimensional electrophoresis according to a conventional method, the presence or absence of protein, the molecular weight, and the isoelectric point can be confirmed.
  • the molecular weight of the protein A is preferably 30,000 to 70,000, more preferably 30,000 to 50,000, and even more preferably 30,000 to 40,000 from the viewpoint of exerting the effect of the present invention.
  • the isoelectric point (pI) of the protein A is preferably 6.00 to 9.00, more preferably 6.00 to 8.50, and even more preferably 6.50 to 8.50.
  • the protein A is preferably annexin.
  • Annexin is a protein with a curved core domain consisting of so-called annexin repeats (about 70 amino acid residues) having 4 or 8 ⁇ -helix structures.
  • the amino-terminal domain is a sequence unique to each annexin (residues 11 to 196), but the carboxy-terminal domain is well conserved among the annexins.
  • the calcium binding site and the phospholipid binding site are located on the domain on the carboxy-terminal side.
  • the molecular weight of annexin VI with 8 annexin repeats is about 66 k.
  • the origin of the annexin contained in the decellularized tissue composition is not particularly limited as long as it is derived from eukaryotes, but annexins derived from mammals or birds are preferable.
  • Mammals include cows, horses, camels, ryamas, donkeys, yaks, sheep, pigs, goats, deer, alpaca, dogs, raccoon dogs, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, mice, squirrels, or raccoons. And so on.
  • birds include inco, parrot, chicken, duck, turkey, goose, guinea fowl, pheasant, ostrich, quail, and emu.
  • annexins A1 to A11 and A13 are present and have a binding site between Ca 2+ and phospholipids.
  • S100A10 having a C-terminal lysine binds to the N-terminal region of the concave surface of annexin A2, and becomes a binding site for tissue-type plasminogen activator (tPA) and plasminogen.
  • the annexin contains annexins having different modifications. That is, it comprises phosphorylated, glycosylated, ubiquitinated, nitrosylated, methylated, or acetylated, or unmodified annexins.
  • the annexin A2 described in the examples is three kinds of annexin A2 having different phosphorylation, and all of them can show the effect of the present invention.
  • the decellularized tissue composition of the present invention can include, but is not limited to, a variant of annexin as long as the effects of the present invention can be obtained.
  • Annexin variants include, for example. (1) One or more (preferably 1 to 10, more preferably 1 to 7) as a whole at one or more of the amino acid sequences of annexin (for example, the amino acid sequence represented by SEQ ID NO: 1). More preferably 1-5), eg, a polypeptide comprising an amino acid sequence in which 1-several amino acids have been deleted, substituted, inserted, and / or added as a whole and have annexin activity, or (2).
  • a polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence of annexin (for example, the amino acid sequence represented by SEQ ID NO: 1) and having annexin activity can be mentioned.
  • the activity of annexin include the ability to bind "Ca 2+ and phospholipids".
  • the decellularized tissue composition of the present invention may be used to improve cell adhesion as compared with a cell-free tissue composition.
  • the variant may be a polypeptide consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence of annexin (eg, SEQ ID NO: 1).
  • the modified polypeptide has a binding ability between Ca 2+ and a phospholipid. That is, a polypeptide that does not show the ability to bind Ca 2+ to a phospholipid is not included in the modified polypeptide.
  • amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added means that the amino acid has been modified by substitution or the like.
  • the number of amino acid modifications can be, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, preferably 1 to 8, more preferably 1 to 6, and even more preferably. Is 1 to 5, most preferably 1 to 2.
  • An example of a variant amino acid sequence that can be used in the present invention is preferably an amino acid sequence in which the amino acid has one or several (preferably 1, 2, 3 or 4) conservative substitutions. be able to.
  • the variant may be a polypeptide consisting of an amino acid sequence having an amino acid sequence identity of 90% or more with respect to the amino acid sequence of annexin (eg, SEQ ID NO: 1).
  • the modified polypeptide has a binding ability between Ca 2+ and a phospholipid. That is, a polypeptide that does not show the ability to bind Ca 2+ to a phospholipid is not included in the modified polypeptide. More preferably, an amino acid sequence having an identity of 95% or more, more preferably an amino acid sequence having an identity of 96% or more, more preferably an amino acid sequence having an identity of 97% or more, and more preferably an amino acid sequence having an identity of 98% or more.
  • Is a polypeptide consisting of an amino acid sequence having an amino acid sequence of 99% or more, and is a polypeptide showing a binding ability between Ca 2+ and a phospholipid.
  • amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added or "amino acid sequence having 90% or more identity” in the amino acid sequence of the annexin (for example, SEQ ID NO: 1).
  • substitution of the amino acid sequence of annexin eg, SEQ ID NO: 1
  • conservative substitution means a substitution in which the superior effect of annexin is not lost. That is, it is a substitution that can maintain the binding ability of Ca 2+ of annexin to a phospholipid even when it is inserted, substituted, deleted, or added.
  • amino acid residue means replacing the amino acid residue with another chemically similar amino acid residue.
  • hydrophobic residue is replaced with another hydrophobic residue
  • polar residue is replaced with another polar residue having the same charge
  • Functionally similar amino acids that can be made by making such substitutions are known in the art for each amino acid.
  • non-polar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine and the like.
  • polar (neutral) amino acid include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
  • positively charged (basic) amino acids include arginine, histidine, and lysine.
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the content of the protein A is preferably 0.01 to 5.0% by mass, more preferably 0.03 to 4.5% by mass, and 0.05 to 4.0% by mass with respect to the decellularized tissue composition.
  • mass% is even more preferable, 0.1 to 3.5% by mass is even more preferable, 0.2 to 3.3% by mass is even more preferable, and 0.3 to 3.0% by mass is most preferable. Thereby, the effect of the present invention can be exhibited.
  • the molecular weight of the protein B is preferably 3,000 to 15,000, more preferably 5,000 to 14,000, and even more preferably 7,000 to 13,000, from the viewpoint of exerting the effect of the present invention.
  • the isoelectric point (pI) of the protein is preferably 3.00 to 5.50, more preferably 3.40 to 5.30, and even more preferably 3.80 to 5.20.
  • the content of the protein B is preferably 0.001 to 3.0% by mass, more preferably 0.003 to 2.0% by mass, and 0.005 to 1.5% by mass with respect to the decellularized tissue composition.
  • the mass% is further preferable, 0.008 to 1.0% by mass is further preferable, 0.009 to 0.5% by mass is further preferable, and 0.01 to 0.1% by mass is most preferable. Thereby, the effect of the present invention can be exhibited.
  • the protein B preferably includes a fragment of fibromodulin.
  • Fibromodulin is a protein that mainly binds to fibrous collagen, and is considered to have a role of binding collagen fibers to each other. It is known that when the expression of fibromodulin increases, the density of collagen fibers increases. Fibromodulin is a protein consisting of 375 amino acids (SEQ ID NO: 2), having a molecular weight of 43,000 and a pI of 5.6.
  • Protein B is a fragment of fibromodulin, and is not limited, but is a fragment consisting of 90 amino acids at the C-terminal (SEQ ID NO: 3) estimated from the molecular weight. The fibromodulin fragment has a molecular weight of 10,000 and a pI of 4.65.
  • the origin of the fibromodulin fragment that can be contained in the decellularized tissue composition is not particularly limited as long as it is derived from eukaryote like annexin, and the same as annexin can be mentioned.
  • the decellularized tissue composition of the present invention can include, but is not limited to, a variant of the fibromodulin fragment as long as the effects of the present invention can be obtained.
  • the fibromodulin fragment variant include, for example. (1) One or more (preferably 1 to 10, more preferably 1 to 1) as a whole at one or more of the amino acid sequences of the fibromodulin fragment (for example, the amino acid sequence represented by SEQ ID NO: 3). 7, and more preferably 1-5), eg, 1-several amino acids as a whole contain deleted, substituted, inserted, and / or added amino acid sequences and have the activity of a fibromodulin fragment.
  • fibromodulin fragment for example, the amino acid sequence represented by SEQ ID NO: 3
  • the activity of the fibromodulin fragment includes, as the decellularized tissue composition of the present invention, improving the adhesion of cells as compared with the one containing no variant.
  • the variant of the fibromodulin fragment exhibits the same amino acid mutation, identity, conservative substitution, etc. as the annexin variant.
  • the fibromodulin fragment is produced from fibromodulin by irradiating the decellularized tissue composition with gamma rays.
  • the intensity of gamma-ray irradiation is not particularly limited as long as the effect of the present invention can be obtained, but is 10 to 50 kGy, preferably 15 to 35 kGy, and more preferably 20 to 30 kGy. Within the above range, fibromodulin fragments can be produced.
  • the reason why the decellularized tissue composition of the present invention exhibits excellent strength and excellent cell adhesion is not completely understood, but can be inferred as follows.
  • the protein A eg, annexin
  • the protein A contained in the decellularized tissue composition of the present invention has 4 or 8 ⁇ -helix structures (anexin repeats) and binds to Ca 2+ and phospholipids. It is considered that the binding ability between Ca 2+ and phospholipids is related to the cell adhesion ability, but not limited to. In addition, it is considered that annexin effectively acts on the strength of decellularized tissue.
  • the protein B (for example, fibromodulin fragment) contained in the decellularized tissue composition of the present invention is produced by cleaving fibromodulin by gamma-ray irradiation.
  • Fibromodjurin has a role of stabilizing collagen fibers, and it is considered that the presence of sufficient fibromodjurin suppresses the renewal of collagen in cells.
  • the decellularized tissue composition of the present invention can be produced by a method for obtaining decellularized tissue.
  • a method for obtaining decellularized tissue in order to efficiently obtain the decellularized tissue composition and to significantly exert the effect of the present invention, it is preferable to use the method by high hydrostatic pressure treatment.
  • the protein can be added externally to the decellularized tissue obtained by any method to obtain the decellularized tissue composition of the present invention.
  • Example 1 a decellularized tissue composition was prepared by high hydrostatic pressure treatment using bovine pericardium.
  • the bovine pericardium was cut open into a sheet to remove fat entirely (hereinafter, this sheet-shaped bovine pericardium is referred to as a "pericardial sheet").
  • High-pressure processing equipment for research and development Karl Fischer Steel Co., Ltd .: Dr. CHEF was used for high hydrostatic pressure treatment at 600 MPa for 10 minutes.
  • the pericardial sheet treated with high hydrostatic pressure was treated with the nucleic acid degrading enzyme DNaseI (125 U / mL), shaken at 4 ° C for 18 hours and 5 minutes or more, and then treated in 80% ethanol at 4 ° C for 1 hour or more. Finally, it was washed with 4 L of water for injection at 4 ° C. The freeze-drying treatment was carried out to obtain the decellularized tissue composition of Example 1.
  • DNaseI 125 U / mL
  • Example 2 a decellularized material was prepared by treatment with a surfactant using bovine pericardium, and annexin A2 was added to obtain a decellularized tissue composition.
  • the pericardial sheet was sterilized by immersing it in a 0.1% (v / v) peracetic acid solution prepared to contain 4% ethanol at 4 ° C for 2 hours, and then sterilized with water for injection at 4 ° C. Washed for hours. Then, shake with 0.25 mass% sodium dodecyl sulfate (hereinafter, SDS) solution (10 mM Tris, pH 8.0) at 4 ° C. for 24 hours, and then 0.5 mass% Triton-X (polyoxy).
  • SDS sodium dodecyl sulfate
  • Ethylene octylphenyl ether TX solution (10 mM Tris, pH 8.0) was shaken at 4 ° C. for 24 hours. Then, after washing with 10 L of water for injection at 4 ° C., the mixture was shaken with a phosphate buffer (PBS, 0.01M, pH 7.4) at 4 ° C. for 1 hour. After freeze-drying treatment, annexin A2 was added so as to be 0.3% by mass based on the weight of the decellularized tissue composition, and the decellularized tissue composition of Example 2 was obtained.
  • PBS phosphate buffer
  • Example 3 a decellularized material was prepared by freeze-thaw treatment using bovine pericardium, and annexin A2 was added to obtain a decellularized tissue composition.
  • the pericardial sheet was frozen in dry ice, stored in a cool box containing dry ice (about ⁇ 78 ° C.) for 20 hours, and then thawed at 25 ° C.
  • the pericardial sheet obtained by repeating this freeze-thaw four times was treated with DNaseI (125 U / mL) as a nucleolytic enzyme and shaken at 4 ° C. for 96 hours. Further, it was shaken in 80% ethanol at 4 ° C.
  • annexin A2 was added so as to be 0.3% by mass based on the weight of the decellularized tissue composition, and the decellularized tissue composition of Example 3 was obtained.
  • Example 4 a decellularized material was prepared by ultrasonic treatment using bovine pericardium, and annexin A2 was added to obtain a decellularized tissue composition.
  • the pericardial sheet was sonicated (intensity: 10 W / cm 2 , frequency: 10 kHz, action time: 2 minutes) in physiological saline.
  • the sonicated bovine pericardium was shaken in 1 mass% TX solution (10 mM Tris, pH 8.0) at 4 ° C. for 96 hours.
  • the TX-treated pericardial sheet was treated with DNaseI (125 U / mL) as a nucleolytic enzyme and shaken at 4 ° C. for 96 hours.
  • annexin A2 was added so as to be 0.3% by mass based on the weight of the decellularized tissue composition, and the decellularized tissue composition of Example 4 was obtained.
  • Example 5 a decellularized material was prepared by high hydrostatic pressure treatment using bovine pericardium, and annexin A2 was added to obtain a decellularized tissue composition.
  • the bovine pericardium was cut open into a sheet and the fat was completely removed to obtain a pericardial sheet.
  • High-pressure processing equipment for research and development Karl Fischer Steel Co., Ltd .: High hydrostatic pressure treatment for 10 minutes was performed at 600 MPa with Dr. CHEF).
  • the pericardial sheet treated with high hydrostatic pressure was treated with the nucleic acid degrading enzyme DNase 125 U / mL, shaken at 4 ° C for 18 hours and 5 minutes or more, then treated in 80% ethanol at 4 ° C for 1 hour or more, and finally 4 Washing was performed with 4 L of water for injection at ° C. Freeze-drying treatment was performed to obtain a decellularized bovine heart cyst. Annexin A2 was added so as to be 1.0% by mass (total content) per weight of the decellularized tissue composition to obtain the decellularized tissue composition of Example 5.
  • Example 6 a decellularized material was prepared by treatment with a surfactant using bovine pericardium, and annexin A2 was added to obtain a decellularized tissue composition.
  • the pericardial sheet was sterilized by immersing it in a 0.1% (v / v) peracetic acid solution prepared to contain 4% ethanol at 4 ° C for 2 hours, and then sterilized with water for injection at 4 ° C. Washed for hours. Then, shake with 0.25% by mass SDS solution (10 mM Tris, pH 8.0) at 4 ° C. for 24 hours, and then with 0.5% by mass TX solution (10 mM Tris, pH 8.0) 4 Shake at ° C for 24 hours.
  • Example 7 a decellularized material was prepared by freeze-thaw treatment using bovine pericardium, and annexin A2 was added to obtain a decellularized tissue composition.
  • the pericardial sheet was frozen in dry ice, stored in a cool box containing dry ice (about ⁇ 78 ° C.) for 20 hours, and then thawed at 25 ° C.
  • the pericardial sheet obtained by repeating this freeze-thaw four times was treated with DNaseI (125 U / mL) as a nucleolytic enzyme and shaken at 4 ° C. for 96 hours. Further, it was shaken in 80% ethanol at 4 ° C.
  • Example 8 a decellularized material was prepared by ultrasonic treatment using bovine pericardium, and annexin A2 was added to obtain a decellularized tissue composition.
  • the pericardial sheet was sonicated (intensity: 10 W / cm 2 , frequency: 10 kHz, action time: 2 minutes) in physiological saline.
  • the sonicated bovine pericardium was shaken in 1 mass% TX solution (10 mM Tris, pH 8.0) at 4 ° C. for 96 hours.
  • the TX-treated pericardial sheet was treated with DNaseI (125 U / mL) as a nucleolytic enzyme and shaken at 4 ° C. for 96 hours.
  • Comparative Example 1 a decellularized material was prepared by treating with a surfactant using bovine pericardium.
  • the pericardial sheet was sterilized by immersing it in a 0.1% (v / v) peracetic acid solution prepared to contain 4% ethanol at 4 ° C for 2 hours, and then sterilized with water for injection at 4 ° C. Washed for hours. Then, shake with 0.25 mass% SDS solution (10 mM Tris, pH 8.0) at 4 ° C. for 24 hours, and then with 0.5 mass% TX solution (10 mM Tris, pH 8.0) for 4 hours. Shake at ° C for 24 hours.
  • a decellularized material was prepared by freeze-thaw treatment using bovine pericardium.
  • the pericardial sheet was frozen in dry ice, stored in a cool box containing dry ice (about ⁇ 78 ° C.) for 20 hours, and then thawed at 25 ° C.
  • the pericardial sheet obtained by repeating this freeze-thaw four times was treated with DNaseI (125 U / mL) as a nucleolytic enzyme and shaken at 4 ° C. for 96 hours. Further, it was shaken in 80% ethanol at 4 ° C. for 96 hours, and then shaken in water for injection at 4 ° C. for 2 hours. Freeze-drying treatment was performed to obtain a decellularized bovine heart cyst (decellularized material) of Comparative Example 2.
  • a decellularized material was prepared by ultrasonic treatment using bovine pericardium.
  • the pericardial sheet was sonicated (intensity: 10 W / cm 2 , frequency: 10 kHz, action time: 2 minutes) in physiological saline.
  • the sonicated bovine pericardium was shaken in 1 mass% TX solution (10 mM Tris, pH 8.0) at 4 ° C. for 96 hours.
  • the TX-treated pericardial sheet was treated with DNaseI (125 U / mL) as a nucleolytic enzyme and shaken at 4 ° C. for 96 hours. Further, it was shaken in 80% ethanol at 4 ° C. for 72 hours, and then shaken in physiological saline at 4 ° C. for 2 hours. Freeze-drying treatment was performed to obtain a decellularized bovine heart cyst (decellularized material) of Comparative Example 3.
  • ⁇ Cell adhesion test The decellularized tissue composition or decellularized material obtained in Examples and Comparative Examples was cut into a disk shape having a diameter of 4 mm using autoclaved scissors and tweezers, and used as a cell insert for 96-well plate. I set it. 175 ⁇ L of DMEM medium was added to the lower side of the cell insert and 15 ⁇ L to the upper side, and the mixture was allowed to stand at 37 ° C. for 24 hours or more to acclimate the decellularized tissue composition or decellularized material. After removing DMEM medium, human skin fibroblasts (NHDF) were seeded (7.0 ⁇ 104 cells / cm 2 ) and cultured at 37 ° C. for 3 hours.
  • NHDF human skin fibroblasts
  • Example 9 a decellularized tissue composition was prepared by high hydrostatic pressure treatment using pig liver.
  • the pig liver was cut open into a sheet to remove fat entirely (hereinafter, this sheet-shaped pig liver is referred to as a "liver sheet").
  • High-pressure processing equipment for research and development (Kobe Steel Co., Ltd .: Dr. .CHEF) was subjected to high hydrostatic pressure treatment at 600 MPa for 10 minutes.
  • the liver sheet treated with high hydrostatic pressure was treated with the nucleic acid degrading enzyme DNaseI (125 U / mL), shaken at 4 ° C for 18 hours and 5 minutes or more, and then treated in 80% ethanol at 4 ° C for 1 hour or more, and finally.
  • the freeze-drying treatment was carried out to obtain the decellularized tissue composition of Example 9.
  • Example 10 a decellularized material was prepared by high hydrostatic pressure treatment using bovine pericardium, and annexin A2 was added to obtain a decellularized tissue composition.
  • the bovine pericardium was cut open into a sheet and the fat was completely removed to obtain a pericardial sheet.
  • High-pressure processing equipment for research and development Karl Fischer Steel Co., Ltd .: High hydrostatic pressure treatment for 10 minutes was performed at 600 MPa with Dr. CHEF).
  • the pericardial sheet treated with high hydrostatic pressure was treated with the nucleic acid degrading enzyme DNase 125 U / mL, shaken at 4 ° C for 18 hours and 5 minutes or more, then treated in 80% ethanol at 4 ° C for 1 hour or more, and finally 4 Washing was performed with 4 L of water for injection at ° C. Freeze-drying treatment was performed to obtain a decellularized bovine heart cyst. Annexin A2 was added so as to be 3.0% by mass (total content) per weight of the decellularized tissue composition to obtain the decellularized tissue composition of Example 10.
  • Example 11 a decellularized tissue composition was prepared by high hydrostatic pressure treatment using bovine pericardium.
  • the bovine pericardium was cut open into a sheet to remove fat entirely (hereinafter, this sheet-shaped bovine pericardium is referred to as a "pericardial sheet").
  • High-pressure processing equipment for research and development Karl Fischer Steel Co., Ltd .: Dr. CHEF was used for high hydrostatic pressure treatment at 600 MPa for 10 minutes.
  • the pericardial sheet treated with high hydrostatic pressure was treated with the nucleic acid degrading enzyme DNaseI (125 U / mL), shaken at 4 ° C for 18 hours and 5 minutes or more, and then treated in 80% ethanol at 4 ° C for 1 hour or more. Finally, it was washed with 4 L of water for injection at 4 ° C. After freeze-drying treatment, gamma-ray irradiation (25 kGy) was performed to obtain a decellularized tissue composition of Example 11.
  • DNaseI 125 U / mL
  • Example 12 a decellularized material was prepared by high hydrostatic pressure treatment using bovine pericardium, and annexin A2 was added to obtain a decellularized tissue composition.
  • the bovine pericardium was cut open into a sheet and the fat was completely removed to obtain a pericardial sheet.
  • High-pressure processing equipment for research and development Karl Fischer Steel Co., Ltd .: High hydrostatic pressure treatment for 10 minutes was performed at 600 MPa with Dr. CHEF).
  • the pericardial sheet treated with high hydrostatic pressure was treated with the nucleic acid degrading enzyme DNase 125 U / mL, shaken at 4 ° C for 18 hours and 5 minutes or more, then treated in 80% ethanol at 4 ° C for 1 hour or more, and finally 4 Washing was performed with 4 L of water for injection at ° C.
  • decellularized bovine heart cyst was obtained by gamma-ray irradiation (25 kGy).
  • Annexin A2 was added so as to be 1.0% by mass (total content) per weight of the decellularized bovine heart membrane to obtain the decellularized tissue composition of Example 12.
  • Example 13 a decellularized material was prepared by high hydrostatic pressure treatment using bovine pericardium, and annexin A2 was added to obtain a decellularized tissue composition.
  • the bovine pericardium was cut open into a sheet and the fat was completely removed to obtain a pericardial sheet.
  • High-pressure processing equipment for research and development Karl Fischer Steel Co., Ltd .: High hydrostatic pressure treatment for 10 minutes was performed at 600 MPa with Dr. CHEF).
  • the pericardial sheet treated with high hydrostatic pressure was treated with the nucleic acid degrading enzyme DNase 125 U / mL, shaken at 4 ° C for 18 hours and 5 minutes or more, then treated in 80% ethanol at 4 ° C for 1 hour or more, and finally 4 Washing was performed with 4 L of water for injection at ° C.
  • decellularized bovine heart cyst was obtained by gamma-ray irradiation (25 kG).
  • Annexin A2 was added so as to be 3.0% by mass (total content) per weight of the decellularized bovine heart membrane to obtain the decellularized tissue composition of Example 13.
  • Example 9 The above-mentioned cell adhesion test was performed on Examples 9 to 13. The results are shown in FIG. It was found that Examples 9 to 13 had a larger number of cell adhesions and had a better cell-inducing effect than Comparative Example 1. In particular, Example 11 had a good cell-inducing effect. In Example 9, it was found that good results were also obtained using pig liver.
  • test piece As the width (mm) of the test piece, the length (40 mm) of the punched blade type cutting surface tube of the parallel portion was used as it was.
  • the cross-sectional area A (mm 2 ) of the test piece was calculated from the thickness and width of the test piece by the following formula.
  • A t ⁇ w (A: test piece cross-sectional area (mm 2 ), t: test piece thickness (mm), w: test piece width mm)) (3)
  • Test procedure The tensile test was carried out as follows in accordance with ISO37. In order to evenly distribute the tensile force over the cross section, the test piece was attached to a mechanical tester (MCT2150, manufactured by AND) so that both ends of the test piece were held in contrast.
  • the distance between the marked lines Lb (mm) for operating the testing machine was measured.
  • the speed of the grip was set to 200 mm / min. Specimen data broken outside between the markings was rejected and repeated tests were performed with additional specimens. The test piece was taken until it was measured correctly 3 times. From the measured values, the breaking strength [MPa] and elastic modulus [MPa] (at 10% elongation and at maximum) were calculated by the following formulas. (4) Result calculation ⁇ Breaking strength> The breaking strength (MPa (N / mm 2 )) was calculated by the following formula.
  • Breaking strength (MPa) maximum load (N) / cross section of test piece (mm 2 ) ⁇ Elastic modulus> The elastic modulus (stress / elongation strain)) was calculated by the following formula.
  • Elastic modulus (MPa) load (MPa (N / mm 2 )) / elongation strain ((LL 0 ) / L 0 )
  • the tear strength test of the decellularized tissue composition or the decellularized material obtained in Examples and Comparative Examples was carried out as follows. (1) Collection and preparation of test pieces A freeze-dried decellularized tissue composition or decellularized material was immersed in physiological saline for 15 minutes or more. It was collected from the swollen decellularized tissue composition or decellularized material so as to be 20 mm ⁇ 15 mm. A hole with a diameter of 6 mm was drilled at a position 2 mm from the top of the long side. A thread was passed through the hole to prepare a test piece. (2) Test procedure One end of the test piece was attached to the grip of the mechanical tester (MCT2150, manufactured by AND), and the thread was attached to the hook type fixator. The speed of the grip was set to 200 mm / min. The test piece was taken until it was measured correctly 3 times. The measured value was calculated as the maximum load [N] at the time of breakage.
  • Examples 1 and 11 required less force to stretch by 10% than Comparative Example 1, that is, they were soft and had good handleability. Further, Examples 1 and 11 have a larger maximum elastic modulus than Comparative Example 1 and are close to the elastic modulus of human pericardium 49.62 ⁇ 3.22 (MPa, see Interactive CardioVascularand Thoric Surgery 22 (2016) 72-84). It showed the value. As a result, when transplanted into a patient, it is easy to become familiar with the patient's tissue, and the structural stability can be maintained with respect to pulse, pulsation, and the like.
  • Example 1 showed a maximum elastic modulus of 53.9 Mpa. Further, it was found that Examples 1 and 11 had higher breaking strength, that is, higher strength and higher strength than Comparative Example 1. Further, it was found that Examples 1 and 11 had higher tear strength than Comparative Example 1, that is, they were resistant to threading and had high suture strength when used in surgery. As described above, it was found that Examples 1 and 11 had better handleability and were stronger than Comparative Example 1.
  • a protein dissolving solution (containing urea and a surfactant) was added to prepare a total volume of 0.34 mL (protein 200 ⁇ g), which was used as a sample solution.
  • the sample solution was placed in a swelling tray, and the first-dimensional electrophoresis precast gel was placed over the solution. Further, a dry strip cover solution was layered and allowed to stand overnight. The swollen precast gel was set in an electrophoresis device and electrophoresed (500 V for 1 minute, 3500 V for 7.5 hours, 20 ° C.). An acrylamide gel with a concentration gradient of 10-20% was used. After preparation, the mixture was allowed to stand for 24 hours to completely polymerize acrylamide.
  • the equilibrated one-dimensional electrophoresis precast gel was placed on an acrylamide gel and fixed with a 1% agarose solution containing 0.125% bromophenol blue. A molecular weight marker was applied to the left end of the gel to make it a standard for molecular weight. The bromophenol blue band was run at 80 V for 17 hours until it was visible at the bottom of the gel.
  • the gel after migration was stained with a fluorescent stain for detecting all proteins (SYPRO Ruby protein gel stain, S21900, Thermo Fisher Scientific Inc.), and the image was preserved using a fluorescent scanner.
  • the image was captured by the scanner with an excitation wavelength of 488 nm, a fluorescence filter of 640 nm Bandpass, and a resolution of 100 micrometer.
  • silver staining was performed using silver nitrate (195-09382, Wako Pure Chemical Industries, Ltd.).
  • the silver-stained image after development was captured using a flatbed scanner. The results of two-dimensional electrophoresis are shown in FIG.
  • Example 1 three spots (1 to 3) were detected at isoelectric points pI 6 to 9 and a molecular weight of 30,000 to 40,000. On the other hand, in Comparative Example 1, the three spots detected in Example 1 were not detected. Also in Comparative Examples 2 and 3, the three spots detected in Example 1 were not detected. Example 1 was found to contain a specific protein not found in Comparative Examples 1-3.
  • spot identification Spots visualized by silver staining were cut out, 100 ⁇ L of 15 mM potassium ferricyanide and 50 mM sodium thiosulfate were added to a gel piece cut into 1 mm square pieces, and the mixture was shaken for 10 minutes. The solution was discarded, Milli-Q water was added, the mixture was shaken, and the gel pieces were washed until the color was removed. Acetonitrile was added to the gel pieces to dehydrate them. The gel pieces were swollen by adding 10 ⁇ L of an enzyme solution of 100 mM ammonium hydrogen carbonate and 0.01 ⁇ g / ⁇ L trypsin, and the gel pieces were kept at 37 ° C. for 16 hours.
  • Table 1 shows the results of identifying the three spots of Example 1 using a mass spectrometer.
  • the spot numbers in Table 1 and Table 2 described later correspond to the three spot numbers in FIG.
  • the three spots were identified as Annexin A2.
  • the protein weight per spot was calculated from the ratio of the weight of the protein subjected to two-dimensional electrophoresis to 200 ⁇ g and each spot signal (spot signal concentration) to the total sum of the spot signals.
  • spot 1 to 0.7 ⁇ g, spot 2 to 2.0 ⁇ g, and spot 3 to 0.8 ⁇ g A total protein weight of 3.5 ⁇ g was calculated from the three spots.
  • Example 1 (Calculation of protein weight of 3 spots contained in decellularized tissue composition) From the relationship between the weight of the purified and recovered protein and the weight of the two-dimensionally electrophoresed protein, the protein weights of the three spots per 20 mg of the decellularized tissue composition were calculated. The results are shown in Table 2. In Example 1, protein weights were calculated from the three spots. Example 1 contained 0.43% by weight of protein A. On the other hand, in Comparative Example 1, the protein weight corresponding to these three spots was not calculated. Also in Comparative Examples 2 and 3, the protein weight corresponding to the three spots was not calculated.
  • the proteins of the decellularized tissue compositions of Examples 9 and 11 to 13 were analyzed in the same manner.
  • the results of the two-dimensional electrophoresis of Example 9 are shown in FIG. 7A. Similar to Example 1, three spots (4 to 6) were detected at isoelectric points pI 6 to 9 and a molecular weight of 30,000 to 40,000.
  • the results of the two-dimensional electrophoresis of Example 11 are shown in FIG. 7B. Three spots (7-9) are detected at an isoelectric point pI6-9 and a molecular weight of 30,000-40,000, and at an isoelectric point pI4-5 and a molecular weight of 8,000-12,000. Spot (10) was detected. Spot 10 was detected only in Examples 11-13.
  • Example 9 The spots of Examples 9 and 11 were identified. The results of Example 9 are shown in Table 3. The spot numbers in Table 3 and Table 5 described later correspond to the spot numbers in FIG. 7A. Spots 4 to 6 of Example 9 were identified as Annexin A2 as in Example 1 (see Spot Nos. 1 to 3 in Table 1).
  • Table 4 shows the results of Example 11.
  • the spot numbers in Table 4 and Table 6 described later correspond to the spot numbers in FIG. 7B.
  • Spots 7-9 of Example 11 were identified as Annexin A2 as in Example 1, and Spot 10 was identified as a fibromodulin-derived peptide.
  • Example 9 the spot signal concentration was calculated and the spot weight was calculated.
  • Example 9 0.14 ⁇ g from spot 4, 0.27 ⁇ g from spot 5, and 0.03 ⁇ g from spot 6 were obtained.
  • a total protein weight of 0.44 ⁇ g was calculated from the three spots.
  • Example 11 the protein weights of 0.7 ⁇ g from the spot 7, 1.6 ⁇ g from the spot 8, 0.4 ⁇ g from the spot 9, and 2.7 ⁇ g in total from the three spots were calculated, and the protein weight was calculated from the spot 10 to 0.
  • a protein weight of 24 ⁇ g was calculated.
  • Example 9 contained 0.05% by weight of protein A.
  • Example 11 contained 0.33% by mass of protein A and 0.03% by mass of protein B.
  • the decellularized tissue composition of the present invention can be used as a tissue for transplantation showing good tissue regeneration.

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CN202180076247.XA CN116615528A (zh) 2020-11-12 2021-11-12 脱细胞化组织组合物
KR1020237018831A KR20230107827A (ko) 2020-11-12 2021-11-12 탈세포화 조직 조성물
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