CN115105636B - 脱细胞人羊膜的制备方法 - Google Patents
脱细胞人羊膜的制备方法 Download PDFInfo
- Publication number
- CN115105636B CN115105636B CN202210835190.3A CN202210835190A CN115105636B CN 115105636 B CN115105636 B CN 115105636B CN 202210835190 A CN202210835190 A CN 202210835190A CN 115105636 B CN115105636 B CN 115105636B
- Authority
- CN
- China
- Prior art keywords
- amniotic membrane
- solution
- freezing
- human amniotic
- membrane
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000001691 amnion Anatomy 0.000 title claims abstract description 97
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000007710 freezing Methods 0.000 claims abstract description 16
- 230000008014 freezing Effects 0.000 claims abstract description 16
- 239000008223 sterile water Substances 0.000 claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 12
- 238000004140 cleaning Methods 0.000 claims abstract description 12
- 238000010257 thawing Methods 0.000 claims abstract description 12
- 102000004882 Lipase Human genes 0.000 claims abstract description 11
- 108090001060 Lipase Proteins 0.000 claims abstract description 11
- 239000004367 Lipase Substances 0.000 claims abstract description 11
- 235000019421 lipase Nutrition 0.000 claims abstract description 11
- 238000004108 freeze drying Methods 0.000 claims abstract description 8
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 7
- 239000005708 Sodium hypochlorite Substances 0.000 claims abstract description 7
- 239000012528 membrane Substances 0.000 claims abstract description 7
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 7
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 7
- 238000002791 soaking Methods 0.000 claims abstract description 6
- 238000003892 spreading Methods 0.000 claims abstract description 6
- 210000002919 epithelial cell Anatomy 0.000 claims abstract description 5
- 239000012535 impurity Substances 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 19
- 238000005406 washing Methods 0.000 claims description 12
- 238000007789 sealing Methods 0.000 claims description 8
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 229930183010 Amphotericin Natural products 0.000 claims description 3
- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 claims description 3
- 229930182555 Penicillin Natural products 0.000 claims description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 229940009444 amphotericin Drugs 0.000 claims description 3
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims description 3
- 210000001136 chorion Anatomy 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 230000005251 gamma ray Effects 0.000 claims description 3
- 229910052742 iron Inorganic materials 0.000 claims description 3
- 229940049954 penicillin Drugs 0.000 claims description 3
- 229920003023 plastic Polymers 0.000 claims description 3
- 238000003825 pressing Methods 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 230000029663 wound healing Effects 0.000 claims description 2
- 230000037314 wound repair Effects 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims 4
- 108090000695 Cytokines Proteins 0.000 abstract description 5
- 102000004127 Cytokines Human genes 0.000 abstract description 5
- 238000000053 physical method Methods 0.000 abstract description 3
- 238000007790 scraping Methods 0.000 abstract description 3
- 230000001988 toxicity Effects 0.000 abstract 1
- 231100000419 toxicity Toxicity 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 29
- 239000003102 growth factor Substances 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 5
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 5
- 206010052428 Wound Diseases 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 210000002744 extracellular matrix Anatomy 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 238000008157 ELISA kit Methods 0.000 description 4
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 4
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 4
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 description 3
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 3
- 108010025020 Nerve Growth Factor Proteins 0.000 description 3
- 102000007072 Nerve Growth Factors Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000002386 leaching Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- GUTLYIVDDKVIGB-OUBTZVSYSA-N Cobalt-60 Chemical compound [60Co] GUTLYIVDDKVIGB-OUBTZVSYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 101000898034 Homo sapiens Hepatocyte growth factor Proteins 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 102000009618 Transforming Growth Factors Human genes 0.000 description 2
- 108010009583 Transforming Growth Factors Proteins 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000000249 desinfective effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940116977 epidermal growth factor Drugs 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000004388 gamma ray sterilization Methods 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 231100001083 no cytotoxicity Toxicity 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 1
- 101001111439 Homo sapiens Beta-nerve growth factor Proteins 0.000 description 1
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 description 1
- 101001076408 Homo sapiens Interleukin-6 Proteins 0.000 description 1
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 description 1
- 101000868152 Homo sapiens Son of sevenless homolog 1 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000007547 Laminin Human genes 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102000002938 Thrombospondin Human genes 0.000 description 1
- 108060008245 Thrombospondin Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000005482 chemotactic factor Substances 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 102000057308 human HGF Human genes 0.000 description 1
- 102000046917 human NGF Human genes 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000037311 normal skin Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
Classifications
-
- 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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
-
- 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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Dermatology (AREA)
- Botany (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Materials Engineering (AREA)
- General Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明公开了一种脱细胞人羊膜的制备方法，包括以下步骤：(1)取新鲜的人羊膜，浸泡于初步消毒溶液中；(2)依次用无菌水、次氯酸钠溶液、无菌水清洗；(3)浸入冻存溶液中，捞出，反复冻融两次；(4)置于TritonX‑100溶液中，摇床孵育，初步去除细胞；然后用PBS溶液震荡清洗羊膜；(5)置于脂肪酶溶液中，摇床孵育，以充分去除上皮细胞及其他杂质，然后用PBS溶液震荡清洗羊膜；(6)将羊膜平铺在硝酸纤维膜上，冷冻；(7)冷冻干燥至含水量为5％～7％。本发明的脱细胞人羊膜的制备方法，不使用物理方法刮擦羊膜，保证了原有的重要细胞因子含量，维持了组织学结构，无细胞毒性，DNA含量符合脱细胞标准。
Description
技术领域
本发明涉及一种脱细胞人羊膜的制备方法，属于生物材料处理技术领域。
背景技术
人羊膜是一张半透明薄膜，位于胎盘最内层，总厚度0.02～0.5mm，结构共五层，包括上皮细胞层、基底膜层、致密层、成纤维细胞层、海绵层。羊膜没有神经、淋巴管和血管，具有低免疫原性，能抑制白细胞与细菌浸润，抑制相应蛋白酶，具有多种因子，这些特性使羊膜被多个临床科室应用。然而，新鲜羊膜在安全性、存储等方面仍具有挑战，研发人员一直在研究稳定的脱细胞方法。脱细胞化过程是去除所有的细胞，降低免疫原性和炎症的同时保留基底膜和细胞外基质(ECM)的主要成分，使脱细胞羊膜成为一种新型、可靠的生物材料，以用作各种器官和组织修复的支架。
成人皮肤和组织受损后，会进行自我修复，但由于缺乏维持正常皮肤弹性的胶原纤维网络及生长因子，愈合后的伤口只能达到其原始组织强度的70％。脱细胞人羊膜含有表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)、角质形成细胞生长因子(KGF)、血管EGF(VEGF)、转化生长因子(TGFs)、神经生长因子(NGF)和许多已知的趋化因子，以及Ⅰ、Ⅲ、Ⅳ、Ⅴ、Ⅶ型胶原、粘连蛋白、层粘连蛋白，这些生长因子及蛋白可以发挥上皮化作用，有利于伤口的愈合，因此脱细胞人羊膜对急性和慢性伤口的愈合都很重要，也能提高患处皮肤移植成功率。
中国发明专利CN 105797212 A公开了一种用于皮肤难愈合创面修复的脱细胞羊膜制备方法及应用，该脱细胞方法涉及到胰蛋白酶与核酸酶，会降解ECM成分和生长因子，导致羊膜结构发生改变，从而影响创伤的修复效果。中国发明专利CN 110946879 A公开了一种包含疏松层的脱细胞生物羊膜及其制备方法，使用过氧乙酸进行病毒灭活，过氧乙酸性质不稳定，易分解，在稀释时很难控制浓度。
发明内容
针对上述现有技术，本发明提供了一种脱细胞人羊膜的制备方法。本发明的方法不使用物理方法刮擦羊膜，结合低温和表面活性剂方法，优化低温冷冻操作，减少冰晶形成，保护羊膜结构和基质完整。
本发明是通过以下技术方案实现的：
一种脱细胞人羊膜的制备方法，包括以下步骤：
(1)取新鲜的人羊膜，浸泡于初步消毒溶液中(浸泡时间20分钟)，对羊膜进行初步消毒，并抑制细菌生长；
所述初步消毒溶液为含有50μg/ml青霉素、50μg/ml链霉素和2.5μg/ml两性霉素的PBS溶液；
(2)取出羊膜，用无菌水清洗(4次，每次10分钟)，再用浓度为0.05％的次氯酸钠溶液清洗10分钟(次氯酸钠灭活病毒，对羊膜无损伤)，再用无菌水清洗(4次，每次10分钟)；
进一步地，清洗过程中进行摇床震荡，羊膜会随晃动而充分展开，清洗效果更佳；
(3)将清洗后的羊膜浸入冻存溶液中，捞出置于滤纸上，充分展开，再覆盖一层滤纸，置于-80℃环境下冻存1小时，然后置于37℃环境下融化0.5小时，再置于-80℃环境下冻存1小时，然后置于37℃环境下融化0.5小时(即反复冻融2次)，以破坏细胞壁，且冻融次数少，甘油可以减少冰晶损伤，滤纸能吸走部分水分；
(4)将反复冻融后的羊膜置于1％的Triton X-100溶液中，37℃摇床(转速70～100rpm/min)孵育24小时，初步去除细胞；然后用PBS溶液震荡清洗羊膜(10分钟)；
(5)将羊膜置于1000U/L的脂肪酶溶液中，37℃摇床(转速70～100rpm/min)孵育15小时，以充分去除上皮细胞及其他杂质，然后用PBS溶液震荡清洗羊膜(3次，每次10分钟)；
所述脂肪酶为购自索莱宝生物科技公司的脂肪酶TYPE II L8070；
(6)将羊膜平铺在硝酸纤维膜上，绒毛膜面朝向硝酸纤维膜，压在铁盘下，置于-40℃环境下冷冻10～15小时；
(7)将冷冻的羊膜在-30℃、0.2Mbar的环境下冷冻干燥至含水量为5％～7％(约6小时)，此时羊膜达到较佳的脱水状态，在后续进行γ射线灭菌时不会产生自由基；
(8)将干燥的羊膜按要求尺寸裁剪，装入塑料袋中真空密封，γ射线(钴60)辐照灭菌。
利用上述方法制备得到的脱细胞人羊膜，DNA含量符合脱细胞标准，经检测保留住了更多的生长因子。
所述脱细胞人羊膜作为支架材料在伤口愈合、创面修复、皮肤移植中的应用。
现有技术中的脱细胞化通常使用胰蛋白酶、EDTA、核酸酶、十二烷基硫酸钠(SDS)、酸碱化学试剂等，或需要额外的超声等机械刮擦，其中有文献报道在不同的浓度和不同的时间情况下使用胰蛋白酶-EDTA脱细胞后，部分生长因子和ECM成分未被识别，包括：纤维连接蛋白、弹性蛋白、VI型胶原、血栓反应蛋白、TGF-a、-b1和-b2受体、bFGF、KGF、EGFR、PDGF和VEGF，说明胰蛋白酶-EDTA易降解ECM成分和生长因子，同时刮擦方式容易对羊膜造成机械损伤，不能保证生产的一致性。本发明的脱细胞人羊膜的制备方法，不使用物理方法刮擦羊膜，结合多种脱细胞方法，经检测发现脱细胞冷冻干燥处理后不仅保证了原有的重要细胞因子含量，还会高于新鲜羊膜细胞因子含量，维持了组织学结构，无细胞毒性，DNA含量符合脱细胞标准。
本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。
附图说明
图1：HE染色结果示意图。
图2：MASSON染色结果示意图。
图3：细胞毒性试验结果示意图。
图4：DNA含量测定结果示意图。
图5：生长因子含量对比结果示意图。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而，本发明的范围并不限于下述实施例。本领域技术人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料等，若无特别说明，均为现有技术中已有的常规仪器、试剂、材料等，可通过正规商业途径获得。下述实施例中所涉及的实验方法，检测方法等，若无特别说明，均为现有技术中已有的常规实验方法，检测方法等。
实施例1脱细胞人羊膜的制备
步骤如下：
(1)取新鲜的人羊膜，浸泡于初步消毒溶液中，浸泡时间20分钟，对羊膜进行初步消毒，并抑制细菌生长；
所述初步消毒溶液为含有50μg/ml青霉素、50μg/ml链霉素和2.5μg/ml两性霉素的PBS溶液；
(2)取出羊膜，用无菌水震荡清洗(4次，每次10分钟)，再用浓度为0.05％的次氯酸钠溶液震荡清洗(次氯酸钠灭活病毒，对羊膜无损伤；10分钟)，再用无菌水震荡清洗(4次，每次10分钟)；
(3)将清洗后的羊膜置于冻存液中，捞出置于滤纸上，充分展开，再覆盖一层滤纸，置于-80℃环境下冻存1小时，然后置于37℃环境下融化0.5小时，再置于-80℃环境下冻存1小时，然后置于37℃环境下融化0.5小时(即反复冻融2次)；
所述冻存液是由甘油和DMEM培养基按体积比1:1混合而成的；
(4)将反复冻融后的羊膜置于1％的Triton X-100溶液中，37℃摇床(转速80rpm/min)孵育24小时，初步去除细胞；然后用PBS溶液震荡清洗羊膜(10分钟)；
(5)将羊膜置于1000U/L的脂肪酶溶液中，37℃摇床(转速80rpm/min)孵育15小时，以充分去除上皮细胞及其他杂质，还能保证羊膜结构的完整，保留了羊膜中的生长因子，占羊膜中生长因子总含量的75％；然后用PBS溶液震荡清洗羊膜(3次，每次10分钟)；
所述脂肪酶为购自索莱宝生物科技公司的脂肪酶TYPE II L8070；
(6)将羊膜平铺在硝酸纤维膜上，(绒毛膜面朝向硝酸纤维膜)，压在铁盘下，置于-40℃环境下冷冻12小时；
(6)将冷冻的羊膜在-30℃、0.2Mbar的环境下冷冻干燥至含水量为6％(约6小时)，此时羊膜达到较佳的脱水状态，在后续进行γ射线灭菌时不会产生自由基；
(7)将干燥的羊膜裁剪后装入塑料袋中真空密封，γ射线(钴60)辐照灭菌。
实施例2脱细胞人羊膜的检测
(一)染色实验：实施例1制备的脱细胞人羊膜，进行HE染色和MASSON染色(具体的染色方法为常规的实验方法)，染色结果如图1、图2所示，由图可见，能成功去除整个细胞碎片，胶原纤维结构得到保留，羊膜的组织结构完整，无损坏。
(二)细胞毒性实验：
步骤如下：
(1)培养293T细胞至融合；加入胰蛋白酶消化细胞，然后将细胞收集到DMEM+10％FBS中；
(2)离心(300g，10min)得细胞悬液，用DMEM培养基重悬细胞，计数；
(3)将细胞置于96孔培养板(Corning公司)，100μl、5000cells/孔，培养4小时至细胞贴壁；
(4)实施例1制备的脱细胞人羊膜，用无菌水浸提，浸提比例为6cm2/mL，浸提温度为37±1℃，浸提时间为24±2h，获得浸提液；
(5)每孔加入100μl浸提液，三个复孔，分别孵育24小时、48小时、72小时；然后每孔加入20μl的MTT溶液，37℃培养2小时；
(6)吸出孔中全部液体，加入100μl的二甲基亚砜溶液，漩涡振荡10min，在570nm处测吸光度值，结果如图3所示。由图可知，冷冻干燥脱细胞羊膜浸提液均无细胞毒性作用。
(三)DNA含量测定：使用AxyPrep DNA提取试剂盒DNA含量测定试剂盒(默克DNAQF)，对新鲜和脱细胞人羊膜样品中残留DNA进行定量分析。
步骤如下：
(A)提取DNA：
(1)冷冻干燥脱细胞羊膜完全浸入无菌水中3～5min复水；取20mg羊膜组织移入预冷的研钵中，快速用力研磨成匀浆；
(2)加入350μl的Buffer PBS和0.9μl的RNase A后温和研磨30s；
(3)收集350μl研磨好的组织匀浆转入2ml离心管；
(4)加入150μl的Buffer C-L和20μl的Proteinase K，立即漩涡振荡1min混合均匀。短暂离心后，将离心管置于56℃水浴10min；
(5)加入350μl的Buffer P-D，漩涡振荡30s混合均匀，12000g离心10min；
(6)将DNA制备管置于2ml离心管中，将上述离心后的上清液移至制备管中，12000g离心1min；
(7)弃滤液，将制备管置回到原来的2ml离心管中，加入500μl的Buffer W1，12000g离心1min；
(8)弃滤液，将制备管置回到原来的2ml离心管中，加入500μl的Buffer W2，12000g离心1min；重复洗涤一次；
(9)弃滤液，将制备管置回原来的2ml离心管中，12000g离心1min；
(10)将DNA制备管置于另一洁净的1.5ml离心管中，加入100μl去离子水，室温静置1min，12000g离心1min洗脱DNA。
(B)测定DNA含量：
(1)打开荧光计并使其预热。将激发波长设置为360nm和发射波长为460nm。
(2)制备0.1mg/ml和1mg/ml bisBenzimide H 10X荧光检测缓冲液的33258溶液，并存放在黑暗直到准备好使用；
(3)将2ml适当的染料溶液移入比色皿；
(4)在环境温度下以360nm激发和460nm发射读取空白；
(5)将适当的DNA溶液添加到比色皿中，混合比色皿中的溶液并读取数值；
(6)清洗比色皿，用剩余的DNA标准品和未知样品用新鲜的染料溶液重复步骤(4)和(5)。
结果如图4所示，由图可知，新鲜羊膜DNA含量为258.324±5.71μg/mg，经脱细胞技术处理后，DNA含量为32.76±16.2μg/mg，DNA残留显著减少。
标准品稀释表如表1所示。
表1
(四)总细胞因子水平测定： Human EGF ELISA kit、/>Human bFGF ELISA kit、/> Human HGF ELISA kit、/> Human TGF-β1ELISA kit、/> Human NGF ELISA kit。
(1)取100ng冷冻干燥羊膜完全浸入无菌水中，复水3～5min；将组织放于预冷研钵中快速用力研磨成匀浆，用500μl无菌水重悬匀浆；
(2)从已平衡至室温的密封袋中取出试验所需板条；
(3)空白孔加标准品，标本通用稀释液，其余相应孔中加标本或不同浓度标准品(100μl/孔)，用封板胶纸封住反应孔，37℃恒温箱，避光孵育90分钟；
(4)提前20分钟准备生物素化抗体工作液；
(5)洗板5次；
(6)空白孔加生物素化抗体稀释液，其余孔加入生物素化抗体工作液(100μl/孔)；用新封板胶纸封住反应孔，37℃恒温箱，避光孵育60分钟；
(7)提前20分钟准备酶结合物工作液，避光室温(22～25℃)放置；
(8)洗板5次；
(9)空白孔加酶结合物稀释液，其余孔加入酶结合物工作液(100μl/孔)；用新封板胶纸封住反应孔，37℃恒温箱，避光孵育30分钟；
(10)打开酶标仪电源，预热仪器，设置好检测程序；
(11)洗板5次；
(12)加入显色底物(TMB)100μl/孔，37℃恒温箱，避光孵育15分钟；
(13)加入反应终止液100μl/孔,混匀后即刻测量OD450值(3分钟内)。
结果如图5所示，由图可知，经过脱细胞及冷冻干燥处理后，细胞因子EGF、bFGF、HGF、TGF-β1、NGF含量还略高于新鲜羊膜细胞因子水平。
给本领域技术人员提供上述实施例，以完全公开和描述如何实施和使用所主张的实施方案，而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
Claims (8)
1.一种脱细胞人羊膜的制备方法，其特征在于，包括以下步骤：
（1）取新鲜的人羊膜，浸泡于初步消毒溶液中，对羊膜进行初步消毒，并抑制细菌生长；
（2）取出羊膜，用无菌水清洗，再用次氯酸钠溶液清洗，次氯酸钠溶液的浓度为0.05%，清洗时间为10分钟；再用无菌水清洗；
（3）将清洗后的羊膜浸入冻存溶液中，捞出置于滤纸上，充分展开，再覆盖一层滤纸，反复冻融两次；反复冻融两次的具体操作为：置于-80℃环境下冻存1小时，然后置于37℃环境下融化0.5小时，再置于-80℃环境下冻存1小时，然后置于37℃环境下融化0.5小时；
（4）将反复冻融后的羊膜置于1%的Triton X-100溶液中，摇床孵育，初步去除细胞，摇床的转速为70～100 rpm，孵育温度为37℃，孵育时间为24小时；然后用PBS溶液震荡清洗羊膜，清洗的时间为10分钟；
（5）将羊膜置于脂肪酶溶液中，摇床孵育，以充分去除上皮细胞及其他杂质，然后用PBS溶液震荡清洗羊膜；所述脂肪酶溶液的浓度为1000 U/L；摇床的转速为70～100 rpm，孵育温度为37℃，孵育时间为24小时；
（6）将羊膜平铺在硝酸纤维膜上，绒毛膜面朝向硝酸纤维膜，压在铁盘下，置于-40℃环境下冷冻10～15小时；
（7）将冷冻的羊膜冷冻干燥至含水量为5%～7%。
2.根据权利要求1所述的脱细胞人羊膜的制备方法，其特征在于：还包括步骤（8）：将干燥的羊膜裁剪，装入塑料袋中真空密封，γ射线辐照灭菌。
3.根据权利要求1所述的脱细胞人羊膜的制备方法，其特征在于：所述步骤（1）中，初步消毒溶液为含有50 μg/mL青霉素、50 μg/mL链霉素和2.5 μg/mL两性霉素的PBS溶液；浸泡时间为20分钟。
4.根据权利要求1所述的脱细胞人羊膜的制备方法，其特征在于：所述步骤（2）中，无菌水清洗的次数为4次；每次清洗的时间为10分钟。
5.根据权利要求1所述的脱细胞人羊膜的制备方法，其特征在于：所述步骤（5）中，脂肪酶为购自索莱宝生物科技公司的脂肪酶 TYPE II L8070；清洗次数为3次，每次清洗的时间为10分钟。
6.根据权利要求1所述的脱细胞人羊膜的制备方法，其特征在于：所述步骤（7）中，冷冻干燥的温度和压力为：-30℃、0.2 Mbar。
7.利用权利要求1～6中任一项所述的方法制备得到的脱细胞人羊膜。
8.权利要求7所述的脱细胞人羊膜在伤口愈合、创面修复、皮肤移植的支架材料中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210835190.3A CN115105636B (zh) | 2022-07-16 | 2022-07-16 | 脱细胞人羊膜的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210835190.3A CN115105636B (zh) | 2022-07-16 | 2022-07-16 | 脱细胞人羊膜的制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115105636A CN115105636A (zh) | 2022-09-27 |
| CN115105636B true CN115105636B (zh) | 2024-03-19 |
Family
ID=83332744
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210835190.3A Active CN115105636B (zh) | 2022-07-16 | 2022-07-16 | 脱细胞人羊膜的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115105636B (zh) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6152142A (en) * | 1997-02-28 | 2000-11-28 | Tseng; Scheffer C. G. | Grafts made from amniotic membrane; methods of separating, preserving, and using such grafts in surgeries |
| KR20110014024A (ko) * | 2009-08-04 | 2011-02-10 | 중앙대학교 산학협력단 | 동결-해동-원심분리를 이용한 무세포 양막의 신규한 제조방법 및 이의 이용 |
| CN102225217A (zh) * | 2011-06-23 | 2011-10-26 | 天津世纪康泰生物医学工程有限公司 | 脱细胞干燥活性羊膜的制备方法 |
| CN102367434A (zh) * | 2011-06-03 | 2012-03-07 | 中国人民解放军第二军医大学 | 模拟表皮干细胞生长niche微环境的羊膜微载体及其皮肤替代物 |
| US9132156B1 (en) * | 2014-06-15 | 2015-09-15 | Amnio Technology Llc | Acellular amnion derived therapeutic compositions |
| CN109568663A (zh) * | 2018-11-27 | 2019-04-05 | 中山大学中山眼科中心 | 角膜支架材料的制备方法及角膜支架材料 |
| CN114712562A (zh) * | 2022-04-08 | 2022-07-08 | 上海亚朋生物技术有限公司 | 一种脱细胞冻干羊膜产品的制备工艺 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170021058A1 (en) * | 2015-07-24 | 2017-01-26 | Musculoskeletal Transplant Foundation | Acellular soft tissue-derived matrices and methods for preparing same |
| KR20180095913A (ko) * | 2015-12-23 | 2018-08-28 | 라이프넷 헬스 | 탈세포화된 태반막 및 이의 제조 방법 및 용도 |
-
2022
- 2022-07-16 CN CN202210835190.3A patent/CN115105636B/zh active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6152142A (en) * | 1997-02-28 | 2000-11-28 | Tseng; Scheffer C. G. | Grafts made from amniotic membrane; methods of separating, preserving, and using such grafts in surgeries |
| KR20110014024A (ko) * | 2009-08-04 | 2011-02-10 | 중앙대학교 산학협력단 | 동결-해동-원심분리를 이용한 무세포 양막의 신규한 제조방법 및 이의 이용 |
| CN102367434A (zh) * | 2011-06-03 | 2012-03-07 | 中国人民解放军第二军医大学 | 模拟表皮干细胞生长niche微环境的羊膜微载体及其皮肤替代物 |
| CN102225217A (zh) * | 2011-06-23 | 2011-10-26 | 天津世纪康泰生物医学工程有限公司 | 脱细胞干燥活性羊膜的制备方法 |
| US9132156B1 (en) * | 2014-06-15 | 2015-09-15 | Amnio Technology Llc | Acellular amnion derived therapeutic compositions |
| CN109568663A (zh) * | 2018-11-27 | 2019-04-05 | 中山大学中山眼科中心 | 角膜支架材料的制备方法及角膜支架材料 |
| CN114712562A (zh) * | 2022-04-08 | 2022-07-08 | 上海亚朋生物技术有限公司 | 一种脱细胞冻干羊膜产品的制备工艺 |
Non-Patent Citations (1)
| Title |
|---|
| 人源脱细胞羊膜与脱细胞羊膜下层组织修复皮肤缺损;王丹;尹晶;王玉英;杜勇;李京玲;俞舜;;中国组织工程研究(34);第5570-5576页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115105636A (zh) | 2022-09-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2021205013B2 (en) | Compositions derived from placenta and methods of producing the same | |
| Funamoto et al. | The use of high-hydrostatic pressure treatment to decellularize blood vessels | |
| Luo et al. | Comparison of various reagents for preparing a decellularized porcine cartilage scaffold | |
| CN106075598A (zh) | 一种光交联丝胶蛋白水凝胶及其制备方法和应用 | |
| AU2013289661B2 (en) | Decellularization method for preparing extracellular matrix support material | |
| AU2016324162B2 (en) | Compositions derived from placenta and methods of producing the same | |
| CN104225667B (zh) | 一种促血管生成的温敏性水凝胶粉及用其制备的温敏性水凝胶 | |
| Pérez et al. | Fast protocol for the processing of split-thickness skin into decellularized human dermal matrix | |
| CN104383601A (zh) | 一种骨骼肌脱细胞基质生物补片及其制备方法 | |
| US20200121828A1 (en) | Bioactive scaffold for inducting tendon regeneration, preparation method therefor and use thereof | |
| CN115105636B (zh) | 脱细胞人羊膜的制备方法 | |
| CN113577389A (zh) | 一种来自猪耳软骨的脱细胞软骨材料及制备方法和应用 | |
| CN107412868A (zh) | 一种脱细胞真皮基质的制备方法及制得的脱细胞真皮基质 | |
| WO2021056964A1 (zh) | 一种生物组织基质材料及其制备方法与应用 | |
| CN105031734A (zh) | 髓核-软骨细胞外基质支架及其制备方法 | |
| CN115414531B (zh) | 一种同种异体骨的脂肪及内毒素去除方法 | |
| CN114870088B (zh) | 一种Wnt信号激活骨细胞脱细胞基质及其表衬的骨修复支架的制备方法与应用 | |
| CN114028613A (zh) | 一种功能性骨修复复合支架、制备方法及应用 | |
| CN113599577A (zh) | 一种来自猪肋软骨的脱细胞软骨材料及制备方法和应用 | |
| CN117258047B (zh) | 一种猪牙髓组织脱细胞外基质水凝胶及其制备方法和应用 | |
| CN113813446B (zh) | 去除同种或异种生物材料的细胞成分和/或核酸物质的方法 | |
| US20230347015A1 (en) | Lyophilized perforated placental membrane tissue grafts | |
| CN112516063B (zh) | 一种抗衰老胎盘细胞外基质的制备及使用方法 | |
| CN116870255A (zh) | 一种细胞外基质高保留的脱细胞半月板及其制备方法 | |
| Goyal et al. | Preparation and Characterization of Decellularized Caprine Periosteum Scaffolds for Fracture Gap Healing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |