CN113564111B - 一种低氧培养脐带来源间充质干细胞的方法 - Google Patents
一种低氧培养脐带来源间充质干细胞的方法 Download PDFInfo
- Publication number
- CN113564111B CN113564111B CN202110965758.9A CN202110965758A CN113564111B CN 113564111 B CN113564111 B CN 113564111B CN 202110965758 A CN202110965758 A CN 202110965758A CN 113564111 B CN113564111 B CN 113564111B
- Authority
- CN
- China
- Prior art keywords
- stem cells
- mesenchymal stem
- umbilical cord
- culture medium
- culture
- 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
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 64
- 210000003954 umbilical cord Anatomy 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 27
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 27
- 239000001301 oxygen Substances 0.000 title claims abstract description 27
- 238000012258 culturing Methods 0.000 title claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 32
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 claims abstract description 24
- WGVKWNUPNGFDFJ-DQCZWYHMSA-N β-tocopherol Chemical compound OC1=CC(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C WGVKWNUPNGFDFJ-DQCZWYHMSA-N 0.000 claims abstract description 24
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 20
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 20
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 20
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000000654 additive Substances 0.000 claims abstract description 14
- 229940087168 alpha tocopherol Drugs 0.000 claims abstract description 12
- 229940066595 beta tocopherol Drugs 0.000 claims abstract description 12
- 229960000984 tocofersolan Drugs 0.000 claims abstract description 12
- 239000002076 α-tocopherol Substances 0.000 claims abstract description 12
- 235000004835 α-tocopherol Nutrition 0.000 claims abstract description 12
- 239000011590 β-tocopherol Substances 0.000 claims abstract description 12
- 235000007680 β-tocopherol Nutrition 0.000 claims abstract description 12
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 10
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims abstract description 10
- 108010024636 Glutathione Proteins 0.000 claims abstract description 10
- 102000004877 Insulin Human genes 0.000 claims abstract description 10
- 108090001061 Insulin Proteins 0.000 claims abstract description 10
- 229930003268 Vitamin C Natural products 0.000 claims abstract description 10
- BZKPWHYZMXOIDC-UHFFFAOYSA-N acetazolamide Chemical compound CC(=O)NC1=NN=C(S(N)(=O)=O)S1 BZKPWHYZMXOIDC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229960000571 acetazolamide Drugs 0.000 claims abstract description 10
- 230000000996 additive effect Effects 0.000 claims abstract description 10
- 229960003180 glutathione Drugs 0.000 claims abstract description 10
- 229940125396 insulin Drugs 0.000 claims abstract description 10
- 235000019154 vitamin C Nutrition 0.000 claims abstract description 10
- 239000011718 vitamin C Substances 0.000 claims abstract description 10
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007640 basal medium Substances 0.000 claims abstract description 6
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 5
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 4
- 210000001519 tissue Anatomy 0.000 claims description 12
- 210000004027 cell Anatomy 0.000 claims description 11
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 6
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 6
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 6
- 210000001691 amnion Anatomy 0.000 claims description 3
- 210000004204 blood vessel Anatomy 0.000 claims description 3
- 238000010008 shearing Methods 0.000 claims description 3
- 206010021143 Hypoxia Diseases 0.000 claims description 2
- 230000007954 hypoxia Effects 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 5
- 210000000130 stem cell Anatomy 0.000 abstract description 5
- 238000004113 cell culture Methods 0.000 abstract description 4
- 230000003321 amplification Effects 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- 239000013589 supplement Substances 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 7
- 230000003833 cell viability Effects 0.000 description 6
- 230000010261 cell growth Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 229940126864 fibroblast growth factor Drugs 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 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 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 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 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000013566 allergen Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 210000003074 dental pulp Anatomy 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 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 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/02—Atmosphere, e.g. low oxygen conditions
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Rheumatology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本申请涉及间充质干细胞培养方法的技术领域,具体公开了一种低氧培养脐带来源间充质干细胞的方法。包括以下步骤:将原代脐带来源间充质干细胞接种在培养基上,置于培养温度37℃,氧气体积分数1‑3%,二氧化碳体积分数2‑2.5%,余量氮气的环境中进行传代扩增96‑108h后,收集间充质干细胞;培养基包括基础培养基和传代添加物;基础培养基为MEM、F12、低糖DMEM和高糖DMEM中的一种或多种组合物;传代添加物以培养基终浓度计,包括以下组分,谷胱甘肽11‑15μg/ml,α‑生育酚0.5‑2.5μg/ml,β‑生育酚0.5‑2.0μg/ml,乙酰唑胺2.5‑3.0ng/ml,胰岛素10‑20μg/ml,维生素C8‑10μg/ml。本申请方法可用于对脐带来源间充质干细胞进行扩增培养,且培养所得的干细胞活性高,增殖倍数高,有利于脐带间充质干细胞生物制品的推广应用。
Description
技术领域
本申请涉及间充质干细胞培养方法的技术领域,更具体地说,它涉及一种低氧培养脐带来源间充质干细胞的方法。
背景技术
间充质干细胞(mesenchymal stem cells,MSC)是中胚层来源的、具有高度自我更新能力和多向分化潜能的多能干细胞。目前,主要从脐带、骨髓、脂肪组织、牙髓和胎盘中提取间充质干细胞,间充质干细胞连续传代培养和冷冻保存后,仍具有多向分化潜能,为临床治疗多种疾病,如神经***疾病、肾病、自身免疫性疾病和恶性肿瘤等的治疗提供了新的途径。
脐带来源间充质干细胞是指存在新生儿脐带组织中的一种多功能干细胞,具有取材方便,来源丰富,生物学特性稳定,免疫原性低的特点,使其成为未来干细胞在医疗应用的理想种子细胞。此外,脐带来源间充质干细胞能在培养过程中可分泌多种干细胞因子,包括成纤维细胞生长因子(FGF)、血管内皮生长因子(VEGF)、表皮生长因子(EGF)等在内的各种生长因子,这些生长因子可促进细胞生长,维持细胞干性,并且在美容护肤领域有广泛的应用。
目前对脐带来源间充质干细胞的培养主要分为两种,其一是血清培养法,大多使用DMEM/F12+血清,或者在此基础上添加一些营养物质助于细胞生长,这一方法适用于脐带来源间充质干细胞的大规模培养,但是这一方法中使用的血清可能包含一些未知的过敏原和病原体,对细胞培养的安全性造成影响;其二是无血清培养法,单纯使用培养基及各种营养物质添加剂对期待来源间充质干细胞进行培养,然而这一方法培养的细胞生长缓慢,且细胞活性差,培养基中细胞生长因子含量较低,影响了脐带来源间充质干细胞生物制品的推广应用。
发明内容
为了改善上述提到的问题,本申请提供一种低氧培养脐带来源间充质干细胞的方法。
本申请提供的一种低氧培养脐带来源间充质干细胞的方法,采用如下的技术方案:
一种低氧培养脐带来源间充质干细胞的方法,所述方法包括以下步骤:将原代脐带来源间充质干细胞接种在培养基上,接种密度为10000-50000个/ml,置于培养温度37℃,氧气体积分数1-3%,二氧化碳体积分数2-2.5%,余量氮气的环境中进行传代扩增96-108h后,收集间充质干细胞;所述培养基是由基础培养基和传代添加物组成;所述基础培养基为F12与MEM、低糖DMEM或高糖DMEM其中一种的组合物;所述传代添加物以培养基终浓度计,各组分的用量为,谷胱甘肽11-15μg/ml,α-生育酚0.5-2.5μg/ml,β-生育酚0.5-2.0μg/ml,乙酰唑胺2.5-3.0ng/ml,胰岛素10-20μg/ml,维生素C8-10μg/ml。
优选的,原代脐带来源间充质干细胞培养时,初始氧气体积分数为1%,氧气体积分数以0.1%/2h的速率增长至3%后,保持培养10h,氧气体积分数以0.1%/5h的速率下降至2.3-2.4%,保持培养至传代培养结束。
优选的,间充质干细胞培养过程中,每2天更换一次培养基。
优选的,所述原代脐带来源间充质干细胞由以下步骤制得:采用组织贴壁法,取离体的脐带,剥离羊膜和血管后,剪碎组织,放入含10%FBS的α-MEM完全培养基进行培养,待细胞从组织块中爬出,得原代脐带来源间充质干细胞。
优选的,所述传代添加物以培养基终浓度计,还包括TGF-β0.8-1.0ng/ml。
优选的,所述传代添加物以培养基终浓度计,各组分的用量为,谷胱甘肽12μg/ml,α-生育酚2.1μg/ml,β-生育酚1.6μg/ml,乙酰唑胺2.8ng/ml,胰岛素16μg/ml,维生素C9μg/ml,TGF-β0.8ng/ml。
综上所述,本申请具有以下有益效果:将间充质干细胞放置在低氧环境中,且在培养基中加入多种传代添加物后,进行干细胞传代扩增培养,能够提高间充质干细胞的增殖倍数和培养所得的干细胞细胞活性。
具体实施方式
以下结合实施例1-4和对比例1-2对本申请作进一步详细说明。
制备例
制备例1
制备扩增用的原代脐带来源间充质干细胞:取新鲜健康的人脐带,用PBS缓冲液冲洗干净后,用镊子剥离羊膜和血管,将分离后的脐带胶质剪碎成1-3mm3的组织块,平铺再培养瓶底部,加入含10%FBS,100U/ml青霉素,100U/ml链霉素的α-MEM培养基中,置于37℃,5%二氧化碳的培养箱中进行培养;培养至第七天时,组织块周边出现脐带间充质干细胞,继续培养,待脐带间充质干细胞达到80%的融合时,弃去脐带组织块和原有培养基,生理盐水洗涤后加入0.25%的胰酶消化,即得到扩增用的原代脐带来源间充质干细胞。
实施例
实施例1
本实施例1中对脐带来源间充质干细胞的培养方法,包括以下步骤:
将制备例1中的原代脐带来源间充质干细胞接种在培养基上,细胞接种密度为30000个/ml,置于培养箱中进行细胞扩增培养,培养箱中的培养温度为37℃,保持培养箱中氧气体积分数为2.3-2.4%,二氧化碳体积分数为2-2.5%,余量均为氮气;将原代脐带来源间充质干细胞在培养箱中培养96-108h,且在培养过程中,每48h(2天)更换一次培养基,培养结束后,收集培养所得的间充质干细胞。
其中,培养基包括基础培养基和传代添加物,基础培养基为MEM和F12的1:1混合物,在其他实施例中,基础培养基为F12与MEM、低糖DMEM或高糖DMEM其中一种的组合物,传代添加物以培养基终浓度计,各组分的用量为,谷胱甘肽13μg/ml,α-生育酚1.6μg/ml,β-生育酚1.1μg/ml,乙酰唑胺2.6ng/ml,胰岛素12μg/ml,维生素C9μg/ml。
实施例2
本实施例2与实施例1的不同之处在于,培养基中传代添加物的组分含量不同,实施例2中传代添加物以培养基终浓度计,各组分的用量为,谷胱甘肽15μg/ml,α-生育酚2.5μg/ml,β-生育酚2.0μg/ml,乙酰唑胺3.0ng/ml,胰岛素20μg/ml,维生素C10μg/ml。
实施例3
本实施例3与实施例1的不同之处在于,培养基中传代添加物还包括TGF-β,传代添加物以培养基终浓度计,各组分的用量为,谷胱甘肽12μg/ml,α-生育酚2.1μg/ml,β-生育酚1.6μg/ml,乙酰唑胺2.8ng/ml,胰岛素16μg/ml,维生素C9μg/ml,TGF-β0.8ng/ml。
实施例4
实施例4与实施例3的不同之处在于,进行原代脐带来源间充质干细胞的培养时,培养箱中初始氧气体积分数为1%,氧气体积分数以0.1%/2h的速率增长至3%后,保持培养10h,氧气体积分数以0.1%/5h的速率下降至2.3-2.4%,保持培养至传代培养结束。
对比例
对比例1
本对比例1与实施例1的不同之处在于,培养基中传代添加物不包括α-生育酚,传代添加物以培养基终浓度计,各组分的用量为,谷胱甘肽13μg/ml,β-生育酚2.7μg/ml,乙酰唑胺2.6ng/ml,胰岛素12μg/ml,维生素C9μg/ml。
对比例2
本对比例2与实施例1的不同之处在于,培养基中传代添加物不包括β-生育酚,传代添加物以培养基终浓度计,各组分的用量为,谷胱甘肽13μg/ml,α-生育酚2.7μg/ml,乙酰唑胺2.6ng/ml,胰岛素12μg/ml,维生素C9μg/ml。
性能检测试验
采用0.4%的台盼蓝对实施例1-4和对比例1-2中扩增培养的间充质干细胞进行染色,细胞计数后,统计实施例1-4和对比例1-2中间充质干细胞的增殖倍数及细胞活率,增殖倍数=培养后细胞总数量/初始接种细胞数,细胞活率=(活细胞数量/培养后细胞总数量)×100%,其结果如表1所示。
表1实施例1-4和对比例1-2中间充质干细胞的增殖倍数和细胞活率
结合实施例1-4和对比例1-2,并结合表1可以看出,本申请中一种低氧培养脐带来源间充质干细胞的方法对间充质干细胞的培养效果更好,培养后的间充质干细胞的增殖倍数和细胞活率都更优于对比例1-2。
结合实施例1-2和实施例3,并结合表1可以看出,在培养基中加入TGF-β后,能够提高细胞培养的增殖倍数和细胞活率。
结合实施例3和实施例4,并结合表1可以看出,对间充质干细胞进行培养时,在低氧环境中,且培养箱中的氧气体积随着培养时间的延长发生改变,最后持续稳定,能够提高间充质干细胞的增殖倍数和细胞活率。
结合实施例1和对比例1-2,并结合表1可以看出,在培养基中单独添加α-生育酚或β-生育酚的效果,没有α-生育酚和β-生育酚复配使用时对间充质干细胞的培养促进效果突出。
本具体实施例仅仅是对本申请的解释,其并不是对本申请的限制,本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改,但只要在本申请的权利要求范围内都受到专利法的保护。
Claims (6)
1.一种低氧培养脐带来源间充质干细胞的方法,其特征在于,所述方法包括以下步骤:
将原代脐带来源间充质干细胞接种在培养基上,接种密度为10000-50000个/ml,置于培养温度37℃,氧气体积分数1-3%,二氧化碳体积分数2-2.5%,余量氮气的环境中进行传代扩增96-108h后,收集间充质干细胞;
所述培养基是由基础培养基和传代添加物组成;
所述基础培养基为F12与MEM、低糖DMEM或高糖DMEM其中一种的组合物;
所述传代添加物以培养基终浓度计,各组分的用量为,谷胱甘肽11-15μg/ml,α-生育酚0.5-2.5μg/ml,β-生育酚0.5-2.0μg/ml,乙酰唑胺2.5-3.0ng/ml,胰岛素10-20μg/ml,维生素C8-10μg/ml。
2.根据权利要求1所述的一种低氧培养脐带来源间充质干细胞的方法,其特征在于:原代脐带来源间充质干细胞培养时初始氧气体积分数为1%,氧气体积分数以0.1%/2h的速率增长至3%后,保持培养10h,氧气体积分数以0.1%/5h的速率下降至2.3-2.4%,保持培养至传代培养结束。
3.根据权利要求1所述的一种低氧培养脐带来源间充质干细胞的方法,其特征在于:间充质干细胞培养过程中,每2天更换一次培养基。
4.根据权利要求1所述的一种低氧培养脐带来源间充质干细胞的方法,其特征在于,所述原代脐带来源间充质干细胞由以下步骤制得:采用组织贴壁法,取离体的脐带,剥离羊膜和血管后,剪碎组织,放入含10%FBS的α-MEM完全培养基进行培养,待细胞从组织块中爬出,得原代脐带来源间充质干细胞。
5.根据权利要求1所述的一种低氧培养脐带来源间充质干细胞的方法,其特征在于:所述传代添加物以培养基终浓度计,还包括TGF-β0.8-1.0ng/ml。
6.根据权利要求5所述的一种低氧培养脐带来源间充质干细胞的方法,其特征在于:所述传代添加物以培养基终浓度计,各组分的用量为,谷胱甘肽12μg/ml,α-生育酚2.1μg/ml,β-生育酚1.6μg/ml,乙酰唑胺2.8ng/ml,胰岛素16μg/ml,维生素C9μg/ml,TGF-β0.8ng/ml。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110965758.9A CN113564111B (zh) | 2021-08-23 | 2021-08-23 | 一种低氧培养脐带来源间充质干细胞的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110965758.9A CN113564111B (zh) | 2021-08-23 | 2021-08-23 | 一种低氧培养脐带来源间充质干细胞的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113564111A CN113564111A (zh) | 2021-10-29 |
CN113564111B true CN113564111B (zh) | 2023-11-21 |
Family
ID=78172372
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110965758.9A Active CN113564111B (zh) | 2021-08-23 | 2021-08-23 | 一种低氧培养脐带来源间充质干细胞的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113564111B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114350603B (zh) * | 2022-01-23 | 2022-08-23 | 上海揽微赛尔生物科技有限公司 | 包含外泌体的间充质干细胞胞外基质及其制备和在细胞修复中的应用 |
CN115521907A (zh) * | 2022-08-25 | 2022-12-27 | 王意忠 | 一种培养间充质干细胞的方法 |
CN116606806A (zh) * | 2023-07-20 | 2023-08-18 | 北京益华生物科技有限公司 | 一种低氧环境下提高骨髓间充质干细胞活率的方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113151165A (zh) * | 2021-05-14 | 2021-07-23 | 河北驰熙科技发展有限公司 | 一种人脐带间充质干细胞扩增用培养基及培养方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015170347A2 (en) * | 2014-05-09 | 2015-11-12 | Reelabs Private Limited | Foetal polymix of mesenchymal stem cells under hypoxic conditions for the treatment of clinical disorders |
-
2021
- 2021-08-23 CN CN202110965758.9A patent/CN113564111B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113151165A (zh) * | 2021-05-14 | 2021-07-23 | 河北驰熙科技发展有限公司 | 一种人脐带间充质干细胞扩增用培养基及培养方法 |
Also Published As
Publication number | Publication date |
---|---|
CN113564111A (zh) | 2021-10-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113564111B (zh) | 一种低氧培养脐带来源间充质干细胞的方法 | |
EP2368974A1 (en) | Methods for isolating mesenchymal stem cells from embryos of human or animals and extracting secretion substances thereof | |
CA2682243C (en) | Methods and means for stem cell proliferation and subsequent generation and expansion of progenitor cells, as well as production of effector cells as clinical therapeutics | |
CN112608894A (zh) | 一种间充质干细胞培养基 | |
CN104988110A (zh) | 脐带间充质干细胞转化为胰岛细胞的方法 | |
CN114480273A (zh) | 用于获得间充质干细胞及其外泌体的培养基及其制备方法 | |
CN100508948C (zh) | 一种美容护肤产品、其制备方法 | |
CN110946129A (zh) | 细胞复苏后高活率冻存液 | |
US11840706B2 (en) | Composition and method for generating a desired cell type and/or tissue type from hair follicular stem cells | |
CN113151165B (zh) | 一种人脐带间充质干细胞扩增用培养基及培养方法 | |
CN102965337A (zh) | 分离提取人皮下脂肪间充质干细胞的方法和提取专用培养基 | |
CN102146359A (zh) | 从胎盘中提取原始间充质干细胞及无血清扩增的方法 | |
CN106701670A (zh) | 一种增强间充质干细胞分泌生物活性因子能力及培养液中活性因子的提取方法 | |
CN111235101A (zh) | 一种人脐带间充质干细胞培养基及人脐带间充质干细胞的培养方法 | |
KR101753557B1 (ko) | 줄기세포 증식 향상 배지 조성물 및 줄기세포의 배양방법 | |
CN104988111A (zh) | 用于将uc-msc转化为胰岛细胞的诱导液及其应用 | |
CN109864964B (zh) | 一种包含干细胞的抗衰老组合物及其应用 | |
CN111849887A (zh) | 一种自体脂肪三维凝胶及其svf干细胞的制备方法及应用 | |
CN203625386U (zh) | 一种用于分离和培养脐带间充质干细胞的试剂盒 | |
CN112680408A (zh) | 一种诱导人间充质干细胞成软骨分化培养基及其制备方法 | |
JP2011211956A (ja) | 幹細胞の未分化維持剤及び増殖促進剤 | |
RU2645255C1 (ru) | Способ получения биобезопасной культуры мезенхимальных стволовых клеток из ворсин хориона человека | |
CN113018316A (zh) | 多能干细胞来源的间充质干细胞在修复皮肤损伤中的应用 | |
CN105039239A (zh) | 细胞转化诱导液及其应用 | |
CN115305232B (zh) | 一种脂肪间充质干细胞复苏培养液及复苏方法 |
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 | ||
CB02 | Change of applicant information | ||
CB02 | Change of applicant information |
Address after: 200333 Room 203, second floor, building 2, No. 518 and No. 538, Chaozhou Road, Putuo District, Shanghai Applicant after: Shanghai Taiyao Biomedical Co.,Ltd. Address before: 200333 Room 203, second floor, building 2, No. 518 and No. 538, Chaozhou Road, Putuo District, Shanghai Applicant before: Shanghai taiantang biomedical Co.,Ltd. |
|
GR01 | Patent grant | ||
GR01 | Patent grant |