CN110923196B - Serum-free medium, preparation method thereof and mesenchymal stem cell culture method - Google Patents
Serum-free medium, preparation method thereof and mesenchymal stem cell culture method Download PDFInfo
- Publication number
- CN110923196B CN110923196B CN201911219419.5A CN201911219419A CN110923196B CN 110923196 B CN110923196 B CN 110923196B CN 201911219419 A CN201911219419 A CN 201911219419A CN 110923196 B CN110923196 B CN 110923196B
- Authority
- CN
- China
- Prior art keywords
- recombinant human
- serum
- mesenchymal stem
- stem cells
- culture medium
- 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 28
- 239000012679 serum free medium Substances 0.000 title claims description 21
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 238000004113 cell culture Methods 0.000 title abstract description 7
- 239000001963 growth medium Substances 0.000 claims abstract description 23
- 239000004017 serum-free culture medium Substances 0.000 claims abstract description 23
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims abstract description 20
- 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
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 20
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims abstract description 20
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 19
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 19
- 239000007640 basal medium Substances 0.000 claims abstract description 11
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229930024421 Adenine Natural products 0.000 claims abstract description 10
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 claims abstract description 10
- 101001027128 Homo sapiens Fibronectin Proteins 0.000 claims abstract description 10
- 101000976075 Homo sapiens Insulin Proteins 0.000 claims abstract description 10
- 101000954805 Homo sapiens Protein Wnt-3a Proteins 0.000 claims abstract description 10
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 claims abstract description 10
- RWSXRVCMGQZWBV-PHDIDXHHSA-N L-Glutathione Natural products OC(=O)[C@H](N)CCC(=O)N[C@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-PHDIDXHHSA-N 0.000 claims abstract description 10
- 239000002211 L-ascorbic acid Substances 0.000 claims abstract description 10
- 235000000069 L-ascorbic acid Nutrition 0.000 claims abstract description 10
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229960000643 adenine Drugs 0.000 claims abstract description 10
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 10
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 10
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 claims abstract description 10
- 229960003957 dexamethasone Drugs 0.000 claims abstract description 10
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims abstract description 10
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 10
- 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 10
- 102000056781 human WNT3A Human genes 0.000 claims abstract description 10
- 229940116978 human epidermal growth factor Drugs 0.000 claims abstract description 10
- 229960000890 hydrocortisone Drugs 0.000 claims abstract description 10
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 claims abstract description 10
- 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 claims abstract description 10
- 229920001993 poloxamer 188 Polymers 0.000 claims abstract description 10
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 claims abstract description 10
- 108700005467 recombinant KCB-1 Proteins 0.000 claims abstract description 10
- 229960001471 sodium selenite Drugs 0.000 claims abstract description 10
- 235000015921 sodium selenite Nutrition 0.000 claims abstract description 10
- 239000011781 sodium selenite Substances 0.000 claims abstract description 10
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000012136 culture method Methods 0.000 claims abstract description 9
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims abstract description 5
- 229920000053 polysorbate 80 Polymers 0.000 claims abstract description 5
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims abstract description 4
- 150000001413 amino acids Chemical class 0.000 claims abstract description 4
- 229940107161 cholesterol Drugs 0.000 claims abstract description 4
- 229960002743 glutamine Drugs 0.000 claims abstract description 4
- 229940068984 polyvinyl alcohol Drugs 0.000 claims abstract description 4
- 229940024606 amino acid Drugs 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 11
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 10
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 239000003797 essential amino acid Substances 0.000 claims description 7
- 235000020776 essential amino acid Nutrition 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 2
- 210000003954 umbilical cord Anatomy 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 12
- 210000000130 stem cell Anatomy 0.000 abstract description 11
- 108010071390 Serum Albumin Proteins 0.000 abstract description 8
- 102000007562 Serum Albumin Human genes 0.000 abstract description 8
- 230000035755 proliferation Effects 0.000 abstract description 8
- 230000009467 reduction Effects 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 19
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 6
- 108091006905 Human Serum Albumin Proteins 0.000 description 5
- 102000008100 Human Serum Albumin Human genes 0.000 description 5
- 238000004090 dissolution Methods 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 238000011177 media preparation Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 3
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 3
- 230000009818 osteogenic differentiation Effects 0.000 description 3
- 102100022464 5'-nucleotidase Human genes 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 102100037241 Endoglin Human genes 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 102000006354 HLA-DR Antigens Human genes 0.000 description 2
- 108010058597 HLA-DR Antigens Proteins 0.000 description 2
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 2
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000009815 adipogenic differentiation Effects 0.000 description 2
- 235000021120 animal protein Nutrition 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 210000002826 placenta Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000010186 staining Methods 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
- 239000012588 trypsin Substances 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 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
- 208000006735 Periostitis Diseases 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 229920002978 Vinylon Polymers 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 description 1
- 150000001241 acetals Chemical class 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000002293 adipogenic effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 208000019061 glycogen storage disease due to GLUT2 deficiency Diseases 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 210000004967 non-hematopoietic stem cell Anatomy 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000000123 paper Substances 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000003460 periosteum Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 210000004003 subcutaneous fat Anatomy 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 210000001258 synovial membrane Anatomy 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 239000012209 synthetic fiber Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
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
- 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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
-
- 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
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
-
- 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
- C12N5/0668—Mesenchymal stem cells from other natural sources
-
- 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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- 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
- C12N2500/32—Amino acids
-
- 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
- C12N2500/36—Lipids
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides or bases
-
- 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
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- 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
- C12N2500/46—Amines, e.g. putrescine
-
- 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
- C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
-
- 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
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- 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
- C12N2501/11—Epidermal growth factor [EGF]
-
- 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
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- 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
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- 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
- C12N2501/998—Proteins 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Rheumatology (AREA)
- Gynecology & Obstetrics (AREA)
- Reproductive Health (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of cell culture, in particular to a serum-free culture medium, a preparation method thereof and a culture method of mesenchymal stem cells. The culture medium consists of the following components: nonessential amino acids, glutamine, hydrocortisone, dexamethasone, polyvinyl alcohol, recombinant human insulin, recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human Wnt-3a protein, recombinant human fibronectin, recombinant human laminin, L-glutathione, L-ascorbic acid, beta-mercaptoethanol, ethanolamine, Pluronic F-68, Tween 80, cholesterol, adenine, sodium selenite and basal medium. The invention discovers that polyvinyl alcohol can replace the carrier effect of serum albumin in a serum-free culture medium, effectively improves the proliferation capacity of mesenchymal stem cells, has more stable performance than the serum albumin, and can effectively solve the problem of the proliferation and the passage capacity reduction of the stem cells.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a serum-free culture medium, a preparation method thereof and a culture method of mesenchymal stem cells.
Background
Research in the field of regenerative medicine represented by stem cells has continuously made a major technical breakthrough in the last two decades, and has led to high attention on industrialization and clinical application of stem cell technology. By 2019, 16 stem cell products are on the market worldwide, and the intermediate stem cells (MSCs) become the most product and most rapidly developed stem cell types due to various advantages. Mesenchymal stem cells are derived from mesoderm in early development, and are a type of non-hematopoietic stem cells widely existing in bone marrow, subcutaneous fat, periosteum, muscle, synovium, synovial fluid, liver, peripheral tissues, umbilical cord blood, placenta and other tissues. The MSCs have high self-renewal capacity and multidirectional differentiation potential, can be cultured and amplified in vitro, can support the growth of hematopoietic stem cells, and also has the function of immune regulation; under different induction conditions, the cells can be differentiated into bones, cartilages, muscles, nerves, cardiac muscles, endothelia, fat and the like in vitro, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ injuries caused by aging and pathological changes. Therefore, the MSCs have wide clinical application prospect, are the first choice seed cells for cell replacement therapy and tissue engineering, and are the research hotspots in the field of transplantation and the treatment of autoimmune diseases. At present, the demand for the mesenchymal stem cell related technology and products which are large-scale, standardized and industrialized and can be clinically applied is increasingly called for in the industry.
In studies in the field of biomedical engineering, the acquisition of large numbers of seed cells is often achieved by in vitro culture and harvesting of stem cells. At present, a serum culture medium or a serum-free culture medium is used for mesenchymal stem cell culture. Serum media mostly contain animal serum, such as the most common Fetal Bovine Serum (FBS). FBS is complex in composition, contains heterogeneous proteins, and is likely to carry viruses or to be infected with mycoplasma. On the other hand, the FBS has large batch difference and unstable source, and has large influence on the process of amplifying the MSCs in vitro on a large scale. Research shows that MSCs phagocytose protein in culture medium during culture, and the culture medium contains bovine serum albumin, so that anti-bovine albumin antibody produced in a recipient can cause immune response, and the MSCs are ineffective or especially ineffective after repeated infusion treatment. Therefore, more and more researchers and enterprises are beginning to develop alternatives to FBSs.
Serum-Free Medium (SFM), as the name implies, means that it is not necessary to add animal or human Serum to the cell culture. However, in order to meet the requirement of cell growth, materials for replacing serum functions, mainly including binding proteins, growth factors, adhesion factors, hormones, trace elements, and the like, are usually added to the culture medium. The development of SFM applied to mesenchymal stem cells has gone through four major stages. The first generation is serum-free culture medium in general meaning, adopts various biological materials capable of replacing serum function, contains a large amount of animal-derived proteins and unidentified components, such as animal or human-derived platelet lysate and the like; compared with a serum-containing culture medium, the method has the advantages of high experimental accuracy, repeatability and stability; however, since the chemical composition of the additive substance is not clear, most of the additive substances contain a large amount of animal-derived proteins, which is not favorable for the separation and purification of target proteins and has high cost. The second generation is serum-free and animal protein-free culture medium, and the added components of the culture medium do not use animal protein at all, but replace various recombinant proteins or animal and plant protein hydrolysates; the method has the advantages that the stability is improved compared with the first generation, the cost is reduced, and the effect is correspondingly improved; but the cost is extremely high, and the method is only suitable for scientific research users with small demand and is difficult to be used by enterprises developing large-scale production. At present, the performance of the product on the market is stable, and most of scientific research users use the second generation SFM products. The third generation is a component-limited medium, also known as a double-no medium; the added components are completely free of serum, free of protein or extremely low in protein content, and the contained protein is definite in components; the third-generation SFM has obvious advantages, the cell culture and the production are easy to be constant, the separation and the purification of target protein are easier, and the cost is greatly reduced; however, at the present stage, the products all have the defects of insufficient cell generation and expansion capacity, which is also a bottleneck to be broken through by SFM research and development enterprises at present; and has high specificity to cultured cells, so that the cell lines suitable for culture are few, and the product development difficulty is great. The fourth generation is a chemical component limited culture medium, the added components do not contain serum or any protein, unstable protein substances are mainly replaced by compounds, and the medium can be sterilized at high temperature and is suitable for a totipotent culture medium for the growth of various cells; at present, no such products are on the market and still in the development stage. Serum-free media are currently on the market, in which bovine serum albumin or human serum albumin is almost an essential raw material in the base. The serum albumin has a large adding proportion in a serum-free culture medium, and has the functions of not only serving as a transport protein, but also increasing the viscosity of the culture medium and protecting cells from mechanical damage. Research shows that bovine or human serum albumin may promote stem cell differentiation, resulting in reduction of stem cell proliferation and passability.
Disclosure of Invention
In view of the above, the invention provides a serum-free medium, a preparation method thereof and a culture method of mesenchymal stem cells. The serum-free culture medium can effectively solve the problems of stem cell proliferation and passage capacity reduction.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a serum-free culture medium, which consists of the following components:
the invention selects a high molecular organic compound-polyvinyl alcohol (PVA), which can replace the carrier effect of serum albumin in a culture medium, effectively improves the proliferation capacity of mesenchymal stem cells, and has more stable performance than the serum albumin. Industrial grade PVA is an important chemical raw material for the manufacture of polyvinyl acetal, gasoline resistant pipelines and vinylon synthetic fibers, fabric treating agents, emulsifiers, paper coatings, adhesives, glues, and the like. The medical PVA is a safe polymer organic matter, has no toxicity to human body, no side effect and excellent biocompatibility, is especially widely used in medical treatment, such as water gel in ophthalmology, wound dressing and artificial joint, and is also used in medicinal film, artificial kidney film and other fields. The safety can be seen from the use in wound skin repair, and eye drop products. Some of these types are also commonly used in cosmetic masks, facial cleansers, lotions and lotions, as a safe film former. The latest international research finds that polyvinyl alcohol contained in common glue can be used for a culture solution of hematopoietic stem cells, so that the culture cost of the hematopoietic stem cells is expected to be greatly reduced, and the glue helps to treat diseases such as leukemia.
The invention provides a high molecular organic compound polyvinyl alcohol which replaces the common serum albumin in the existing serum-free medium product and can effectively improve the defects of insufficient passage and amplification capacity of mesenchymal stem cells.
Preferably, the amounts of the components in the culture medium are as follows:
preferably, the amounts of the components in the culture medium are as follows:
more preferably, the media is prepared with the following components:
preferably, the basal medium is one of DMEM/F12, high-sugar DMEM, and a-MEM.
Preferably, penicillin and streptomycin can also be added into the serum-free medium.
The invention also provides a preparation method of the serum-free culture medium, which comprises the steps of dissolving non-essential amino acid, glutamine, hydrocortisone, dexamethasone, polyvinyl alcohol, recombinant human insulin, recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human Wnt-3a protein, recombinant human fibronectin, recombinant human laminin, L-glutathione, L-ascorbic acid, beta-mercaptoethanol, ethanolamine, Pluronic F-68, Tween 80, cholesterol, adenine and sodium selenite in a basal culture medium, filtering and sterilizing.
Preferably, the pore size of the filter used for the filter sterilization is not more than 0.22. mu.m.
Preferably, the pore size of the filter used for the filter sterilization is 0.22. mu.m.
The invention also provides a culture method of the mesenchymal stem cells, and the serum-free culture medium is adopted to culture the mesenchymal stem cells.
Preferably, the mesenchymal stem cell is one of umbilical cord mesenchymal stem cell, bone marrow mesenchymal stem cell, adipose mesenchymal stem cell, amniotic fluid mesenchymal stem cell and placenta mesenchymal stem cell.
Preferably, the culture conditions are 5% CO2At 37 ℃, the density inoculation is (0.1-10) multiplied by 104one/mL.
The invention provides a serum-free culture medium, a preparation method thereof and a culture method of mesenchymal stem cells. The culture medium consists of the following components: nonessential amino acids, glutamine, hydrocortisone, dexamethasone, polyvinyl alcohol, recombinant human insulin, recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human Wnt-3a protein, recombinant human fibronectin, recombinant human laminin, L-glutathione, L-ascorbic acid, beta-mercaptoethanol, ethanolamine, Pluronic F-68, Tween 80, cholesterol, adenine, sodium selenite and basal medium. The invention has the technical effects that:
according to the invention, the polyvinyl alcohol is added into the serum-free culture medium, and the polyvinyl alcohol can replace the carrier effect of serum albumin in the culture medium, so that the proliferation capacity of the mesenchymal stem cells is effectively improved, and the performance of the mesenchymal stem cells is more stable than that of the serum albumin.
The serum-free culture medium provided by the invention can effectively solve the problems of stem cell proliferation and passage capacity reduction.
Drawings
FIG. 1 shows the morphology of UC-MSCs in each group (100X);
FIG. 2 shows growth curves for various groups of UC-MSCs;
FIG. 3 shows the adipogenic differentiation effect of UC-MSCs in each group (400X);
FIG. 4 is a graph showing the osteogenic differentiation effect of UC-MSCs in each group (40X).
Detailed Description
The invention discloses a serum-free culture medium, a preparation method thereof and a culture method of mesenchymal stem cells, and the method can be realized by appropriately improving process parameters by taking the contents into reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In the invention, the components and reagents are conventional commercial products. Such as DMEM/F12 basal medium (cat 11330), an optional amino acid solution (cat 11140-. Other components are commercially available from sigma, MP, Gibco, and the like.
The invention is further illustrated by the following examples:
example 1 serum-free medium preparation:
the serum-free medium composition of the present example includes:
non-essential amino acids: the volume ratio is 1%
Glutamine (b): 2mM
Hydrocortisone: 50 mu g/L
Dexamethasone: 10 mu g/L
Polyvinyl alcohol: 2g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 mu g/L
Recombinant human basic fibroblast growth factor: 20 mu g/L
Recombinant human Wnt-3a protein: 20 mu g/L
Recombinant human fibronectin: 50 mu g/L
Recombinant human laminin: 20 mu g/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68:500mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
DMEM/F12 basal medium: and (4) the balance.
Dissolving the components according to respective dissolution characteristics, filtering and sterilizing by a 0.22 mu m filter membrane, adding into a basic culture medium, and mixing uniformly.
Example 2 serum-free medium preparation:
the serum-free medium composition of the present example includes:
non-essential amino acids: the volume ratio is 1%
Glutamine (b): 2mM
Hydrocortisone: 50 mu g/L
Dexamethasone: 10 mu g/L
Polyvinyl alcohol: 4g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 mu g/L
Recombinant human basic fibroblast growth factor: 20 mu g/L
Recombinant human Wnt-3a protein: 20 mu g/L
Recombinant human fibronectin: 50 mu g/L
Recombinant human laminin: 20 mu g/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68:500mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
DMEM/F12 basal medium: and (4) the balance.
Dissolving the components according to respective dissolution characteristics, filtering and sterilizing by a 0.22 mu m filter membrane, adding into a basic culture medium, and mixing uniformly.
Example 3 serum-free medium preparation:
the serum-free medium composition of the present example includes:
non-essential amino acids: the volume ratio is 1%
Glutamine (b): 2mM
Hydrocortisone: 50 mu g/L
Dexamethasone: 10 mu g/L
Polyvinyl alcohol: 10g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 mu g/L
Recombinant human basic fibroblast growth factor: 20 mu g/L
Recombinant human Wnt-3a protein: 20 mu g/L
Recombinant human fibronectin: 50 mu g/L
Recombinant human laminin: 20 mu g/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68:500mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
DMEM/F12 basal medium: and (4) the balance.
Dissolving the components according to respective dissolution characteristics, filtering and sterilizing by a 0.22 mu m filter membrane, adding into a basic culture medium, and mixing uniformly.
Comparative example 1 serum-free medium preparation:
compared with the example 1, the comparison example adopts the recombinant human serum albumin to replace the polyvinyl alcohol, and the serum-free culture medium of the comparison example comprises the following components:
non-essential amino acids: the volume ratio is 1%
Glutamine (b): 2mM
Hydrocortisone: 50 mu g/L
Dexamethasone: 10 mu g/L
Recombinant human serum albumin: 2g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 mu g/L
Recombinant human basic fibroblast growth factor: 20 mu g/L
Recombinant human Wnt-3a protein: 20 mu g/L
Recombinant human fibronectin: 50 mu g/L
Recombinant human laminin: 20 mu g/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68:500mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
DMEM/F12 basal medium: and (4) the balance.
Dissolving the components according to respective dissolution characteristics, filtering and sterilizing by a 0.22 mu m filter membrane, adding into a basic culture medium, and mixing uniformly.
Comparative example 2 serum-free medium preparation:
compared with the comparative example 1 and the examples, the comparative example does not add the recombinant human serum albumin or the polyvinyl alcohol, and the serum-free culture medium of the comparative example comprises the following components:
non-essential amino acids: the volume ratio is 1%
Glutamine (b): 2mM
Hydrocortisone: 50 mu g/L
Dexamethasone: 10 mu g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 mu g/L
Recombinant human basic fibroblast growth factor: 20 mu g/L
Recombinant human Wnt-3a protein: 20 mu g/L
Recombinant human fibronectin: 50 mu g/L
Recombinant human laminin: 20 mu g/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68:500mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
DMEM/F12 basal medium: and (4) the balance.
Dissolving the components according to respective dissolution characteristics, filtering and sterilizing by a 0.22 mu m filter membrane, adding into a basic culture medium, and mixing uniformly.
Test examples Effect detection
The following experiments were carried out with the serum-free media of examples 1 to 3 of the present invention set as experimental group 1 to 3, the complete medium containing 10% FBS set as control group 1, the medium of comparative example 1 set as control group 2, and the medium of comparative example 2 set as control group 3.
(I) UC-MSCs morphology observation and activity detection
Selecting P3 UC-MSCs for experiment, wherein UC-MSCs is 1 × 104/cm2The density was inoculated in T25 flasks, with 3 replicates per group set up. Placing in 5% CO2The culture was carried out in an incubator at 37 ℃. UC-MSCs images were collected after 48h of culture, and the results are shown in FIG. 1. Each group of UC-MSCs grows in a monolayer adherent manner, and most cells are in a long fusiform shape and have irregular shapes.
After 72h of culture, digesting and collecting UC-MSCs in 0.05% trypsin solution, calculating the cell number and cell viability of each group, and showing that the UC-MSCs activity of the experimental group is higher than that of the three control groups (Table 1).
TABLE 1 UC-MSCs Activity assay results for each group
(II) UC-MSCs proliferation rate detection
Selecting P3 UC-MSCs for experiment, wherein UC-MSCs is 1 × 104one/mL of the seed was inoculated in a 24-well plate and charged with 5% CO2The culture was carried out in an incubator at 37 ℃. Cells were collected daily for cell counting, 3 wells were counted for each random collection, and cell growth curves were plotted for 7 consecutive days, with the results shown in tables 2-3, FIG. 2. As can be seen from the results in tables 2-3 and FIG. 2, the UC-MSCs cultured in the human serum-free medium of the present invention have higher proliferation activity than those in the control group 1 (containing serum), the control group 2 and the control group 3. After three days of culture, the proliferation fold of each experimental group is significantly higher than that of the control group 1, the control group 2 and the control group 3.
According to the multiplication time calculation formula: DT ═ t × lg2/(lgNt-lgNo) ], where t is the incubation time; no is the number of cells counted for the first time; nt is the number of cells after t time.
The result shows that the doubling time of the UC-MSCs in the experimental group is shorter than that of the control group 1, the control group 2 and the control group 3, which indicates that the proliferation rate of the UC-MSCs cultured by the serum-free culture medium is higher.
TABLE 2 7-day cell count results for UC-MSCs in each group
TABLE 3 UC-MSCs doubling time results for each group
Experimental groups | Control group 1 | Control group 2 | Control group 3 | Experimental group 1 | Experimental group 2 | Experimental group 3 |
Doubling time (h) | 29.18 | 32.79 | 34.27 | 27.12 | 22.72 | 26.24 |
(III) UC-MSCs surface marker detection
Selecting P3 UC-MSCs for experiment, wherein UC-MSCs is 1 × 104/cm2Inoculating into 10cm culture dish at density, and placing in 5% CO2The culture was carried out in an incubator at 37 ℃. After 3 days, each group of UC-MSCs is digested by 0.05% trypsin solution and detected by flow cytometry on the surface markers such as CD105, CD73, CD90, CD34, CD45, HLA-DR and the like. The results are shown in Table 4. The detection result shows that the UC-MSCs of the experimental group, the control group 1 and the control group 2 have surface markerCD105, CD73 and CD90 positive expression (the expression rate is more than or equal to 95 percent), and have negative expression (the expression rate is less than or equal to 2 percent) of CD34, CD45 and HLA-DR, and the groups have no significant difference. The expression rates of CD105 and CD34 in control group 3 did not meet the standard. The result shows that the UC-MSCs cultured by the serum-free culture medium does not influence the expression of the surface markers.
TABLE 4 detection results of UC-MSCs surface marker of each group
(IV) detection of UC-MSCs multidirectional differentiation potential
Selecting UC-MSCs of P3 generation for experiment, culturing UC-MSCs of experiment group 2, control group 1 and control group 2 to passage of P5 generation at 1 × 105The cells were seeded in 6-well plates at a density of one mL/mL and 5% CO was added2The culture was carried out in an incubator at 37 ℃. When the fusion degree of UC-MSCs in each group reaches more than 80%, a control hole and an induction hole are respectively arranged to induce the UC-MSCs to form bone and fat differentiation. And (3) carrying out oil red O staining on the cells in the adipogenic differentiation experiment group after 14 days, and carrying out alizarin red staining on the cells in the osteogenic differentiation experiment group after 21 days. The experimental result shows that the UC-MSCs cultured by the serum-free culture medium does not influence the osteogenic differentiation potential of the adipogenic bone marrow and maintains the dryness (figure 3 and figure 4).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A method for culturing mesenchymal stem cells is characterized in that a serum-free culture medium is adopted to culture the mesenchymal stem cells;
the mesenchymal stem cells are umbilical cord mesenchymal stem cells;
the serum-free culture medium consists of the following components:
non-essential amino acids: 1vt% of
Glutamine (b): 2mM
Hydrocortisone: 50 mug/L
Dexamethasone: 10 microgram/L
Polyvinyl alcohol: 2 to 10g/L
Recombinant human insulin: 10mg/L
Recombinant human transferrin: 5.5mg/L
Recombinant human epidermal growth factor: 20 microgram/L
Recombinant human basic fibroblast growth factor: 20 microgram/L
Recombinant human Wnt-3a protein: 20 microgram/L
Recombinant human fibronectin: 50 mug/L
Recombinant human laminin: 20 microgram/L
L-glutathione: 4mg/L
L-ascorbic acid: 50mg/L
Beta-mercaptoethanol: 2.5mg/L
Ethanolamine: 0.2g/L
Pluronic F-68: 500 mg/L
Tween 80: 22 mg/L
Cholesterol: 2.2mg/L
Adenine: 10mg/L
Sodium selenite: 0.00067mg/L
Basic culture medium: and (4) the balance.
2. The culture method according to claim 1, wherein the basal medium is one of DMEM/F12, high-sugar DMEM, and a-MEM.
3. The culture method according to claim 1 or 2, wherein the serum-free medium is prepared by: dissolving nonessential amino acids, glutamine, hydrocortisone, dexamethasone, polyvinyl alcohol, recombinant human insulin, recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human Wnt-3a protein, recombinant human fibronectin, recombinant human laminin, L-glutathione, L-ascorbic acid, beta-mercaptoethanol, ethanolamine, Pluronic F-68, Tween 80, cholesterol, adenine and sodium selenite in a basal medium, and filtering and sterilizing.
4. The culture method according to claim 3, wherein the filter used for the filter sterilization has a pore size of 0.22 μm.
5. The method according to claim 1 or 2, wherein the culturing is performed under conditions of 5% CO2At 37 ℃, the density inoculation is (0.1-10) multiplied by 104one/mL.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911219419.5A CN110923196B (en) | 2019-12-03 | 2019-12-03 | Serum-free medium, preparation method thereof and mesenchymal stem cell culture method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911219419.5A CN110923196B (en) | 2019-12-03 | 2019-12-03 | Serum-free medium, preparation method thereof and mesenchymal stem cell culture method |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110923196A CN110923196A (en) | 2020-03-27 |
CN110923196B true CN110923196B (en) | 2021-07-09 |
Family
ID=69848601
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911219419.5A Active CN110923196B (en) | 2019-12-03 | 2019-12-03 | Serum-free medium, preparation method thereof and mesenchymal stem cell culture method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110923196B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113736729A (en) * | 2021-08-25 | 2021-12-03 | 生物岛实验室 | Composition, stem cell serum-free culture medium containing composition and stem cell culture method |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111484970B (en) * | 2020-04-30 | 2022-09-16 | 广州再生医学与健康广东省实验室 | Serum-free and feeder-layer-free embryo and pluripotent stem cell culture medium with low protein content |
CN111621476B (en) * | 2020-05-12 | 2021-03-09 | 天信和(苏州)生物科技有限公司 | Serum-free culture medium for mesenchymal stem cells and preparation method thereof |
CN111593019B (en) * | 2020-06-02 | 2021-06-25 | 广州同康生物科技有限公司 | Serum-free culture medium for placenta mesenchymal stem cells |
CN112048463A (en) * | 2020-08-18 | 2020-12-08 | 赵峻岭 | Serum substitute for cell culture |
CN112210532A (en) * | 2020-10-15 | 2021-01-12 | 生物岛实验室 | Serum-free medium and application thereof in subculture of mesenchymal stem cells |
CN113234672B (en) * | 2021-06-04 | 2022-08-19 | 普华赛尔生物医疗科技有限公司 | Human mesenchymal stem cell culture medium and application thereof |
CN114350603B (en) * | 2022-01-23 | 2022-08-23 | 上海揽微赛尔生物科技有限公司 | Mesenchymal stem cell extracellular matrix containing exosome, preparation method thereof and application thereof in cell repair |
CN114807028B (en) * | 2022-06-02 | 2022-11-01 | 深圳格泰赛尔生物科技有限公司 | Serum-free mesenchymal stem cell culture medium and stem cell culture method |
CN115537388A (en) * | 2022-10-12 | 2022-12-30 | 杭州极麋生物科技有限公司 | Bovine adipose-derived mesenchymal stem cell serum-free medium suitable for cell culture of meat and preparation method thereof |
CN116396930B (en) * | 2023-06-08 | 2023-09-22 | 北京华龛生物科技有限公司 | Mesenchymal stem cell serum-free medium and application thereof |
CN117551600B (en) * | 2024-01-04 | 2024-04-02 | 成都云测医学生物技术有限公司 | Culture medium for promoting differentiation of induced mesenchymal stem cells into dermal papilla cells and induction method |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007228815A (en) * | 2006-02-27 | 2007-09-13 | Gifu Univ | Method for maintaining embryonic stem cell |
CN101984048A (en) * | 2010-11-24 | 2011-03-09 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Culture medium for culturing mesenchymal stem cells |
WO2011100286A2 (en) * | 2010-02-09 | 2011-08-18 | The Johns Hopkins University | Compositions and methods of generating a differentiated mesodermal cell |
CN102827807A (en) * | 2012-09-19 | 2012-12-19 | 北京京蒙高科干细胞技术有限公司 | Serum-free culture medium for mesenchymal stem cells |
CN104894064A (en) * | 2015-07-08 | 2015-09-09 | 河南中科干细胞基因工程有限公司 | Culture medium for culturing mesenchymal stem cells |
CN106479971A (en) * | 2016-12-28 | 2017-03-08 | 深圳江淼医疗有限公司 | A kind of serum-free medium for cultivating mescenchymal stem cell and method |
CN107475184A (en) * | 2016-06-07 | 2017-12-15 | 广州美萨生物科技有限公司 | A kind of low blood serum medium for being used to cultivate human mesenchymal stem cell |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2846004B1 (en) * | 2002-10-16 | 2006-06-23 | Maco Pharma Sa | COMPOSITION FOR CULTIVATION OF CELLS, IN PARTICULAR ANIMAL OR TISSUE, COMPRISING POLYETHYLENE GLYCOL |
US10894944B2 (en) * | 2009-04-10 | 2021-01-19 | Monash University | Cell culture media |
-
2019
- 2019-12-03 CN CN201911219419.5A patent/CN110923196B/en active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007228815A (en) * | 2006-02-27 | 2007-09-13 | Gifu Univ | Method for maintaining embryonic stem cell |
WO2011100286A2 (en) * | 2010-02-09 | 2011-08-18 | The Johns Hopkins University | Compositions and methods of generating a differentiated mesodermal cell |
CN101984048A (en) * | 2010-11-24 | 2011-03-09 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Culture medium for culturing mesenchymal stem cells |
CN102827807A (en) * | 2012-09-19 | 2012-12-19 | 北京京蒙高科干细胞技术有限公司 | Serum-free culture medium for mesenchymal stem cells |
CN104894064A (en) * | 2015-07-08 | 2015-09-09 | 河南中科干细胞基因工程有限公司 | Culture medium for culturing mesenchymal stem cells |
CN107475184A (en) * | 2016-06-07 | 2017-12-15 | 广州美萨生物科技有限公司 | A kind of low blood serum medium for being used to cultivate human mesenchymal stem cell |
CN106479971A (en) * | 2016-12-28 | 2017-03-08 | 深圳江淼医疗有限公司 | A kind of serum-free medium for cultivating mescenchymal stem cell and method |
Non-Patent Citations (1)
Title |
---|
Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation;Adam C. Wilkinson1等;《NATURE》;20190704;第571卷;第117-135页 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113736729A (en) * | 2021-08-25 | 2021-12-03 | 生物岛实验室 | Composition, stem cell serum-free culture medium containing composition and stem cell culture method |
CN113736729B (en) * | 2021-08-25 | 2023-05-02 | 生物岛实验室 | Composition, serum-free medium containing composition and stem cell culture method |
Also Published As
Publication number | Publication date |
---|---|
CN110923196A (en) | 2020-03-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110923196B (en) | Serum-free medium, preparation method thereof and mesenchymal stem cell culture method | |
CN103805562B (en) | Cultivate the serum free medium of placenta mesenchyma stem cell | |
KR101211913B1 (en) | Medium for Culturing Mesenchymal Stem Cells Derived from Amnion and Method for Culturing Mesenchymal Stem Cells Derived from Amnion Using thereof | |
CN113736729B (en) | Composition, serum-free medium containing composition and stem cell culture method | |
CN104877963A (en) | Serum-free human umbilical cord mesenchymal stem cell culture medium and preparation method thereof | |
CN112048470B (en) | Method for preparing clinical grade mesenchymal stem cell preparation by using human induced pluripotent stem cells | |
CN104877962A (en) | Serum-free adipose-derived stem cell culture medium and preparation method thereof | |
CN104877961A (en) | Serum-free human amniotic mesenchymal stem cell culture medium and preparation method thereof | |
US20220251512A1 (en) | Serum-free polypeptide composition for promoting proliferation of mesenchymal stem cells | |
CN109706115B (en) | Construction method of mouse bone marrow mesenchymal stem cell line | |
CN114540298A (en) | Stem cell serum-free medium and preparation method thereof | |
CN107418930A (en) | A kind of preparation method purified with amplification human marrow mesenchymal stem cell | |
CN113201489A (en) | Preparation method of adipose-derived mesenchymal stem cell working cell bank | |
CN109628388B (en) | Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition | |
CN111484970A (en) | Serum-free and feeder-layer-free embryo and pluripotent stem cell culture medium with low protein content | |
CN113249314B (en) | Culture method for promoting proliferation and differentiation of mesenchymal stem cells and serum-free culture medium | |
CN110699317A (en) | Human umbilical cord mesenchymal stem cell serum-free medium and preparation method and application thereof | |
CN112210532A (en) | Serum-free medium and application thereof in subculture of mesenchymal stem cells | |
CN111235100B (en) | Culture method of human umbilical cord blood mesenchymal stem cells | |
CN112608892B (en) | Method for serum-free separation and subculturing of umbilical cord mesenchymal stem cells by using platelet lysate | |
CN116218772A (en) | Urine source mixed cell culture method | |
WO2023044443A1 (en) | Cell expansion methods and compositions for use therein | |
CN113249313A (en) | Primary culture method of stem cells with high cell adherence capacity | |
CN113215095B (en) | Compositions, media supplements, and stem cell media and methods of culture | |
CN114807028B (en) | Serum-free mesenchymal stem cell culture medium and stem cell culture method |
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 |