CN113801837A - Stem cell culture medium and preparation method thereof - Google Patents
Stem cell culture medium and preparation method thereof Download PDFInfo
- Publication number
- CN113801837A CN113801837A CN202111069332.1A CN202111069332A CN113801837A CN 113801837 A CN113801837 A CN 113801837A CN 202111069332 A CN202111069332 A CN 202111069332A CN 113801837 A CN113801837 A CN 113801837A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- growth factor
- stem cell
- mixture
- cell 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.)
- Pending
Links
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 34
- 239000006143 cell culture medium Substances 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title description 11
- 239000001963 growth medium Substances 0.000 claims abstract description 40
- 239000003102 growth factor Substances 0.000 claims abstract description 29
- 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 22
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims abstract description 22
- 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 22
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims abstract description 22
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 21
- 239000000654 additive Substances 0.000 claims abstract description 19
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 16
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims abstract description 11
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims abstract description 11
- YIKYNHJUKRTCJL-UHFFFAOYSA-N Ethyl maltol Chemical compound CCC=1OC=CC(=O)C=1O YIKYNHJUKRTCJL-UHFFFAOYSA-N 0.000 claims abstract description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 11
- 102000004877 Insulin Human genes 0.000 claims abstract description 11
- 108090001061 Insulin Proteins 0.000 claims abstract description 11
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims abstract description 11
- 102000004338 Transferrin Human genes 0.000 claims abstract description 11
- 108090000901 Transferrin Proteins 0.000 claims abstract description 11
- 229930003268 Vitamin C Natural products 0.000 claims abstract description 11
- 150000001413 amino acids Chemical class 0.000 claims abstract description 11
- 229910001424 calcium ion Inorganic materials 0.000 claims abstract description 11
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 11
- 229940093503 ethyl maltol Drugs 0.000 claims abstract description 11
- 229930195729 fatty acid Natural products 0.000 claims abstract description 11
- 239000000194 fatty acid Substances 0.000 claims abstract description 11
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 11
- 229960000304 folic acid Drugs 0.000 claims abstract description 11
- 235000019152 folic acid Nutrition 0.000 claims abstract description 11
- 239000011724 folic acid Substances 0.000 claims abstract description 11
- 239000008103 glucose Substances 0.000 claims abstract description 11
- 229940125396 insulin Drugs 0.000 claims abstract description 11
- 229960005322 streptomycin Drugs 0.000 claims abstract description 11
- 239000012581 transferrin Substances 0.000 claims abstract description 11
- 235000019154 vitamin C Nutrition 0.000 claims abstract description 11
- 239000011718 vitamin C Substances 0.000 claims abstract description 11
- 230000000996 additive effect Effects 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims description 45
- 238000003756 stirring Methods 0.000 claims description 25
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 20
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 20
- 230000001954 sterilising effect Effects 0.000 claims description 20
- 239000011159 matrix material Substances 0.000 claims description 16
- 238000009630 liquid culture Methods 0.000 claims description 15
- 238000004659 sterilization and disinfection Methods 0.000 claims description 15
- 102100032528 C-type lectin domain family 11 member A Human genes 0.000 claims description 14
- 101710167766 C-type lectin domain family 11 member A Proteins 0.000 claims description 14
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 14
- 101800003838 Epidermal growth factor Proteins 0.000 claims description 14
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 14
- 229940116977 epidermal growth factor Drugs 0.000 claims description 14
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 14
- 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 claims description 14
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 11
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 10
- 229910052742 iron Inorganic materials 0.000 claims description 10
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 claims description 9
- -1 iron ion Chemical class 0.000 claims description 9
- 229910001414 potassium ion Inorganic materials 0.000 claims description 9
- 229910001415 sodium ion Inorganic materials 0.000 claims description 9
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 7
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 claims description 5
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 claims description 5
- 235000001014 amino acid Nutrition 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 235000001727 glucose Nutrition 0.000 claims description 5
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims 1
- 239000011591 potassium Substances 0.000 claims 1
- 229910052700 potassium Inorganic materials 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 229910052725 zinc Inorganic materials 0.000 claims 1
- 239000011701 zinc Substances 0.000 claims 1
- 230000010261 cell growth Effects 0.000 abstract description 12
- 210000002966 serum Anatomy 0.000 abstract description 12
- 210000004027 cell Anatomy 0.000 abstract description 9
- 241001465754 Metazoa Species 0.000 abstract description 6
- 239000007640 basal medium Substances 0.000 abstract description 5
- 239000002253 acid Substances 0.000 abstract description 2
- 238000010923 batch production Methods 0.000 abstract description 2
- 239000002609 medium Substances 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract 1
- 102000018386 EGF Family of Proteins Human genes 0.000 description 6
- 108010066486 EGF Family of Proteins Proteins 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 4
- 241000233866 Fungi Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002676 xenobiotic agent Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000014670 detection of bacterium Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000007793 ph indicator Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000926 separation method 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
-
- 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
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
-
- 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
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/14—Calcium; Ca chelators; Calcitonin
-
- 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
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/16—Magnesium; Mg chelators
-
- 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
- C12N2500/22—Zinc; Zn chelators
-
- 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
-
- 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
-
- 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
- C12N2500/34—Sugars
-
- 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
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
-
- 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
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
Landscapes
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of cell culture media, in particular to a stem cell culture medium, which comprises a basal medium and an additive, wherein the basal medium is a DMEM/F12 liquid medium, and the additive components and the concentration thereof added into the basal medium are as follows based on the basal medium: 9-12 mu g/mL of transferrin, 1-3mg/mL of insulin, 2-4 mu g/mL of growth factor, 8-10mg/mL of vitamin C, 2-5mg/mL of folic acid, 1-3mg/mL of glucose, 6-8 mu g/mL of metal ions, 3-5 mu g/mL of calcium ions, 0.1-0.2mg/mL of streptomycin, 1-3mg/mL of fatty acid, 1-3mg/mL of amino acid and 1-3mg/mL of ethyl maltol. The stem cell culture medium has determined components, is convenient for batch production and standardization, does not add animal serum, can avoid the heterozygote in the animal serum, can improve the stability of cell growth on acid and heat, can ensure that the cell growth is continuously and stably released in the culture medium, and ensures that the cell is stably amplified.
Description
Technical Field
The invention relates to the technical field of cell culture media, in particular to a stem cell culture medium and a preparation method thereof.
Background
Stem cells do not survive in simple basal media, and certain trace nutrients and growth factors must be provided in cell culture to allow the cells to grow and maintain their growth state. The basal medium used is therefore often supplemented with serum, such as horse serum or fetal bovine serum; because serum is an extremely complex mixture of many biomolecules of different sizes, it provides growth factors, hormones, binding proteins, and provides protection for cells cultured in vitro; moreover, the serum is rich in mitotic factors, and is also beneficial to cell lines and primary culture.
However, the serum culture medium has certain disadvantages, so that the xenobiotics in animal serum cannot be avoided, and human xenobiotics rejection and conflict can exist if cells cultured by using the animal serum culture medium. Especially in the process of stem cell culture, a large amount of complex proteins in serum bring difficulties to the standardization of cell culture, wherein the complex proteins and multiple cytokines can cause the stem cells which are easy to differentiate to generate a plurality of completely different cell phenotypes, thereby generating a plurality of completely different cell differentiation trends, and also bringing great difficulties to the separation and purification of cell culture expression products and the repeatability of results.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a stem cell culture medium and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a stem cell culture medium comprises a matrix culture medium and additives, wherein the base culture medium is a DMEM/F12 liquid culture medium, and the additives are added into the matrix culture medium according to the matrix culture medium, and the concentrations of the additives are as follows: 9-12 mu g/mL of transferrin, 1-3mg/mL of insulin, 2-4 mu g/mL of growth factor, 8-10mg/mL of vitamin C, 2-5mg/mL of folic acid, 1-3mg/mL of glucose, 6-8 mu g/mL of metal ions, 3-5 mu g/mL of calcium ions, 0.1-0.2mg/mL of streptomycin, 1-3mg/mL of fatty acid, 1-3mg/mL of amino acid and 1-3mg/mL of ethyl maltol.
Preferably, the growth factors comprise fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor to the epidermal growth factor to the stem cell growth factor is 4:3: 3.
Preferably, the metal ions include equal amounts of iron ions, sodium ions, potassium ions, zinc ions, and magnesium ions.
A preparation method of a stem cell culture medium comprises the following steps:
s1, taking the additive components with the concentration and DMEM/F12 liquid culture medium;
s2, adding transferrin, insulin, vitamin C, folic acid, glucose, fatty acid, amino acid and ethyl maltol into a DMEM/F12 liquid culture medium, mixing and stirring for 20-40min, then adding growth factors, stirring for 10-15min again, finally adding streptomycin, stirring for 10-15min, and standing for 3-4h to obtain a mixture A;
s3, mixing and stirring the metal ion solutions and the calcium ion solution for 20-30min, and standing for 1-2h after stirring to obtain a mixture B;
s4, dropwise adding the mixture B into the mixture A through a rubber head dropper, oscillating in the dropwise adding process, and standing after the dropwise adding is finished to obtain a mixture C;
s5, adding a sodium carbonate solution with the mass fraction of 6-8% into the mixture C, and adjusting the pH of the mixture C to 7.0-7.4;
s6, sterilizing the mixture C after the pH is adjusted, wherein the sterilization temperature is 110-120 ℃, the sterilization time is 10-15min, and the stem cell culture medium is obtained after sterilization.
Preferably, the metal ion solution in S3 includes an iron ion solution, a sodium ion solution, a potassium ion solution, a zinc ion solution, and a magnesium ion solution in equal amounts.
Preferably, the growth factors in S2 include fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor, the epidermal growth factor and the stem cell growth factor is 4:3: 3.
Preferably, a 7% sodium carbonate solution is added to the mixture C in S5, and the pH of the mixture C is adjusted to 7.2.
The invention has the beneficial effects that:
the stem cell culture medium has determined components, is convenient for batch production and standardization, does not add animal serum, can avoid the heterozygote in the animal serum, can improve the stability of cell growth on acid and heat, can ensure that the cell growth is continuously and stably released in the culture medium, and ensures that the cell is stably amplified.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
In example 1
A stem cell culture medium and a preparation method thereof comprise a matrix culture medium and additives, wherein the matrix culture medium is a DMEM/F12 liquid culture medium, and the additives and the concentrations thereof added into the matrix culture medium are as follows: 9. mu.g/mL transferrin, 1mg/mL insulin, 2. mu.g/mL growth factor, 8mg/mL vitamin C, 2mg/mL folic acid, 1mg/mL glucose, 6. mu.g/mL metal ions, 3. mu.g/mL calcium ions, 0.1mg/mL streptomycin, 1mg/mL fatty acids, 1mg/mL amino acids, and 1mg/mL ethyl maltol.
Furthermore, the growth factors comprise fibroblast growth factors, epidermal growth factors and stem cell growth factors, and the concentration ratio of the fibroblast growth factors to the epidermal growth factors to the stem cell growth factors is 4:3: 3.
Further, the metal ions include iron ions, sodium ions, potassium ions, zinc ions, and magnesium ions in equal amounts.
A preparation method of a stem cell culture medium comprises the following steps:
s1, taking the additive components with the concentration and DMEM/F12 liquid culture medium;
s2, adding transferrin, insulin, vitamin C, folic acid, glucose, fatty acid, amino acid and ethyl maltol into a DMEM/F12 liquid culture medium, mixing and stirring for 20-40min, then adding growth factors, stirring again for 10min, finally adding streptomycin, stirring for 10min, and standing for 3h to obtain a mixture A;
s3, mixing and stirring the metal ion solutions and the calcium ion solution for 20min, and standing for 1h after stirring to obtain a mixture B;
s4, dropwise adding the mixture B into the mixture A through a rubber head dropper, oscillating in the dropwise adding process, and standing after the dropwise adding is finished to obtain a mixture C;
s5, adding a sodium carbonate solution with the mass fraction of 6-8% into the mixture C, and adjusting the pH of the mixture C to 7.0;
s6, sterilizing the mixture C after the pH is adjusted, wherein the sterilization temperature is 110 ℃, the sterilization time is 10min, and the stem cell culture medium is obtained after sterilization.
Further, the metal ion solution in S3 includes an iron ion solution, a sodium ion solution, a potassium ion solution, a zinc ion solution, and a magnesium ion solution in equal amounts.
Further, the growth factors in S2 include fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor, the epidermal growth factor and the stem cell growth factor is 4:3: 3.
In example 2
A stem cell culture medium and a preparation method thereof comprise a matrix culture medium and additives, wherein the matrix culture medium is a DMEM/F12 liquid culture medium, and the additives and the concentrations thereof added into the matrix culture medium are as follows: 10. mu.g/mL transferrin, 2mg/mL insulin, 3. mu.g/mL growth factor, 9mg/mL vitamin C, 3mg/mL folic acid, 2mg/mL glucose, 7. mu.g/mL metal ions, 4. mu.g/mL calcium ions, 0.15mg/mL streptomycin, 2mg/mL fatty acids, 2mg/mL amino acids, and 2mg/mL ethyl maltol.
Furthermore, the growth factors comprise fibroblast growth factors, epidermal growth factors and stem cell growth factors, and the concentration ratio of the fibroblast growth factors to the epidermal growth factors to the stem cell growth factors is 4:3: 3.
Further, the metal ions include iron ions, sodium ions, potassium ions, zinc ions, and magnesium ions in equal amounts.
A preparation method of a stem cell culture medium comprises the following steps:
s1, taking the additive components with the concentration and DMEM/F12 liquid culture medium;
s2, adding transferrin, insulin, vitamin C, folic acid, glucose, fatty acid, amino acid and ethyl maltol into a DMEM/F12 liquid culture medium, mixing and stirring for 30min, then adding growth factors, stirring again for 12min, finally adding streptomycin, stirring for 12min, and standing for 3.5h to obtain a mixture A;
s3, mixing and stirring the metal ion solutions and the calcium ion solution for 25min, and standing for 1.5h after stirring to obtain a mixture B;
s4, dropwise adding the mixture B into the mixture A through a rubber head dropper, oscillating in the dropwise adding process, and standing after the dropwise adding is finished to obtain a mixture C;
s5, adding a sodium carbonate solution with the mass fraction of 7% into the mixture C, and adjusting the pH of the mixture C to 7.2;
s6, sterilizing the mixture C after the pH is adjusted, wherein the sterilization temperature is 115 ℃, the sterilization time is 12min, and the stem cell culture medium is obtained after sterilization.
Further, the metal ion solution in S3 includes an iron ion solution, a sodium ion solution, a potassium ion solution, a zinc ion solution, and a magnesium ion solution in equal amounts.
Further, the growth factors in S2 include fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor, the epidermal growth factor and the stem cell growth factor is 4:3: 3.
In example 3
A stem cell culture medium and a preparation method thereof comprise a matrix culture medium and additives, wherein the matrix culture medium is a DMEM/F12 liquid culture medium, and the additives and the concentrations thereof added into the matrix culture medium are as follows: transferrin at 12. mu.g/mL, insulin at 3mg/mL, growth factor at 4. mu.g/mL, vitamin C at 10mg/mL, folic acid at 5mg/mL, glucose at 3mg/mL, metal ions at 8. mu.g/mL, calcium ions at 5. mu.g/mL, streptomycin at 0.2mg/mL, fatty acid at 3mg/mL, amino acid at 3mg/mL, and ethyl maltol at 3 mg/mL.
Furthermore, the growth factors comprise fibroblast growth factors, epidermal growth factors and stem cell growth factors, and the concentration ratio of the fibroblast growth factors to the epidermal growth factors to the stem cell growth factors is 4:3: 3.
Further, the metal ions include iron ions, sodium ions, potassium ions, zinc ions, and magnesium ions in equal amounts.
A preparation method of a stem cell culture medium comprises the following steps:
s1, taking the additive components with the concentration and DMEM/F12 liquid culture medium;
s2, adding transferrin, insulin, vitamin C, folic acid, glucose, fatty acid, amino acid and ethyl maltol into a DMEM/F12 liquid culture medium, mixing and stirring for 40min, then adding growth factors, stirring again for 15min, finally adding streptomycin, stirring for 15min, and standing for 4h to obtain a mixture A;
s3, mixing and stirring the metal ion solutions and the calcium ion solution for 30min, and standing for 2h after stirring to obtain a mixture B;
s4, dropwise adding the mixture B into the mixture A through a rubber head dropper, oscillating in the dropwise adding process, and standing after the dropwise adding is finished to obtain a mixture C;
s5, adding a sodium carbonate solution with the mass fraction of 6-8% into the mixture C, and adjusting the pH of the mixture C to 7.4;
s6, sterilizing the mixture C after the pH is adjusted, wherein the sterilization temperature is 120 ℃, the sterilization time is 15min, and the stem cell culture medium is obtained after sterilization.
Further, the metal ion solution in S3 includes an iron ion solution, a sodium ion solution, a potassium ion solution, a zinc ion solution, and a magnesium ion solution in equal amounts.
Further, the growth factors in S2 include fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor, the epidermal growth factor and the stem cell growth factor is 4:3: 3.
Detection of bacteria, fungi, pH indicators according to GMP standards, as shown in table 1:
bacteria | Fungi | pH | |
Example 1 | Negative of | Negative of | 7.0 |
Example 2 | Negative of | Negative of | 7.2 |
Example 3 | Negative of | Negative of | 7.4 |
The culture media prepared in examples 1-3 were tested to meet the criteria.
The cell expansion fold of the medium in examples 1-3 was monitored for three weeks as shown in table 2:
first week | Second week | The third week | |
Example 1 | 144.24 | 1356.92 | 6358.57 |
Example 2 | 143.85 | 1349.84 | 6299.75 |
Example 3 | 145.17 | 1360.77 | 6433.43 |
According to the monitoring of cell expansion in the culture media of examples 1-3, the culture media of the present invention allow the cells to grow and be released continuously and smoothly in the culture media, and allow the cells to be expanded smoothly.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A stem cell culture medium comprises a matrix culture medium and additives, and is characterized in that the matrix culture medium is a DMEM/F12 liquid culture medium, and the additives are added into the matrix culture medium according to the matrix culture medium, wherein the additives comprise the following components in concentration: 9-12 mu g/mL of transferrin, 1-3mg/mL of insulin, 2-4 mu g/mL of growth factor, 8-10mg/mL of vitamin C, 2-5mg/mL of folic acid, 1-3mg/mL of glucose, 6-8 mu g/mL of metal ions, 3-5 mu g/mL of calcium ions, 0.1-0.2mg/mL of streptomycin, 1-3mg/mL of fatty acid, 1-3mg/mL of amino acid and 1-3mg/mL of ethyl maltol.
2. The stem cell culture medium according to claim 1, wherein the growth factors comprise fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor to the epidermal growth factor to the stem cell growth factor is 4:3: 3.
3. The stem cell culture medium of claim 1, wherein the metal ions comprise equal amounts of iron, sodium, potassium, zinc and magnesium ions.
4. A method of preparing the stem cell culture medium of claim 1, comprising the steps of:
s1, taking the additive components with the concentration and DMEM/F12 liquid culture medium;
s2, adding transferrin, insulin, vitamin C, folic acid, glucose, fatty acid, amino acid and ethyl maltol into a DMEM/F12 liquid culture medium, mixing and stirring for 20-40min, then adding growth factors, stirring for 10-15min again, finally adding streptomycin, stirring for 10-15min, and standing for 3-4h to obtain a mixture A;
s3, mixing and stirring the metal ion solutions and the calcium ion solution for 20-30min, and standing for 1-2h after stirring to obtain a mixture B;
s4, dropwise adding the mixture B into the mixture A through a rubber head dropper, oscillating in the dropwise adding process, and standing after the dropwise adding is finished to obtain a mixture C;
s5, adding a sodium carbonate solution with the mass fraction of 6-8% into the mixture C, and adjusting the pH of the mixture C to 7.0-7.4;
s6, sterilizing the mixture C after the pH is adjusted, wherein the sterilization temperature is 110-120 ℃, the sterilization time is 10-15min, and the stem cell culture medium is obtained after sterilization.
5. The method according to claim 4, wherein the metal ion solution in S3 includes equal amounts of iron ion solution, sodium ion solution, potassium ion solution, zinc ion solution and magnesium ion solution.
6. The method according to claim 4, wherein the growth factors in S2 include fibroblast growth factor, epidermal growth factor and stem cell growth factor, and the concentration ratio of the fibroblast growth factor, the epidermal growth factor and the stem cell growth factor is 4:3: 3.
7. The method for preparing the stem cell culture medium according to claim 4, wherein a 7% sodium carbonate solution is added to the mixture C in the S5 by mass fraction, and the pH of the mixture C is adjusted to 7.2.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111069332.1A CN113801837A (en) | 2021-09-13 | 2021-09-13 | Stem cell culture medium and preparation method thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111069332.1A CN113801837A (en) | 2021-09-13 | 2021-09-13 | Stem cell culture medium and preparation method thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113801837A true CN113801837A (en) | 2021-12-17 |
Family
ID=78940952
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111069332.1A Pending CN113801837A (en) | 2021-09-13 | 2021-09-13 | Stem cell culture medium and preparation method thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113801837A (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106434549A (en) * | 2016-12-20 | 2017-02-22 | 江西宜信堂医疗科技有限公司 | Serum-free stem cell culture medium and preparation method thereof |
CN107043699A (en) * | 2017-04-25 | 2017-08-15 | 徐子雁 | A kind of kit of low-energy laser inducing mesenchymal stem cell vascularization |
CN112313329A (en) * | 2018-06-27 | 2021-02-02 | 味之素株式会社 | Additive for stem cell culture, culture medium for stem cell culture, and culture method |
-
2021
- 2021-09-13 CN CN202111069332.1A patent/CN113801837A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106434549A (en) * | 2016-12-20 | 2017-02-22 | 江西宜信堂医疗科技有限公司 | Serum-free stem cell culture medium and preparation method thereof |
CN107043699A (en) * | 2017-04-25 | 2017-08-15 | 徐子雁 | A kind of kit of low-energy laser inducing mesenchymal stem cell vascularization |
CN112313329A (en) * | 2018-06-27 | 2021-02-02 | 味之素株式会社 | Additive for stem cell culture, culture medium for stem cell culture, and culture method |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP3329387B2 (en) | Medium concentrate technology | |
US5122469A (en) | Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins | |
CN102827804B (en) | Culture medium and method suitable for the amplification culture of Vero cell microcarrier suspension | |
US4657866A (en) | Serum-free, synthetic, completely chemically defined tissue culture media | |
CN109337861B (en) | CHO cell serum-free medium supporting high expression of product | |
CN102317440B (en) | Improved culture media additive and process for using it | |
CN112795531B (en) | CHO cell serum-free and protein-free culture medium and application thereof | |
CN107460159A (en) | Serum-free, without albumen supplemented medium and preparation method thereof and use | |
CN104357379B (en) | Stem cell media | |
CN109234223B (en) | Low-protein serum-free cell culture medium | |
CN101760442A (en) | Serum-free medium for MDCK cell large-scale adherent culture and single-cell suspension culture | |
JPH06511389A (en) | animal cell culture | |
KR20070058584A (en) | Culturing human embryonic stem cells | |
CN111518749A (en) | In-vitro culture solution for immature oocyte, preparation method and application thereof | |
CN101418330A (en) | Non protein culture medium adapted to large-scale culture of NSO cell and production of antibody | |
CN105838675A (en) | Hematopoietic stem cell serum-free culture medium | |
CN113801837A (en) | Stem cell culture medium and preparation method thereof | |
CN117511854B (en) | Immature oocyte culture solution and preparation method thereof | |
US20200263120A1 (en) | Method for the culture of photosynthetic organisms using a co2 source | |
CN113913368A (en) | Serum-free medium suitable for CHO cell large-scale suspension amplification culture and preparation and application thereof | |
CN114540274B (en) | Serum-free medium, preparation method thereof and method for culturing 2BS cells by using serum-free medium | |
CN110669730B (en) | Human peripheral blood lymphocyte culture medium | |
JPS5974982A (en) | Cell growth medium replenishing agent and method of growing cell in vitro | |
CN113943694B (en) | Universal serum-free culture medium supporting adherence or suspension culture of various vaccine cells and preparation method thereof | |
CN102653729B (en) | Culture medium used for Chinese hamster ovary cells |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211217 |