CN110117573A - 一种无血清细胞培养基及其应用 - Google Patents
一种无血清细胞培养基及其应用 Download PDFInfo
- Publication number
- CN110117573A CN110117573A CN201910299846.2A CN201910299846A CN110117573A CN 110117573 A CN110117573 A CN 110117573A CN 201910299846 A CN201910299846 A CN 201910299846A CN 110117573 A CN110117573 A CN 110117573A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- serum
- cell culture
- free cell
- content
- 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
- 239000006143 cell culture medium Substances 0.000 title claims abstract description 43
- 239000000654 additive Substances 0.000 claims abstract description 15
- 230000000996 additive effect Effects 0.000 claims abstract description 15
- 239000001963 growth medium Substances 0.000 claims abstract description 14
- 150000001413 amino acids Chemical class 0.000 claims abstract description 12
- 150000003839 salts Chemical class 0.000 claims abstract description 12
- 239000002773 nucleotide Substances 0.000 claims abstract description 11
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 11
- 229940088594 vitamin Drugs 0.000 claims abstract description 11
- 229930003231 vitamin Natural products 0.000 claims abstract description 11
- 235000013343 vitamin Nutrition 0.000 claims abstract description 11
- 239000011782 vitamin Substances 0.000 claims abstract description 11
- 150000003722 vitamin derivatives Chemical class 0.000 claims abstract description 11
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 10
- 239000008367 deionised water Substances 0.000 claims abstract description 6
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 28
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 12
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 12
- 229940024606 amino acid Drugs 0.000 claims description 11
- 235000001014 amino acid Nutrition 0.000 claims description 11
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 10
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 10
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- VCJMYUPGQJHHFU-UHFFFAOYSA-N iron(3+);trinitrate Chemical compound [Fe+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O VCJMYUPGQJHHFU-UHFFFAOYSA-N 0.000 claims description 10
- -1 2- ethoxy Chemical group 0.000 claims description 9
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 9
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 claims description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 8
- 229940031098 ethanolamine Drugs 0.000 claims description 8
- 239000011781 sodium selenite Substances 0.000 claims description 8
- 235000015921 sodium selenite Nutrition 0.000 claims description 8
- 229960001471 sodium selenite Drugs 0.000 claims description 8
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 claims description 7
- 210000004408 hybridoma Anatomy 0.000 claims description 7
- 229960003531 phenolsulfonphthalein Drugs 0.000 claims description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 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 description 6
- 102000002070 Transferrins Human genes 0.000 claims description 6
- 108010015865 Transferrins Proteins 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 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 description 6
- 229940054269 sodium pyruvate Drugs 0.000 claims description 6
- KWTQSFXGGICVPE-UHFFFAOYSA-N 2-amino-5-(diaminomethylideneamino)pentanoic acid;hydron;chloride Chemical compound Cl.OC(=O)C(N)CCCN=C(N)N KWTQSFXGGICVPE-UHFFFAOYSA-N 0.000 claims description 5
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims description 5
- XJZYXTWSMDRIJE-UHFFFAOYSA-N 3,7-dihydropurin-6-one;sodium Chemical compound [Na].O=C1N=CNC2=C1NC=N2 XJZYXTWSMDRIJE-UHFFFAOYSA-N 0.000 claims description 5
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 235000019743 Choline chloride Nutrition 0.000 claims description 5
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 5
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 5
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 5
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 5
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 claims description 5
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 5
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 5
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 5
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 claims description 5
- 229960005261 aspartic acid Drugs 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 239000011575 calcium Substances 0.000 claims description 5
- 229910052791 calcium Inorganic materials 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 229960003178 choline chloride Drugs 0.000 claims description 5
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 5
- 210000004907 gland Anatomy 0.000 claims description 5
- 229960000367 inositol Drugs 0.000 claims description 5
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- 229960005190 phenylalanine Drugs 0.000 claims description 5
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 5
- 229960000344 thiamine hydrochloride Drugs 0.000 claims description 5
- 235000019190 thiamine hydrochloride Nutrition 0.000 claims description 5
- 239000011747 thiamine hydrochloride Substances 0.000 claims description 5
- 239000004474 valine Substances 0.000 claims description 5
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 5
- 229960001763 zinc sulfate Drugs 0.000 claims description 5
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 5
- QZNNVYOVQUKYSC-JEDNCBNOSA-N (2s)-2-amino-3-(1h-imidazol-5-yl)propanoic acid;hydron;chloride Chemical compound Cl.OC(=O)[C@@H](N)CC1=CN=CN1 QZNNVYOVQUKYSC-JEDNCBNOSA-N 0.000 claims description 4
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 claims description 4
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 4
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 4
- 229930182816 L-glutamine Natural products 0.000 claims description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 4
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- 241000218636 Thuja Species 0.000 claims description 4
- ASIYFCYUCMQNGK-JZGIKJSDSA-L disodium L-tyrosinate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CC1=CC=C([O-])C=C1 ASIYFCYUCMQNGK-JZGIKJSDSA-L 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 claims description 4
- 235000020778 linoleic acid Nutrition 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 229960002477 riboflavin Drugs 0.000 claims description 4
- 235000019192 riboflavin Nutrition 0.000 claims description 4
- 239000002151 riboflavin Substances 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 claims description 3
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 claims description 3
- 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 description 3
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 3
- 229930003779 Vitamin B12 Natural products 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 claims description 3
- 229960000304 folic acid Drugs 0.000 claims description 3
- 235000019152 folic acid Nutrition 0.000 claims description 3
- 239000011724 folic acid Substances 0.000 claims description 3
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 3
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 3
- 229960003966 nicotinamide Drugs 0.000 claims description 3
- 235000005152 nicotinamide Nutrition 0.000 claims description 3
- 239000011570 nicotinamide Substances 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- 239000011715 vitamin B12 Substances 0.000 claims description 3
- 235000019163 vitamin B12 Nutrition 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 2
- 239000005864 Sulphur Substances 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 2
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims 1
- 210000000496 pancreas Anatomy 0.000 claims 1
- 230000001737 promoting effect Effects 0.000 claims 1
- 230000006798 recombination Effects 0.000 claims 1
- 238000005215 recombination Methods 0.000 claims 1
- 239000000306 component Substances 0.000 abstract description 11
- 210000002966 serum Anatomy 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 10
- 238000004113 cell culture Methods 0.000 abstract description 9
- 230000004663 cell proliferation Effects 0.000 abstract description 6
- 230000001413 cellular effect Effects 0.000 abstract description 4
- 239000012534 cell culture medium component Substances 0.000 abstract description 2
- 238000009472 formulation Methods 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 13
- 238000001514 detection method Methods 0.000 description 11
- 238000000034 method Methods 0.000 description 6
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 4
- 229930195722 L-methionine Natural products 0.000 description 4
- 239000004473 Threonine Substances 0.000 description 4
- 239000011790 ferrous sulphate Substances 0.000 description 4
- 235000003891 ferrous sulphate Nutrition 0.000 description 4
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 4
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 4
- 229960004452 methionine Drugs 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 229960002898 threonine Drugs 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- LFDGRWDETVOGDT-UHFFFAOYSA-N 1h-pyrrole;hydrochloride Chemical compound Cl.C=1C=CNC=1 LFDGRWDETVOGDT-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940046414 biotin 1 mg Drugs 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- WGIAYBNWTYECJD-UHFFFAOYSA-N 1-ethoxypiperazine Chemical compound CCON1CCNCC1 WGIAYBNWTYECJD-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- BRPMGWKECPTJGE-RGMNGODLSA-N Cl.C(CC)N[C@@H](CCO)C(=O)O Chemical compound Cl.C(CC)N[C@@H](CCO)C(=O)O BRPMGWKECPTJGE-RGMNGODLSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 101100285000 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) his-3 gene Proteins 0.000 description 1
- 241001416149 Ovis ammon Species 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229940057742 cysteine 50 mg Drugs 0.000 description 1
- 235000015177 dried meat Nutrition 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 229940083563 folic acid 1 mg Drugs 0.000 description 1
- 229940106305 folic acid 20 mg Drugs 0.000 description 1
- 229940026527 glycine 50 mg Drugs 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229940000065 linoleic acid 10 mg Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940001087 magnesium sulfate 10 mg Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229940088175 niacinamide 10 mg Drugs 0.000 description 1
- 229940089784 niacinamide 20 mg Drugs 0.000 description 1
- 229940015378 potassium chloride 500 mg Drugs 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- 229940052714 riboflavin 1 mg Drugs 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229940087379 sodium bicarbonate 500 mg Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 229940084609 vitamin b 12 0.5 mg Drugs 0.000 description 1
- 229940084606 vitamin b 12 1 mg Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer 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
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
-
- 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
-
- 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
- C12N2500/38—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
- C12N2500/46—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
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及细胞培养技术领域，具体公开一种无血清细胞培养基及其应用，培养基中各组分含量为：碳水化合物、氨基酸、核苷酸、维生素、无机盐、添加物和去离子水。本发明的无血清细胞培养基组分明确、配方成本低，细胞增殖速度快、活性高，得到的细胞产物产量和纯度高，可完全替代传统含血清的细胞培养基。
Description
技术领域
本发明涉及细胞培养技术领域，尤其涉及一种无血清细胞培养基及其应用。
背景技术
细胞培养技术已经经历了几十年的发展，其广泛应用于生物、医疗和药品等技术领域，具有较高的应用价值，且随着产品分类越来越细化，对细胞培养基的分类和要求也越来越精细，其中通过细胞培养来获得蛋白药物和抗体已经成为医药行业发展的重要方向，而细胞培养基的选择不仅影响细胞的生长和增殖速度，还影响着细胞产物的合成和分离。目前细胞培养用的培养基都普遍添加有动物血清，而动物血清中的成分复杂并且含有外源IgG，导致目标产物分离纯化困难，对产物的品质产生影响，且增加目标产物的生产成本。
发明内容
针对现有细胞培养基细胞增殖速度慢、目标产物合成效率低以及因添加动物血清影响目标产物品质等问题，本发明提供一种无血清细胞培养基及其应用。
为达到上述发明目的，本发明实施例采用了如下的技术方案：
一种无血清细胞培养基，培养基中各组分含量为：2000-5500mg/L的碳水化合物、300-4800mg/L的氨基酸、0.1-55mg/L的核苷酸、8-150mg/L维生素、1060-23000mg/L无机盐、2100-11200mg/L添加物和去离子水；
所述添加物包括酚红、N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸、重组胰岛素、转铁蛋白、牛血清白蛋白、亚硒酸钠和乙醇胺。
相对于现有技术，本发明提供的无血清细胞培养基组分明确、配方成本低，细胞培养过程中，细胞体外增殖速度快、活性高，得到的细胞产物产量和纯度高，可完全替代传统含血清的细胞培养基，同时又避免了因血清的加入而导致得到的细胞产物无法分离进而影响产物品质及增加产物的分离成本。
添加剂中重组胰岛素、转铁蛋白和乙醇胺结合配合添加物中的N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸和亚硒酸钠可代替培养基中血清提供的营养物质，其细胞培养中的细胞增殖效果和产物合成效率甚至优于血清的加入。
优选地，所述碳水化合物中包含的组分及各组分在培养基中的含量为：D-葡萄糖2000-5000mg/L、丙酮酸钠5-300mg/L、亚油酸0.01-20mg/L。
D-葡萄糖作为主要的碳源，配合加入少量的丙酮酸钠和亚油酸，可以加快细胞的增殖速度，保持细胞的生长活性。
优选地，所述氨基酸包含的种类及各种类在培养基中的含量为：甘氨酸10-100mg/L、L-丙氨酸1-20mg/L、L-精氨酸盐酸盐10-500mg/L、L-天冬酰胺1-20mg/L、L-天冬氨酸1-20mg/L、L-半胱氨酸盐酸盐10-100mg/L、L-胱氨酸10-100mg/L、L-谷氨酸1-20mg/L、L-谷氨酰胺100-2000mg/L、L-组氨酸盐酸盐10-100mg/L、L-异亮氨酸20-200mg/L、L-亮氨酸20-200mg/L、L-赖氨酸盐酸盐20-200mg/L、L-蛋氨酸10-100mg/L、L-苯丙氨酸20-200mg/L、L-脯氨酸10-100mg/L、L-丝氨酸10-100mg/L、L-苏氨酸20-200mg/L、L-色氨酸1-30mg/L、L-酪氨酸二钠盐20-200mg/L、L-缬氨酸20-200mg/L。
上述氨基酸的种类和含量的设置，可使细胞在培养基中高效合成活性产物。
优选地，所述核苷酸包含的种类及各种类在培养基中的含量为：腺嘧啶脱氧核苷酸0.001-1mg/L、次黄嘌呤钠0.1-50mg/L。
优选地，所述维生素包含的种类及各种类在培养基中的含量为：生物素0.001-1mg/L、氯化胆碱1-20mg/L、D-泛酸钙1-20mg/L、叶酸1-20mg/L、烟酰胺1-20mg/L、盐酸吡哆醇1-20mg/L、核黄素0.001-1mg/L、盐酸硫胺素1-20mg/L、维生素B12 0.001-1mg/L、肌醇1-20mg/L。
优选地，所述无机盐包含的组分及各组分在培养基中的含量为：氯化钙10-500mg/L、硫酸铜0.001-1mg/L、硝酸铁0.001-1mg/L、硫酸亚铁0.001-1mg/L、氯化镁10-500mg/L、硫酸镁10-500mg/L、氯化钾10-500mg/L、碳酸氢钠500-10000mg/L、氯化钠500-10000mg/L、磷酸氢二钠10-500mg/L、磷酸二氢钠10-500mg/L、硫酸锌0.001-1mg/L。
优选地，所述添加物中各组分在培养基中的含量为：酚红1-50mg/L、N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸2000-8000mg/L、重组胰岛素1-50mg/L、转铁蛋白1-50mg/L、牛血清白蛋白50-3000mg/L、亚硒酸钠1-50μg/L、乙醇胺1-30mg/L。
本发明还提供所述的无血清细胞培养基在促进细胞产生单克隆抗体中的应用。
相对于现有技术，本发明提供的无血清细胞培养基在瘤细胞培养过程中，可保证瘤细胞增殖的稳定性，促进瘤细胞短时间内产生大量单克隆抗体。
优选地，所述无血清细胞培养基用于培养骨髓瘤细胞以促进其产生单克隆抗体。
优选地，所述无血清细胞培养基用于培养杂交瘤细胞以促进其产生单克隆抗体。
附图说明
图1是本发明实施例1的无血清细胞培养基培养5H2细胞过程中培养不同天数检测的抗体浓度；
图2是本发明实施例2的无血清细胞培养基培养5H2细胞过程中培养不同天数检测的抗体浓度；
图3是本发明实施例3的无血清细胞培养基培养5H2细胞过程中培养不同天数检测的抗体浓度；
图4是对比例1中H-SFM无血清细胞培养基培养5H2细胞过程中培养不同天数检测的抗体浓度。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例1
配置1L无血清细胞培养基，分别加入2000mg的碳水化合物、300mg的氨基酸、0.1mg的核苷酸、8mg维生素、1060mg无机盐、2100mg添加物和余量的去离子水。
其中碳水化合物中D-葡萄糖2000mg、丙酮酸钠5mg、亚油酸0.01mg；
氨基酸中甘氨酸10mg、L-丙氨酸1mg、L-精氨酸盐酸盐10mg、L-天冬酰胺1mg、L-天冬氨酸1mg、L-半胱氨酸盐酸盐10mg、L-胱氨酸10mg、L-谷氨酸1mg、L-谷氨酰胺100mg、L-组氨酸盐酸盐10mg、L-异亮氨酸20mg、L-亮氨酸20mg、L-赖氨酸盐酸盐20mg、L-蛋氨酸10mg、L-苯丙氨酸20mg、L-脯氨酸10mg、L-丝氨酸10mg、L-苏氨酸20mg、L-色氨酸1mg、L-酪氨酸二钠盐20mg、L-缬氨酸20mg；
核苷酸中腺嘧啶脱氧核苷酸0.001mg、次黄嘌呤钠0.1mg；
维生素中生物素1mg/L、氯化胆碱1mg、D-泛酸钙1mg、叶酸1mg、烟酰胺1mg、盐酸吡哆醇1mg、核黄素0.001mg、盐酸硫胺素1mg、维生素B120.001mg、肌醇1mg；
无机盐中氯化钙10mg、硫酸铜0.001mg、硝酸铁0.001mg、硫酸亚铁0.001mg、氯化镁10mg、硫酸镁10mg、氯化钾10mg、碳酸氢钠500mg、氯化钠500mg、磷酸氢二钠10mg、磷酸二氢钠10mg、硫酸锌0.001mg；
添加物中酚红1mg、N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸2000mg、重组胰岛素1mg、转铁蛋白50mg、牛血清白蛋白50mg、亚硒酸钠1μg、乙醇胺1mg。
将上述各组分混合均匀并充分溶解，得到无血清细胞培养基。
将生长状态良好、处于对数期的杂交瘤细胞株(5H2)以3×105个/ml接种浓度分别接种到实施例1的无血清细胞培养基中，初始接种体积为100ml，培养于37℃、5％CO2培养箱中，2天后补充培养基50ml，4天后补充培养基50ml，7天收取全部培养基，每天取样用细胞计数仪进行细胞数量和活力的分析，同时测定培养基上清中抗体含量。
其中，抗体含量的测定方法为：
(1)准备待测样品：用缓冲溶液将待测样品稀释200倍，每孔加入100μl，37℃放置45min；
(2)加酶标二抗：每孔加入100μl的l:10000稀释的HRP酶标记的山羊抗鼠IgG，37℃放置30min；
(3)显色：每孔加底物显色液100μl，37℃保温避光反应15min；
(4)终止反应：每孔加入50μl终止液；
(5)测定OD450nm值：用检测波长为450nm的酶标仪读取各孔光密度值，根据标准曲线来确定待测样品的抗体含量，培养不同天数得到的抗体浓度折线图如图1所示。
检测具体结果如下表所示：
实施例2
配置1L无血清细胞培养基，分别加入2610mg的碳水化合物、2100mg的氨基酸、25.5mg的核苷酸、71.5mg维生素、11200mg无机盐、7090mg添加物和余量的去离子水。
其中碳水化合物中D-葡萄糖2500mg、丙酮酸钠100mg、亚油酸10mg；
氨基酸中甘氨酸50mg、L-丙氨酸10mg、L-精氨酸盐酸盐50mg、L-天冬酰胺10mg、L-天冬氨酸10mg、L-半胱氨酸盐酸盐50mg、L-胱氨酸50mg、L-谷氨酸10mg、L-谷氨酰胺1000mg、L-组氨酸盐酸盐50mg、L-异亮氨酸100mg、L-亮氨酸100mg、L-赖氨酸盐酸盐50mg、L-蛋氨酸50mg、L-苯丙氨酸100mg、L-脯氨酸50mg、L-丝氨酸50mg、L-苏氨酸100mg、L-色氨酸10mg、L-酪氨酸二钠盐100mg、L-缬氨酸100mg；
核苷酸中腺嘧啶脱氧核苷酸0.5mg、次黄嘌呤钠25mg；
维生素中生物素0.5mg/L、氯化胆碱10mg、D-泛酸钙10mg、叶酸10mg、烟酰胺10mg、盐酸吡哆醇10mg、核黄素0.5mg、盐酸硫胺素10mg、维生素B12 0.5mg、肌醇10mg；
无机盐中氯化钙200mg、硫酸铜0.5mg、硝酸铁0.5mg、硫酸亚铁0.5mg、氯化镁200mg、硫酸镁200mg、氯化钾200mg、碳酸氢钠5000mg、氯化钠5000mg、磷酸氢二钠200mg、磷酸二氢钠200mg、硫酸锌0.5mg；
添加物中酚红25mg、N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸5000mg、重组胰岛素25mg、转铁蛋白25mg、牛血清白蛋白2000mg、亚硒酸钠25μg、乙醇胺15mg。
将上述各组分混合均匀并充分溶解，得到无血清细胞培养基。
用实施例1中的细胞培养方法和检测方法对实施例2得到的无血清细胞培养基培养的杂交瘤细胞株(5H2)的细胞活性及培养基中抗体含量进行检测，培养不同天数得到的抗体浓度折线图如图2所示，检测具体结果如下表所示：
| 发酵时间d | 细胞数10<sup>5</sup>个/ml | 细胞活率 | 抗体浓度ug/ml |
| 0 | 3 | 93.71％ | 0 |
| 1 | 5.81 | 89.40％ | 5.17 |
| 2 | 12.11 | 88.79％ | 12.36 |
| 3 | 12.95 | 87.44％ | 14.88 |
| 4 | 18.97 | 84.01％ | 21.43 |
| 5 | 19.09 | 74.15％ | 29.92 |
| 6 | 20.07 | 65.44％ | 40.96 |
| 7 | 19.86 | 54.27％ | 59.16 |
实施例3
配置1L无血清细胞培养基，分别加入5320mg的碳水化合物、4710mg的氨基酸、51mg的核苷酸、143mg维生素、23000mg无机盐、11130mg添加物和余量的去离子水。
其中碳水化合物中D-葡萄糖5000mg、丙酮酸钠300mg、亚油酸20mg；
氨基酸中甘氨酸100mg、L-丙氨酸20mg、L-精氨酸盐酸盐500mg、L-天冬酰胺20mg、L-天冬氨酸20mg、L-半胱氨酸盐酸盐100mg、L-胱氨酸100mg、L-谷氨酸20mg、L-谷氨酰胺2000mg、L-组氨酸盐酸盐100mg、L-异亮氨酸200mg、L-亮氨酸200mg、L-赖氨酸盐酸盐200mg、L-蛋氨酸100mg、L-苯丙氨酸200mg、L-脯氨酸100mg、L-丝氨酸100mg、L-苏氨酸200mg、L-色氨酸30mg、L-酪氨酸二钠盐200mg、L-缬氨酸200mg；
核苷酸中腺嘧啶脱氧核苷酸1mg、次黄嘌呤钠50mg；
维生素中生物素1mg/L、氯化胆碱20mg、D-泛酸钙20mg、叶酸20mg、烟酰胺20mg、盐酸吡哆醇20mg、核黄素1mg、盐酸硫胺素20mg、维生素B12 1mg、肌醇20mg；
无机盐中氯化钙500mg、硫酸铜1mg、硝酸铁1mg、硫酸亚铁1mg、氯化镁500mg、硫酸镁500mg、氯化钾500mg、碳酸氢钠10000mg、氯化钠10000mg、磷酸氢二钠500mg、磷酸二氢钠500mg、硫酸锌1mg；
添加物中酚红50mg、N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸8000mg、重组胰岛素50mg、转铁蛋白1mg、牛血清白蛋白3000mg、亚硒酸钠50μg、乙醇胺30mg。
将上述各组分混合均匀并充分溶解，得到无血清细胞培养基。
用实施例1中的细胞培养方法和检测方法对实施例2得到的无血清细胞培养基培养的杂交瘤细胞株(5H2)的细胞活性及培养基中抗体含量进行检测，培养不同天数得到的抗体浓度折线图如图3所示，检测具体结果如下表所示：
| 发酵时间d | 细胞数10<sup>5</sup>个/ml | 细胞活率 | 抗体浓度ug/ml |
| 0 | 3.29 | 91.44％ | 0 |
| 1 | 5.33 | 82.61％ | 5.11 |
| 2 | 10.88 | 84.66％ | 11.36 |
| 3 | 13.10 | 85.97％ | 12.33 |
| 4 | 18.98 | 82.01％ | 22.51 |
| 5 | 19.39 | 72.58％ | 35.22 |
| 6 | 19.87 | 65.35％ | 40.28 |
| 7 | 16.17 | 50.17％ | 58.31 |
实施例4
用实施例1中的无血清细胞培养基和购买的CDM4、EX-CELL、H-SFM三种培养基来对5H2细胞进行无血清培养，在这4种培养基中均无胎牛血清的加入，将生长状态良好、处于对数期的5H2细胞以1.5×105个/ml接种浓度接种到含上述四种培养基的细胞培养板中，培养于37℃、5％CO2培养箱中。用细胞计数仪进行细胞增殖活力的分析，结果见下表：
从表中数据可以看出，在不加血清时，细胞不能在EX-CELL培养基中正常存活，其他3种培养基均能支持5H2细胞的生长与增殖，但实施例1的无血清细胞培养基的效果在4种培养基中是最好的，细胞的密度最大，经过6天培养细胞密度可达到12.88×105个/ml。
对比例1
用实施例1中的检测方法对目前常用的H-SFM无血清细胞培养基培养的杂交瘤细胞株(5H2)的细胞活性及培养基中抗体含量进行检测，培养不同天数得到的抗体浓度折线图如图4所示，检测具体结果如下表所示：
以上培养结果表明，本发明实施例1-3的无血清细胞培养基培养的杂交瘤细胞株(5H2)的细胞增殖速度、细胞活性以及产生的抗体含量都明显高于H-SFM培养基，达到甚至优于含血清的细胞培养基。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换或改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种无血清细胞培养基，其特征在于：培养基中各组分含量为：2000-5500mg/L的碳水化合物、300-4800mg/L的氨基酸、0.1-55mg/L的核苷酸、8-150mg/L维生素、1060-23000mg/L无机盐、2100-11200mg/L添加物和去离子水；
所述添加物包括酚红、N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸、重组胰岛素、转铁蛋白、牛血清白蛋白、亚硒酸钠和乙醇胺。
2.如权利要求1所述的无血清细胞培养基，其特征在于：所述碳水化合物中包含的组分及各组分在培养基中的含量为：D-葡萄糖2000-5000mg/L、丙酮酸钠5-300mg/L、亚油酸0.01-20mg/L。
3.如权利要求1所述的无血清细胞培养基，其特征在于：所述氨基酸包含的种类及各种类在培养基中的含量为：甘氨酸10-100mg/L、L-丙氨酸1-20mg/L、L-精氨酸盐酸盐10-500mg/L、L-天冬酰胺1-20mg/L、L-天冬氨酸1-20mg/L、L-半胱氨酸盐酸盐10-100mg/L、L-胱氨酸10-100mg/L、L-谷氨酸1-20mg/L、L-谷氨酰胺100-2000mg/L、L-组氨酸盐酸盐10-100mg/L、L-异亮氨酸20-200mg/L、L-亮氨酸20-200mg/L、L-赖氨酸盐酸盐20-200mg/L、L-蛋氨酸10-100mg/L、L-苯丙氨酸20-200mg/L、L-脯氨酸10-100mg/L、L-丝氨酸10-100mg/L、L-苏氨酸20-200mg/L、L-色氨酸1-30mg/L、L-酪氨酸二钠盐20-200mg/L、L-缬氨酸20-200mg/L。
4.如权利要求1所述的无血清细胞培养基，其特征在于：所述核苷酸包含的种类及各种类在培养基中的含量为：腺嘧啶脱氧核苷酸0.001-1mg/L、次黄嘌呤钠0.1-50mg/L。
5.如权利要求1所述的无血清细胞培养基，其特征在于：所述维生素包含的种类及各种类在培养基中的含量为：生物素0.001-1mg/L、氯化胆碱1-20mg/L、D-泛酸钙1-20mg/L、叶酸1-20mg/L、烟酰胺1-20mg/L、盐酸吡哆醇1-20mg/L、核黄素0.001-1mg/L、盐酸硫胺素1-20mg/L、维生素B12 0.001-1mg/L、肌醇1-20mg/L。
6.如权利要求1所述的无血清细胞培养基，其特征在于：所述无机盐包含的组分及各组分在培养基中的含量为：氯化钙10-500mg/L、硫酸铜0.001-1mg/L、硝酸铁0.001-1mg/L、硫酸亚铁0.001-1mg/L、氯化镁10-500mg/L、硫酸镁10-500mg/L、氯化钾10-500mg/L、碳酸氢钠500-10000mg/L、氯化钠500-10000mg/L、磷酸氢二钠10-500mg/L、磷酸二氢钠10-500mg/L、硫酸锌0.001-1mg/L。
7.如权利要求1所述的无血清细胞培养基，其特征在于：所述添加物中各组分在培养基中的含量为：酚红1-50mg/L、N-(2-羟乙基)哌嗪-N'-2-乙烷磺酸2000-8000mg/L、重组胰岛素1-50mg/L、转铁蛋白1-50mg/L、牛血清白蛋白50-3000mg/L、亚硒酸钠1-50μg/L、乙醇胺1-30mg/L。
8.权利要求1-7所述的无血清细胞培养基在促进细胞产生单克隆抗体中的应用。
9.如权利要求8所述的应用，其特征在于：所述无血清细胞培养基用于培养骨髓瘤细胞以促进其产生单克隆抗体。
10.如权利要求8所述的应用，其特征在于：所述无血清细胞培养基用于培养杂交瘤细胞以促进其产生单克隆抗体。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910299846.2A CN110117573A (zh) | 2019-04-15 | 2019-04-15 | 一种无血清细胞培养基及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910299846.2A CN110117573A (zh) | 2019-04-15 | 2019-04-15 | 一种无血清细胞培养基及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110117573A true CN110117573A (zh) | 2019-08-13 |
Family
ID=67521005
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910299846.2A Pending CN110117573A (zh) | 2019-04-15 | 2019-04-15 | 一种无血清细胞培养基及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110117573A (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226416A (zh) * | 2020-10-26 | 2021-01-15 | 通山县金瑞生物科技研发中心 | 一种杂交瘤细胞培养用培养基添加剂 |
| CN112300985A (zh) * | 2020-11-03 | 2021-02-02 | 中国人民解放军陆军军医大学第一附属医院 | 一种快速提升哺乳类动物骨髓间充质干细胞增殖效率的增殖培养基及增殖培养方法 |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1229851A (zh) * | 1999-03-02 | 1999-09-29 | 华东理工大学 | 杂交瘤细胞无血清培养基 |
| CN1778902A (zh) * | 2005-09-29 | 2006-05-31 | 华东理工大学 | 适用于多种动物细胞大规模培养的无血清培养基 |
| CN101195817A (zh) * | 2007-12-28 | 2008-06-11 | 天津百若克医药生物技术有限责任公司 | 一种杂交瘤细胞扩增培养基及其用途 |
| CN101613676A (zh) * | 2009-07-27 | 2009-12-30 | 中国科学院生物物理研究所 | 一种鼠骨髓瘤细胞nso及其制备方法与应用 |
| CN104087558A (zh) * | 2014-07-08 | 2014-10-08 | 西藏天虹科技股份有限责任公司 | 一种杂交瘤细胞无血清培养基 |
| CN105462924A (zh) * | 2015-12-04 | 2016-04-06 | 广州赛莱拉干细胞科技股份有限公司 | Nk细胞的培养方法及无血清培养基组合 |
| CN106190985A (zh) * | 2015-04-29 | 2016-12-07 | 李昂 | 杂交瘤细胞无血清培养液 |
| CN107099508A (zh) * | 2017-06-23 | 2017-08-29 | 曲宝赤 | 一种无血清杂交瘤细胞培养基 |
| CN107881142A (zh) * | 2017-12-12 | 2018-04-06 | 成都源泉生物科技有限公司 | 一种杂交瘤细胞无血清培养基 |
| CN108330101A (zh) * | 2017-11-21 | 2018-07-27 | 山东农业大学 | 一种杂交瘤细胞株及其产生的单克隆抗体和应用 |
| CN109234223A (zh) * | 2018-11-21 | 2019-01-18 | 南京基蛋生物医药有限公司 | 低蛋白无血清细胞培养基 |
| CN109265539A (zh) * | 2018-10-12 | 2019-01-25 | 浙江正熙生物医药有限公司 | 利用杂交瘤细胞生产单克隆抗体的方法及其生产的单克隆抗体 |
-
2019
- 2019-04-15 CN CN201910299846.2A patent/CN110117573A/zh active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1229851A (zh) * | 1999-03-02 | 1999-09-29 | 华东理工大学 | 杂交瘤细胞无血清培养基 |
| CN1778902A (zh) * | 2005-09-29 | 2006-05-31 | 华东理工大学 | 适用于多种动物细胞大规模培养的无血清培养基 |
| CN101195817A (zh) * | 2007-12-28 | 2008-06-11 | 天津百若克医药生物技术有限责任公司 | 一种杂交瘤细胞扩增培养基及其用途 |
| CN101613676A (zh) * | 2009-07-27 | 2009-12-30 | 中国科学院生物物理研究所 | 一种鼠骨髓瘤细胞nso及其制备方法与应用 |
| CN104087558A (zh) * | 2014-07-08 | 2014-10-08 | 西藏天虹科技股份有限责任公司 | 一种杂交瘤细胞无血清培养基 |
| CN106190985A (zh) * | 2015-04-29 | 2016-12-07 | 李昂 | 杂交瘤细胞无血清培养液 |
| CN105462924A (zh) * | 2015-12-04 | 2016-04-06 | 广州赛莱拉干细胞科技股份有限公司 | Nk细胞的培养方法及无血清培养基组合 |
| CN107099508A (zh) * | 2017-06-23 | 2017-08-29 | 曲宝赤 | 一种无血清杂交瘤细胞培养基 |
| CN108330101A (zh) * | 2017-11-21 | 2018-07-27 | 山东农业大学 | 一种杂交瘤细胞株及其产生的单克隆抗体和应用 |
| CN107881142A (zh) * | 2017-12-12 | 2018-04-06 | 成都源泉生物科技有限公司 | 一种杂交瘤细胞无血清培养基 |
| CN109265539A (zh) * | 2018-10-12 | 2019-01-25 | 浙江正熙生物医药有限公司 | 利用杂交瘤细胞生产单克隆抗体的方法及其生产的单克隆抗体 |
| CN109234223A (zh) * | 2018-11-21 | 2019-01-18 | 南京基蛋生物医药有限公司 | 低蛋白无血清细胞培养基 |
Non-Patent Citations (4)
| Title |
|---|
| 周维松等: "用于骨髓瘤细胞和杂交瘤细胞增殖以及杂交瘤细胞抗体产生的无血清培养基", 《中国农业科学》 * |
| 李黎等: "无血清培养基在杂交瘤细胞培养中的应用", 《国际生物制品学杂志》 * |
| 杜世彧: "一种"无血清"培养基对肿瘤细胞生长的支持", 《生物技术》 * |
| 王祎: "动物无血清细胞培养基的研究现状及进展", 《科教文汇(下旬刊)》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226416A (zh) * | 2020-10-26 | 2021-01-15 | 通山县金瑞生物科技研发中心 | 一种杂交瘤细胞培养用培养基添加剂 |
| CN112300985A (zh) * | 2020-11-03 | 2021-02-02 | 中国人民解放军陆军军医大学第一附属医院 | 一种快速提升哺乳类动物骨髓间充质干细胞增殖效率的增殖培养基及增殖培养方法 |
| CN112300985B (zh) * | 2020-11-03 | 2022-07-01 | 中国人民解放军陆军军医大学第一附属医院 | 一种快速提升哺乳类动物骨髓间充质干细胞增殖效率的增殖培养基及增殖培养方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2644651C2 (ru) | Среда для культивирования клеток | |
| CN105462924B (zh) | Nk细胞的培养方法及无血清培养基组合 | |
| CN104450614B (zh) | 一种无动物蛋白源的免疫细胞无血清培养基及其使用方法 | |
| JP2783294B2 (ja) | 増強された細胞増殖、培養寿命および生産物発現のための細胞培養培地 | |
| Kishishita et al. | Optimization of chemically defined feed media for monoclonal antibody production in Chinese hamster ovary cells | |
| CN102858953B (zh) | 改进的细胞培养基 | |
| CN113088480B (zh) | 一种用于cho细胞的培养基及其用途 | |
| CN101760442A (zh) | 适于mdck细胞大规模贴壁培养和单细胞悬浮培养的无血清培养基 | |
| CN109294976A (zh) | 一种支持hek293细胞悬浮培养的无血清培养基 | |
| JPS63267269A (ja) | 細胞培養のための基礎栄養培地 | |
| KR101594046B1 (ko) | 개선된 배양 배지 첨가제 및 이를 이용한 방법 | |
| Kang et al. | Utilization of tyrosine‐and histidine‐containing dipeptides to enhance productivity and culture viability | |
| CN1962857A (zh) | 一种哺乳动物细胞无血清培养基 | |
| CN104073463B (zh) | 一种支持cho高密度悬浮培养的无血清无蛋白培养基 | |
| RU2013125777A (ru) | Эффективный и экономичный скрининг добавок для создания сред определенного химического состава при культивировании клеток | |
| CN110117573A (zh) | 一种无血清细胞培养基及其应用 | |
| CN101418330A (zh) | 适于nso细胞大规模培养及抗体生产的无蛋白培养基 | |
| Linz et al. | Stoichiometry, kinetics, and regulation of glucose and amino acid metabolism of a recombinant BHK cell line in batch and continuous cultures | |
| Brinster et al. | Requirements for blastocyst development in vitro | |
| CN107881143A (zh) | 一种cho细胞无血清培养基 | |
| JPS5863385A (ja) | 免疫系由来の細胞用培地 | |
| Sanfeliu et al. | Analysis of nutritional factors and physical conditions affecting growth and monoclonal antibody production of the hybridoma KB‐26.5 cell line | |
| CN107236708A (zh) | 一种支持HeLa细胞贴壁培养的无血清培养基 | |
| CN108949696A (zh) | 免疫细胞培养基与其应用 | |
| CN105087460A (zh) | 一种st细胞培养基 |
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 | ||
| CB03 | Change of inventor or designer information |
Inventor after: Cheng Hua Inventor after: Sun Jinchong Inventor after: Wu Xiangli Inventor after: Han Wei Inventor before: Cheng Hua Inventor before: Sun Jinchong Inventor before: Wu Xiangli Inventor before: Han Wei |
|
| CB03 | Change of inventor or designer information | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190813 |
|
| RJ01 | Rejection of invention patent application after publication |