IE921020A1 - Serum-free medium for culturing mammalian cells - Google Patents
Serum-free medium for culturing mammalian cellsInfo
- Publication number
- IE921020A1 IE921020A1 IE102092A IE921020A IE921020A1 IE 921020 A1 IE921020 A1 IE 921020A1 IE 102092 A IE102092 A IE 102092A IE 921020 A IE921020 A IE 921020A IE 921020 A1 IE921020 A1 IE 921020A1
- Authority
- IE
- Ireland
- Prior art keywords
- medium according
- medium
- culturing
- mammalian cells
- erythropoietin
- Prior art date
Links
- 210000004962 mammalian cell Anatomy 0.000 title claims abstract description 15
- 239000012679 serum free medium Substances 0.000 title claims abstract description 10
- 238000012258 culturing Methods 0.000 title claims description 25
- 239000002609 medium Substances 0.000 claims abstract description 41
- 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 32
- 102000004877 Insulin Human genes 0.000 claims abstract description 16
- 108090001061 Insulin Proteins 0.000 claims abstract description 16
- 229940125396 insulin Drugs 0.000 claims abstract description 16
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims abstract description 15
- 239000000463 material Substances 0.000 claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 150000002506 iron compounds Chemical class 0.000 claims abstract description 12
- 241001465754 Metazoa Species 0.000 claims abstract description 9
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 17
- 102000003951 Erythropoietin Human genes 0.000 claims description 12
- 108090000394 Erythropoietin Proteins 0.000 claims description 12
- 229940105423 erythropoietin Drugs 0.000 claims description 12
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims description 12
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 9
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 9
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 102000004338 Transferrin Human genes 0.000 claims description 6
- 108090000901 Transferrin Proteins 0.000 claims description 6
- 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 description 6
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 claims description 6
- 229920000609 methyl cellulose Polymers 0.000 claims description 6
- 239000001923 methylcellulose Substances 0.000 claims description 6
- 235000010981 methylcellulose Nutrition 0.000 claims description 6
- 239000007793 ph indicator Substances 0.000 claims description 6
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 claims description 6
- 239000012581 transferrin Substances 0.000 claims description 6
- 235000001014 amino acid Nutrition 0.000 claims description 5
- 229940024606 amino acid Drugs 0.000 claims description 5
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 235000018102 proteins Nutrition 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 5
- 235000013343 vitamin Nutrition 0.000 claims description 5
- 239000011782 vitamin Substances 0.000 claims description 5
- 229930003231 vitamin Natural products 0.000 claims description 5
- 229940088594 vitamin Drugs 0.000 claims description 5
- 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 4
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 4
- 229930195729 fatty acid Natural products 0.000 claims description 4
- 239000000194 fatty acid Substances 0.000 claims description 4
- 150000004665 fatty acids Chemical class 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 150000002772 monosaccharides Chemical class 0.000 claims description 4
- 229940054269 sodium pyruvate Drugs 0.000 claims description 4
- 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 3
- 239000005700 Putrescine Substances 0.000 claims description 3
- 229930003779 Vitamin B12 Natural products 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
- 239000008103 glucose Substances 0.000 claims description 3
- 229960000890 hydrocortisone Drugs 0.000 claims description 3
- 239000002777 nucleoside Substances 0.000 claims description 3
- 125000003835 nucleoside group Chemical group 0.000 claims description 3
- -1 potassium ferricyanide Chemical compound 0.000 claims description 3
- 239000011573 trace mineral Substances 0.000 claims description 3
- 235000013619 trace mineral Nutrition 0.000 claims description 3
- 235000019163 vitamin B12 Nutrition 0.000 claims description 3
- 239000011715 vitamin B12 Substances 0.000 claims description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 2
- RBMGJIZCEWRQES-DKWTVANSSA-N (2s)-2,4-diamino-4-oxobutanoic acid;hydrate Chemical compound O.OC(=O)[C@@H](N)CC(N)=O RBMGJIZCEWRQES-DKWTVANSSA-N 0.000 claims description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 2
- IFQSXNOEEPCSLW-DKWTVANSSA-N L-cysteine hydrochloride Chemical compound Cl.SC[C@H](N)C(O)=O IFQSXNOEEPCSLW-DKWTVANSSA-N 0.000 claims description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 2
- 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 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 2
- 239000006035 Tryptophane Substances 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- 229940088710 antibiotic agent Drugs 0.000 claims description 2
- 229940033655 asparagine monohydrate Drugs 0.000 claims description 2
- 235000003704 aspartic acid Nutrition 0.000 claims 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 claims description 2
- 229960002685 biotin Drugs 0.000 claims description 2
- 235000020958 biotin Nutrition 0.000 claims description 2
- 239000011616 biotin Substances 0.000 claims description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 2
- 229930182817 methionine Natural products 0.000 claims description 2
- 229960004799 tryptophan Drugs 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 239000005569 Iron sulphate Substances 0.000 claims 1
- 238000007792 addition Methods 0.000 claims 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 claims 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims 1
- 239000004615 ingredient Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 4
- 239000004017 serum-free culture medium Substances 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 229960001471 sodium selenite Drugs 0.000 description 2
- 235000015921 sodium selenite Nutrition 0.000 description 2
- 239000011781 sodium selenite Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 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 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 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 description 1
- 244000309466 calf Species 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 229960002413 ferric citrate Drugs 0.000 description 1
- 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 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000012913 medium supplement Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 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
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0043—Medium free of human- or animal-derived 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
-
- 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
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (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)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A serum-free medium for the cultivation of mammalian cells without protein material of animal origin contains, besides conventional ingredients in place of animal insulin and transferring recombinant insulin from prokaryotes and a water-soluble iron compound. A medium of this type is used according to the invention for the cultivation of mammalian cells, especially CHO cells.
Description
The present invention is concerned with a serumfree medium for culturing mammalian cells without protein material of animal origin, as well as with a process for the culturing of mammalian cells and especially of CHO cells with the use of such a medium and, finally, with a process for the gene-technological production of erythropoietin in which the correspondingly transformed CHO cells grow in the culture medium according to the present invention.
For the growth of animal cells in a culture, as a medium supplement in general serum and mostly foetal calf serum is a prerequisite. To a base medium containing certain base components, for example inorganic salts, amino acids, monosaccharides, vitamins and the like, is thereby admixed an addition of a particular amount of serum, the cells thereby being provided with conditions which are as favourable as possible for growth in the cell culture.
Attempts have already been made to exchange the serum by replacement materials in known serum-free media. Thus, for example from DE-OS 37 33 453, a serumfree medium is known for the culturing of hybridoma and myeloma cells which, in a synthetic culturing medium, contains a water-soluble iron compound besides other known additive materials. In the description, it is, in this regard, stated that this water-soluble iron compound represents a replacement for the transferrin
-3contained in the serum. According to this publication, the there-mentioned media can be used for culturing hybridoma and myeloma cells.
However, further materials are contained in serum 5 which can influence the growth of mammalian cells.
Besides transferrin, insulin and albumin are also to be especially mentioned. Therefore, quite generally, for the culturing of mammalian cells, in the case of the use of a serum-free medium, care should be taken to come as close as possible to the conditions which are present in the case of the addition of serum. However, for the production of therapeutics, the materials used are critical. In the case of cell culture products, the authorities make, inter alia, an especial note of proteins as medium additive materials which are of animal or human origin because, via these components, for example pathogenic viruses can also get into the cell culture media and possibly into the end product.
Therefore, it is an object of the present invention to provide a serum-free culture medium for mammalian cells of quite general kind which comes very close to the culturing conditions with the use of a medium containing serum but which medium contains no protein material of animal origin which could represent a danger of viral contamination.
•Ε 921020
-4Thus, according to the present invention, there is provided a serum-free medium for culturing mammalian cells without protein material of animal origin containing, besides usual component materials, instead of animal insulin and transferrin, recombinant insulin from prokaryotes and a water-soluble iron compound .
Due to the maintenance of the insulin in the medium according to the present invention, the culture conditions are distinctly more similar to culture conditions with the use of serum than is the case with known serum-free culture media. Furthermore, due to the use of recombinant insulin from prokaryotes in the medium according to the present invention, the danger of viral contaminations is avoided.
Usual component materials of culture media are known to the expert and are, for example, the component materials which are contained in the media such as Dulbecco's modified Eagle's medium (DMEM) (Virology _8,
396/1959) or in medium F12 (Ham's F12 medium, Proc.
Natl. Acad. Sci. USA 53 , 288/1965). Such component materials include, inter alia, amino acids and vitamins, as well as other components, such as glucose, sodium pyruvate, certain fatty acids and inorganic salts.
In a preferred embodiment of the present invention, the medium contains insulin in an amount of 0.1 to 20 mg/1 and the water-soluble iron compound in
-5- 5 - 2 a concentration of 10 to 10 mol/1. As watersoluble iron compound, there are especially used ferric citrate, ferric sulphate, ferric chloride and/or potassium ferricyanide.
For a facilitation in the case of the culturing, it is, in addition, preferred additionally to add a pH indicator to the medium. Such pH indicators are known to the expert. For example, phenol red may be mentioned .
An especially preferred serum-free medium according to the present invention comprises amino acids in a concentration of 3 to 700 mg/1, vitamins of 0.001 to 50 mg/1, monosaccharides of 0.3 to 10 g/1, inorganic salts of 0.1 to 10,000 mg/1, trace elements of 0.001 to 0.1 mg/1, nucleosides of 0.1 to 50 mg/1, fatty acids of 0.001 to 10 mg/1, biotin of 0.01 to 1 mg/1, hydrocortisone of 0.1 to 20 pg/l, insulin of 0.1 to 20 mg/1, vitamin B12 of 0.1 to 10 mg/1, putrescine of 0.01 to 1 mg/1, sodium pyruvate of 10 to 500 mg/1, the water20 soluble iron compound, as well as optionally a pH indicator and antibiotics, said compounds being present dissolved in water. The statements of concentration thereby refer in each case to the individual components and not to the sum of the mentioned compounds. Thus, according to the present invention, there can be used, for example, mixtures of DMEM and F12 medium. Other commercially obtainable media which correspond to the
-6compositions as are given above can also be used as base media and are then correspondingly supplemented with the special components recombinant insulin and water-soluble iron compound.
Furthermore, in the scope of the present invention, it is especially preferred that the medium additionally contains polyvinyl alcohol and/or methyl cellulose. The use of polyvinyl alcohol is described, for examp],e, in European Patent Application 0 248 656, as well as by B.D. Bavister in The Journal of
Experimental Zoology, 217, 45-51/1981 and Shintani et. al in Appl. Microbiol. Biotechnol. , 27 , 533-537/1988.
The addition of polyvinyl alcohol and/or of methyl cellulose is also advantageous for the medium according to the present invention. The compounds are thereby advantageously used in a concentration of 0.1 to 20 g/1.
The medium according to the present invention is especially characterised in that it is particularly suitable for culturing CHO cells. The particularly good suitability of the medium according to the present invention has shown itself especially in the case of culturing CHO cells which contain and express a foreign gene. In the scope of the present invention, the foreign gene to be expressed in CHO cells is preferably the gene for erythropoietin.
Therefore, the present invention also provides a process for culturing mammalian cells, especially of
-ΊCHO cells, in a medium according to the present invention. A manner of procedure has thereby proved to be preferred in which there is used a culture medium according to the present invention impoverished in certain substances and then, during the culturing, there are added twice, preferably after 48 and 96 hours, in each case 2% by volume of a mixture of 0.1 to 1 g/1 of recombinant insulin, 40 to 200 g/1 glucose and 0.4 to 3 g/1 of each of aspartic acid, asparagine monohydrate, histidine, methionine, proline and serine, 1 to 5 g/1 cysteine monohydrochloride monohydrate, to 8 g/1 glutamine and 0.2 to 2 g/1 tryptophane, whereafter the corresponding concentrations of the medium according to the present invention are achieved.
In the case of this preferred carrying out of the process, it has been shown that the growth can be maintained over a long period of time at a relatively high rate and thus cell amounts can be obtained which are higher than in the case of immediate use of the culture medium and the consumption of the corresponding compounds taking place from the beginning.
The process according to the present invention is again preferably employed for culturing CHO cells and especially CHO cells which secrete erythropoietin.
Therefore, the present invention also provides a process for the gene-technological production of erythropoietin in which CHO cells containing the gene
-8for erythropoietin are cultured in a medium according to the present invention or are cultured according to a process of the present invention, erythropoietin being isolated from the culture medium.
The following Examples are given for the purpose of illustrating the present invention, reference being made to the accompanying drawings in which
Fig. 1 shows the living cell density in dependence upon the period of culturing in the case of using different media and
Fig. 2 shows the total cell density in dependence upon the period of culturing.
Examples .
Description of the medium used in the Examples.
The medium of the Examples given hereinafter contains amino acids in a concentration of 3 to 700 mg/1, vitamins of 0.001 to 50 mg/1, monosaccharides of 0.3 to 10 g/1, inorganic salts of 0.1 to 10000 mg/1, trace elements of 0.001 to 0.1 mg/1, nucleosides of 0.1 to
50 mg/1 and fatty acids of 0.01 to 10 mg/1, as well as sodium pyruvate (10 to 500 mg/1) and a pH indicator.
The medium was prepared on the basis of a mixture consisting of equal parts by volume of the DMEM and F12 media (Dulbecco's modified Eagle's medium and nutrient mixture Hams F-12). As special components, there are present
-9biotin hydrocortisone insulin (recombinant) putrescine vitamin B12
Furthermore, the medium of transferrin (human) sodium selenite polyvinyl alcohol
Furthermore, the medium of iron citrate polyvinyl alcohol
Furthermore, the medium of iron citrate methyl cellulose
In all three Example!
under the usual conditions
Example 1.
0.2036 mg/1 3.6 pg/l 5.0 mg/1 0.1 mg/1 0.78 mg/1
Example 1 contains:
mg/1 5 ug/1 1 g/1
Example 2 contains:
124 mg/1 g/1
Example 3 contains:
124 mg/1
0.5 g/1 , CHO cells were cultured (37°C, 57, carbon dioxide).
In the case of the use of a medium which contains 5 mg/1 transferrin, 5 pg/l sodium selenite and 1 g/1 polyvinyl alcohol, as maximum values for the living cell density were achieved 8.1 x 10^/ml (after 189 hours culture period) and for the total cell density
.8 x 10^/rnl (after 197 hours culture period).
Example 2.
In the case of the use of a medium which contains
124 mg/1 iron citrate and 1 g/1 polyvinyl alcohol, as
-10maximum values for the living cell density were achieved
.3 x 10^/mg (after 164 hours culture period) and for the total cell density 25.7 x 10^/ml (after 164 hours culture period).
Example 3.
In the case of the use of a medium which contains 124 mg/1 iron citrate and 0.5 g/1 methyl cellulose, as maximum values for the living cell density were achieved
14.4 x 10^/ml (after 185 hours culture period) and for 10 the total cell density 26.0 x 10^/ml (after 193 hours culture period).
The values obtained for the living and total cell densities of Examples 1 to 3 are illustrated graphically in Figures 1 and 2 of the accompanying drawings.
Claims (19)
1. Serum-free medium for culturing mammalian cells without protein material of animal origin, containing besides usual component materials, instead of animal 5 insulin and transferrin, recombinant insulin from prokaryotes and a water-soluble iron compound.
2. Medium according to claim 1, wherein it contains insulin in an amount of from 0.1 to 20 mg/1 and the water-soluble iron compound in a concentration of 10 10“ 5 to 10 2 mol/1.
3. Medium according to claim 1 or 2, wherein, as water-soluble iron compound, it contains iron citrate, iron sulphate, iron chloride and/or potassium ferricyanide . 15
4. Medium according to any of the preceding claims, wherein it additionally contains a pH indicator.
5. Serum-free medium according to any of the preceding claims, wherein it comprises amino acids in a concentration of 3 to 700 mg/1, vitamins of 0.001 to 20 50 mg/1, monosaccharides of 0.3 to 10 g/1, inorganic salts of 0.1 to 10,000 mg/1, trace elements of 0.001 to 0.1 mg/1, nucleosides of 0.1 to 50 mg/1, fatty acids of 0.001 to 10 mg/1, biotin of 0.01 to 1 mg/1, hydrocortisone of 0.1 to 20 pg/l, insulin of 0.1 to 20 mg/1, 25 vitamin B12 of 0.1 to 10 mg/1, putrescine of 0.01 to 1 mg/1, sodium pyruvate of 10 to 500 mg/1, the watersoluble iron compound, as well as optionally a pH indicator and antibiotics, dissolved in water. -126. Medium according to any of the preceding claims, wherein it additionally contains polyvinyl alcohol and/or methyl cellulose.
6. 7. Medium according to claim 6, wherein it contains 5 polyvinyl alcohol and/or methyl cellulose in a concentration of 0.1 to 20 g/1.
7. 8. Medium according to any of the preceding claims, wherein it is used for culturing CHO cells.
8. 9. Medium according to claim 8, wherein it is used
9. 10 for culturing CHO cells which contain and express a foreign gene. 10. Medium according to claim 9, wherein the foreign gene to be expressed is the gene for erythropoietin.
10. 11. Serum-free medium according to claim 1 for 15 culturing mammalian cells, substantially as hereinbefore described and exemplified.
11. 12. Process for culturing mammalian cells and especially CHO cells, wherein the culturing is carried out in a medium according to any of claims 1 to 11. 20
12. 13. Process according to claim 12, wherein there is used an impoverished culture medium and, during the culturing, there are added twice in each case 2% by volume of a mixture of 0.1 to 1 g/1 of recombinant insulin, 40 to 200 g/1 glucose and 0.4 to 3 g/1 of 25 each of aspartic acid, asparagine monohydrate, histidine, methionine, proline and serine, 1 to 5 g/1 cysteine monohydrochloride monohydrate, 1 to 8 g/1 glutamine and -130.2 to 2 g/1 tryptophane, whereafter the corresponding concentrations of a medium according to any of claims 1 to 11 are reached.
13. 14. Process according to claim 13, wherein the two 5 additions are made after 48 and 96 hours.
14. 15. Process according to any of claims 12 to 14, wherein CHO cells are cultured which secrete erythropoietin.
15. 16. Process for the gene-technological production of 10 erythropoietin, wherein CHO cells containing the gene for erythropoietin are cultured in a medium according to any of claims 1 to 11 or according to a process according to any of claims 12 to 15 and the erythropoietin is isolated from the culture medium. 15
16. 17. Process according to claim 12 for culturing mammalian cells, substantially as hereinbefore described and exemplified.
17. 18. Mammalian cells, whenever cultured by the process according to any of claims 12 to 17.
18. 20
19. Erythropoietin, whenever produced by the process according to any of claims 12 to 17.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4115722A DE4115722A1 (en) | 1991-05-14 | 1991-05-14 | SERUM-FREE MEDIUM FOR CULTIVATING SUGAR CELLS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| IE921020A1 true IE921020A1 (en) | 1992-11-18 |
Family
ID=6431637
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IE102092A IE921020A1 (en) | 1991-05-14 | 1992-03-30 | Serum-free medium for culturing mammalian cells |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0513738A3 (en) |
| JP (1) | JPH05252942A (en) |
| DE (1) | DE4115722A1 (en) |
| IE (1) | IE921020A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11661579B2 (en) | 2007-04-26 | 2023-05-30 | Chugai Seiyaku Kabushiki Kaisha | Cell culture method using amino acid-enriched medium |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9022545D0 (en) | 1990-10-17 | 1990-11-28 | Wellcome Found | Culture medium |
| US5548064A (en) * | 1993-05-24 | 1996-08-20 | Biotech Australia Pty Limited | Vitamin B12 conjugates with EPO, analogues thereof and pharmaceutical compositions |
| EP0699944B1 (en) | 1994-08-26 | 2000-06-07 | Eastman Kodak Company | Tabular grain emulsions with sensitization enhancements |
| IL118201A (en) * | 1995-05-11 | 2004-12-15 | Roche Diagnostics Gmbh | Preparation comprising a protein with human erythropoietin activity which is free of serum and non-recombinant mammalian protein and process for the preparation thereof |
| US5952226A (en) * | 1996-11-05 | 1999-09-14 | Modex Therapeutiques | Hypoxia responsive EPO producing cells |
| SE9700983D0 (en) * | 1997-03-18 | 1997-03-18 | Ascendia Ab | Stimulation, culture and preservation of pancreatic and other cells |
| BR9905868A (en) | 1998-11-06 | 2001-01-23 | Bio Sidus S A | Mass culture procedure for mammalian cells to obtain recombinant human erythropoietin and recombinant human erythropoietin obtained with such procedure |
| DE10011998A1 (en) * | 2000-03-11 | 2001-09-20 | Forschungszentrum Juelich Gmbh | Combination of active ingredients and methods for cultivating cells and methods for producing the combination of active ingredients |
| CN100528235C (en) | 2000-12-20 | 2009-08-19 | 霍夫曼-拉罗奇有限公司 | Conjugates of erythropoietin |
| GB0304799D0 (en) | 2003-03-03 | 2003-04-09 | Glaxosmithkline Biolog Sa | Novel method |
| US7972810B2 (en) | 2005-12-08 | 2011-07-05 | Amgen Inc. | Production of glycoproteins using manganese |
| PT2054074E (en) | 2006-08-04 | 2014-11-07 | Prolong Pharmaceuticals Llc | Modified erythropoietin |
| WO2009127098A1 (en) * | 2008-04-18 | 2009-10-22 | 上海中信国健药业有限公司 | A concentrated culture solution and application method thereof |
| MX2012003558A (en) | 2009-09-23 | 2012-07-03 | Biogenerix Ag | Process for the purification of recombinant human erythropoietin (epo), epo thus purified and pharmaceutical compositions comprising same. |
| RU2663794C2 (en) * | 2011-10-21 | 2018-08-09 | Пфайзер Инк. | Adding iron to improve cell culture |
| US9217168B2 (en) | 2013-03-14 | 2015-12-22 | Momenta Pharmaceuticals, Inc. | Methods of cell culture |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ210501A (en) * | 1983-12-13 | 1991-08-27 | Kirin Amgen Inc | Erythropoietin produced by procaryotic or eucaryotic expression of an exogenous dna sequence |
| DE3785086T2 (en) * | 1986-06-04 | 1993-07-08 | Agency Ind Science Techn | PREPARATION FOR CELL CULTURE AND THEIR USE. |
| WO1988000967A1 (en) * | 1986-08-04 | 1988-02-11 | The University Of New South Wales | Serum free tissue culture medium containing polymeric cell-protective agent |
| IL87737A (en) * | 1987-09-11 | 1993-08-18 | Genentech Inc | Method for culturing polypeptide factor dependent vertebrate recombinant cells |
| DE68917642T2 (en) * | 1988-05-25 | 1995-02-09 | Teijin Ltd | Process for the continuous cultivation of adherent animal cells. |
| FR2635008B1 (en) * | 1988-08-05 | 1990-11-16 | Sanofi Sa | ACTIVATOR FOR THE SPECIFIC PRODUCTIVITY OF RECOMBINANT ANIMAL CELLS BASED ON POLYVINYLPYRROLIDONE AND DEFINED CULTURE MEDIUM CONTAINING THE SAME |
| JPH0279969A (en) * | 1988-09-19 | 1990-03-20 | Shigeyoshi Fujimoto | Method for inducing and activating cytotoxic t-cell |
| GB9022545D0 (en) * | 1990-10-17 | 1990-11-28 | Wellcome Found | Culture medium |
-
1991
- 1991-05-14 DE DE4115722A patent/DE4115722A1/en not_active Withdrawn
-
1992
- 1992-03-30 IE IE102092A patent/IE921020A1/en not_active Application Discontinuation
- 1992-05-11 JP JP11727592A patent/JPH05252942A/en active Pending
- 1992-05-12 EP EP19920107997 patent/EP0513738A3/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11661579B2 (en) | 2007-04-26 | 2023-05-30 | Chugai Seiyaku Kabushiki Kaisha | Cell culture method using amino acid-enriched medium |
Also Published As
| Publication number | Publication date |
|---|---|
| DE4115722A1 (en) | 1992-11-19 |
| EP0513738A2 (en) | 1992-11-19 |
| EP0513738A3 (en) | 1993-05-05 |
| JPH05252942A (en) | 1993-10-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| IE921020A1 (en) | Serum-free medium for culturing mammalian cells | |
| US5122469A (en) | Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins | |
| Gregory Hamilton et al. | Clonal growth of Chinese hamster cell lines in protein-free media | |
| Evans et al. | Chemically defined media for cultivation of long-term cell strains from four mammalian species | |
| KR101264940B1 (en) | Medium and culture of embryonic stem cells | |
| Xie et al. | Integrated approaches to the design of media and feeding strategies for fed-batch cultures of animal cells | |
| Darlington | [3] Liver cell lines | |
| Yamane et al. | Primary culture of human diploid cells and its long-term transfer in a serum-free medium | |
| CN101418330B (en) | Non protein culture medium adapted to large-scale culture of NSO cell and production of antibody | |
| JPH0670757A (en) | Medium | |
| IE84174B1 (en) | Culture medium for CHO cells and adapted CHO cells | |
| Sebald et al. | Electrophoretic pattern of and amino acid incorporation in vitro into the insoluble mitochondrial protein of neurospora crassa wild type and mi‐1 mutant | |
| DK0802257T3 (en) | Immortalized cell line from human colon epithelial cells | |
| Higuchi | Cultivation of animal cells in chemically defined media, a review | |
| CN101195817A (en) | Hybrid tumor cell amplification culture medium and uses thereof | |
| EP0659880B1 (en) | Medium for culturing animal cells or antibody-producing cells | |
| CN1962857A (en) | Serum-free medium for mammalian cell | |
| CA1221651A (en) | Cell growth medium supplement | |
| CN102021139A (en) | Chinese hamster ovary culture medium as well as preparation method and application thereof | |
| AU662491B2 (en) | Medium for culture of mammalian cells | |
| US5871999A (en) | Cell culture process and medium for the growth of adherent animal cells | |
| US20050287666A1 (en) | Cell culture medium comprising transition metals or trace elements | |
| US5126261A (en) | High calcium chemically defined culture medium | |
| US5512477A (en) | Serum-free medium supplement | |
| WO1993000423A1 (en) | Iron chelate culture medium additive |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FA9A | Application withdrawn section 33(1) |