Method for culturing Chinese hamster ovary cells
AssigneeGlaxo Wellcome Inc.
InventorsMichael J. Keen, Nicholas T. Rapson
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Abstract
A biochemically defined culture medium for culturing engineered Chinese hamster ovary (CHO) cell lines, which is essentially free from protein, lipid and carbohydrate isolated from an animal source, having water, an osmolality regulator, a buffer, an energy source, amino acids including L-glutamine, an inorganic or recombinant iron source, and a synthetic or recombinant growth factor, and optionally non-ferrous metal ions vitamins and cofactors. Also cells adapted to grow in such a culture medium.
Claims18
A method for growing CHO cells which comprises culturing CHO cells under cell growing conditions in the absence of serum in a medium comprising water, an osmolality regulator, a buffer, an energy source, L-glutamine and at least one additional amino acid, an inorganic, organic or recombinant iron source and a recombinant or synthetic growth factor wherein each component of said medium is obtained from a source other than directly from an animal source.
A method for culturing CHO cells which comprises culturing and growing Chinese hamster ovary cells in the absence of serum in a medium comprising
- an osmolality regulator to maintain the osmolality of the medium within the range of about 200-350 mOsm,
- a buffer to maintain the pH of the medium within the range of about 6.5 to 7.5,
- about 1000-10,000 mg of a monosaccharide,
- about 400-600 mg of L-glutamine,
- about 10 -200 mg of at least one amino acid selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine,
- about 0.25-5 mg of an inorganic or recombinant iron source,
- about 5 .mu.g-5 mg of a recombinant or synthetic insulin and sufficient water to provide one liter of medium.
A method for culturing CHO cells which comprises culturing and growing Chinese hamster ovary cells in the absence of serum in a medium comprising
- a base medium containing the amino acids, non-ferrous metal ions, vitamins and cofactors essentially as set forth in Table 1,
- an osmolality regulator selected from NaCl, KCl, and KNO.sub.3 in an amount sufficient to maintain the osmolality of the medium within the range of about 200-350 mOsm,
- at least one buffer selected from CaCl.sub.2.2H.sub.2 O, MgSO.sub.4.7H.sub.2 O, NaH.sub.2 PO.sub.4.2H.sub.2 O, sodium pyruvate, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid] (HEPES) and 3-[N-morpholino]-propanesulfonic acid (MOPS) in an amount sufficient to maintain the medium within the pH range of about 6.5-7.5,
- about 1000-10,000 mg of mannose, fructose, glucose or maltose,
- about 5 ml of 200 mM L-glutamine,
- about 50 mg each of L-proline, L-threonine, L-methionine, L-cysteine and L-tyrosine,
- about 20-50 mg of L-ascorbic acid,
- about 0.01-0.5 mg each of Vitamin B6 and Vitamin B12,
- about 0.25 -5 mg of a ferric or ferrous salt,
- about 1 mg of zinc sulfate,
- about 2.5 .mu.g of copper sulfate,
- about 10,000-100,000 IU of at least one antibiotic selected from the group consisting of polymyxin, neomycin, penicillin and streptomycin,
- about 3 .mu.l of ethanolamine,
- about 0.01-1.0 mg of putrescine,
- about 5 .mu.g-5 mg of recombinant insulin and sufficient water to comprise one liter of medium;
- wherein each component of said medium is obtained from a source other than directly from an animal source.
Dependencies are inferred from the claim text.