Serum-free mammalian cell culture medium, and uses thereof

Abstract

The present invention provides a cell culture medium formulation that supports the in vitro cultivation, particularly in suspension, of mammalian cells, particularly epithelial cells and fibroblast cells, and methods for cultivating mammalian cells in suspension in vitro using these media. The media comprise a basal medium and a polyanionic or polyanionic compound, preferably a polysulfonated or polysulfated compound, and more preferably dextran sulfate. The present invention also provides chemically defined, protein-free eukaryotic cell culture media comprising an iron chelate and zinc, which is capable of supporting the growth (and particularly the high-density growth of mammalian cells) in suspension culture, increasing the level of expression of recombinant protein in cultured cells, and/or increasing virus production in cultured cells.

1 - 78 . (canceled) 
     
     
         79 . A method of producing a polypeptide comprising
 (a) obtaining a mammalian cell that has been genetically engineered to produce a polypeptide; and   (b) cultivating said cell in a serum-free and protein-free cell culture medium comprising an insulin substitute and a transferrin substitute, under conditions favoring expression of said polypeptide by said mammalian cell.   
     
     
         80 . The method of  claim 79 , wherein said mammalian cell is an epithelial cell. 
     
     
         81 . The method of  claim 79 , wherein said mammalian cell is a human cell. 
     
     
         82 . The method of  claim 81 , wherein said human cell is a 293 embryonic kidney epithelial cell. 
     
     
         83 . A polypeptide produced according to the method of  claim 79 . 
     
     
         84 . A eukaryotic cell culture medium comprising a Fe 2+  chelate and a Zn 2+  salt, wherein said medium is capable of supporting the high-density growth of mammalian cells in suspension culture and/or the expression of recombinant protein. 
     
     
         85 . (canceled) 
     
     
         86 . The eukaryotic cell culture medium according to  claim 84 , wherein said mammalian cells are Chinese hamster ovary cells. 
     
     
         87 . The eukaryotic cell culture medium according to  claim 84 , wherein said medium is a 1× medium formulation. 
     
     
         88 . The eukaryotic cell culture medium according to  claim 84 , wherein said medium is a concentrated medium formulation. 
     
     
         89 . The eukaryotic cell culture medium according to  claim 88 , wherein said medium is a 10× medium formulation. 
     
     
         90 . The eukaryotic cell culture medium according to  claim 88 , wherein said medium formulation is greater than 10×. 
     
     
         91 - 97 . (canceled) 
     
     
         98 . The eukaryotic cell culture medium according to  claim 84 , further comprising one or more ingredients selected from the group consisting of L-arginine, L-asparagine. H 2 O, L-aspartic acid, L-glutamic acid, L-histidine, hydroxy-L-proline, L-isoleucine, L-leucine, L-lysine.HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cystine.2HCl, Na 2 HPO 4 , pyridoxine. HCl, thiamine. HCl, glutathione, cupric sulfate.7H 2 O, cadmium chloride.5H 2 O, cobalt chloride.2H 2 O, stannous chloride. 2H 2 O, manganous sulfate. H 2 O, nickel sulfate. 6H 2 O, sodium metavanadate, ammonium molybdate. 4H 2 O, barium acetate, potassium bromide, potassium iodide, chromium sulfate, sodium fluoride, silver nitrate, rubidium chloride, zirconyl chloride, aluminum chloride, germanium dioxide, titanium tetrachloride, sodium metasilicate, magnesium chloride (anhydrous), D-calcium pantothenate, calcium nitrate. 4H 2 O, potassium chloride, ascorbic acid magnesium salt phosphate, pluronic F68 10% solution, Na 2 HPO 4 , D-glucose, folic acid, riboflavin, biotin, choline chloride, niacinamide, i-inositol, sodium pyruvate, vitamin B-12, β-mercaptoethanol, para-aminobenzoic acid, β-glycerophosphate, sodium selenite, ethanolamine.HCl, spermine, putrescine.2HCl, monothioglycerol, and sodium bicarbonate, wherein each of said ingredients is present in an amount which supports the high-density growth of Chinese hamster ovary cells in suspension culture and/or the expression of recombinant protein. 
     
     
         99 . A eukaryotic cell culture medium obtained by combining ferrous sulfate.EDTA and ZnSO 4 .7H 2 O, together with a eukaryotic medium, wherein said ferrous sulfate.EDTA and said ZnSO 4 .7H 2 O are each present in an amount which supports the high-density growth of Chinese hamster ovary cells in suspension culture. 
     
     
         100 . The eukaryotic cell culture medium according to  claim 99 , further comprising a polyanionic or polycationic compound, wherein said polyanionic or polycationic compound is present in an amount sufficient to prevent cell clumping and/or increase the level of recombinant protein expression. 
     
     
         101 . The eukaryotic cell culture medium according to  claim 100 , wherein said polyanionic compound is a polysulfonated compound or a polysulfated compound. 
     
     
         102 . The eukaryotic cell culture medium according to  claim 101 , wherein said polysulfonated or polysulfated compound is selected from the group consisting of dextran sulfate, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, pentosan sulfate and a proteoglycan. 
     
     
         103 . The eukaryotic cell culture medium according to  claim 102 , wherein said polysulfonated or polysulfated compound is dextran sulfate. 
     
     
         104 . The eukaryotic cell culture medium according to  claim 103 , wherein said dextran sulfate has an average molecular weight of 5,000 daltons. 
     
     
         105 . The eukaryotic cell culture medium obtained according to  claim 99  or  100 , further comprising one or more ingredients selected from the group consisting of L-arginine, L-asparagine.H 2 O, L-aspartic acid, L-glutamic acid, L-histidine, hydroxy-L-proline, L-isoleucine, L-leucine, L-lysine.HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cystine.2HCl, Na 2 HPO 4 , pyridoxine.HCl, thiamine.HCl, glutathione, cupric sulfate.5H 2 O, cadmium chloride.5H 2 O, cobalt chloride.2H 2 O, stannous chloride.2H 2 O, manganous sulfate.H 2 O, nickel sulfate.6H 2 O, sodium metavanadate, ammonium molybdate.4H 2 O, barium acetate, potassium bromide, potassium iodide, chromium sulfate, sodium fluoride, silver nitrate, rubidium chloride, zirconyl chloride, aluminum. chloride, germanium dioxide, titanium tetrachloride, sodium metasilicate, magnesium chloride (anhydrous), D-calcium pantothenate, calcium nitrate. 4H 2 O, potassium chloride, ascorbic acid magnesium salt phosphate, pluronic F68 10% solution, Na 2 HP0 4 , D-glucose, folic acid, riboflavin, biotin, choline chloride, niacinamide, i-inositol, sodium pyruvate, vitamin B-12, β-mercaptoethanol, para-aminobenzoic acid, β-glycerophosphate, sodium selenite, ethanolamine.HCl, monothioglycerol, and sodium bicarbonate, spermine, putrescine.2HCl, wherein each of said ingredients is present in an amount which supports the high-density growth of Chinese hamster ovary cells in suspension culture and/or the expression of recombinant protein. 
     
     
         106 - 128 . (canceled) 
     
     
         129 . A kit for the cultivation of a mammalian epithelial cell in suspension in vitro, said kit comprising one or more containers, wherein a first container contains a medium comprising a Fe 2+  chelate and a Zn 2+  salt, wherein said medium is capable of supporting the high-density growth of mammalian cells in suspension culture and/or the expression of recombinant protein. 
     
     
         130 - 139 . (canceled)