Culture medium for cell growth and transfection

Abstract

The present invention is directed generally to cell culture media (particularly serum free, non animal derived, and/or chemically defined media) which are useful for introducing macromolecules and compounds (e.g., nucleic acid molecules) into cells (e.g., eukaryotic cells). According to the invention, such introduction can take place in the presence of said medium. Cells containing such introduced materials can then be cultured in the medium and the effect of the introduced materials on the cells can be measured or determined. In particular, the invention allows introduction of nucleic acid molecules (e.g., vectors) into cells (particularly eukaryotic cells) and expression of proteins encoded by the nucleic acid molecules in the cells. The invention obviates the need to change the cell culture medium each time a different procedure is performed with the cells (e.g., culturing cells vs. transfecting cells). The invention thus provides efficient and high throughput methods to transform/transfect culture and cells avoiding the need for multiple manipulations and transfers of cells during transfection and expression studies. The invention also relates to compositions and kits useful for culturing and transforming/transfecting cells.

What is claimed is:  
     
         1 . A cell culture medium, wherein said medium supports introduction of one or more macromolecules into at least one eukaryotic cell in culture and supports cultivation of said at least one cell subsequent to said introduction, 
 wherein it is not necessary to remove medium used during said introduction from the presence of said at least one cell to support growth of said at least one cell, and/or    wherein after said introduction growth can be accomplished in cultivation in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which said introduction occurred.    
     
     
         2 . The cell culture medium of  claim 1 , wherein said medium also can support cultivation of said at least one cell prior to said introduction.  
     
     
         3 . The cell culture medium of  claim 2 , wherein said medium can support cultivation prior to said introduction and wherein the medium does not need to be removed between said cultivation prior to said introduction.  
     
     
         4 . The cell culture medium of  claim 1 , wherein replenishing, replacing or supplementing said medium with fresh medium after said introduction does not improve said cultivation, relative to cultivation in said medium without said replenishing, replacing or supplementing.  
     
     
         5 . The cell culture medium of  claim 1 , wherein replenishing, replacing or supplementing with fresh medium does not improve the protein yield of said at least one cell, relative to the protein yield in said medium without said replenishing, replacing or supplementing.  
     
     
         6 . The cell culture medium of  claim 1 , wherein said culture is a suspension culture.  
     
     
         7 . The cell culture medium of  claim 1 , wherein said one or more macromolecules includes at least one nucleic acid molecule.  
     
     
         8 . The cell culture medium of  claim 7 , wherein said medium supports the expression of said nucleic acid in said at least one cell into which said at least one nucleic acid is introduced.  
     
     
         9 . The cell culture medium of  claim 7 , wherein said at least one nucleic acid molecule is deoxyribonucleic acid (DNA).  
     
     
         10 . The cell culture medium of  claim 1 , wherein said cultivation includes the production of at least one protein product.  
     
     
         11 . The cell culture medium of  claim 10 , wherein said at least one protein product includes at least one polypeptide or protein.  
     
     
         12 . The cell culture medium of  claim 10 , wherein said at least one protein product includes at least one molecule that is a reactant product catalyzed by at least one protein.  
     
     
         13 . The cell culture medium of  claim 1 , wherein said medium is a serum-free medium.  
     
     
         14 . The cell culture medium of  claim 1 , wherein said medium is a chemically defined medium.  
     
     
         15 . The cell culture medium of  claim 1 , wherein the medium is a low protein or protein-free medium.  
     
     
         16 . The cell culture medium of  claim 1 , wherein said medium substantially lacks or does not contain materials obtained from an animal.  
     
     
         17 . The cell culture medium of  claim 1 , wherein said medium substnatially lacks or does not contain animal derived materials.  
     
     
         18 . The cell culture medium of  claim 1 , said medium comprising at least one compound selected from the group consisting of a polyol, a hydroxypyridine derivative, 1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene, ethylenedi-amine-N,N′-tetramethylenephosphonic acid, trisuccin, an acidic saccharide (e.g. ferrous gluconate), a glycosaminoglycan, diethylenetriaminepentaacetic acid, nitrilotriacetic acid mono-, bis-, or tris-substituted 2,2′-bipyridine, a hydroxamate derivative (e.g. acetohydroxamic acid), an amino acid, deferoxamine, ferrioxamine, iron basic porphine, porphyrin and derivatives thereof, DOTA-lysine, a texaphyrin, a sapphyrin, a polyaminocarboxylic acid, an α-hydroxycarboxylic acid, a polyethylenecarbamate, picolinic acid, 4-pyridoxic acid, 3-hydroxy-2-pyridinemaltol, ethyl maltol, Ustilago ferrichrome, nicotinic acid-N-oxide and IRC011.  
     
     
         19 . The cell culture medium of  claim 18 , wherein said medium comprises a hydroxypyridine derivative.  
     
     
         20 . The cell culture medium of  claim 19 , wherein said hydroxypyridine derivative is selected from the group consisting of 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one, 1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4-one, 1-methyl-3-hydroxypyrid-2-one, 3-hydroxy-2(1H)-pyridinone, pyridoxal isonicotinyl hydrazone, nicotinic acid-N-oxide, and 2-hydroxy-nicotinic acid.  
     
     
         21 . The cell culture medium of  claim 20 , wherein said hydroxypyridine derivative is 2-hydroxypyridine-N-oxide.  
     
     
         22 . The cell culture medium of  claim 1 , said medium comprising one or more ingredients selected from the group consisting of at least one amino acid, at least one vitamin, at least one inorganic salt, at least or organic salt, at least one trace metal, at least one nucleotide, at least one buffering salt, at least one sugar, at least one lipid and at least one hormone.  
     
     
         23 . The cell culture medium of  claim 1 , further comprising at least one ingredient selected from the group consisting of an amino acid, a sugar, a fatty acid, a vitamin, a pH buffer, a surfactant, a trace metal or salt or hydrate thereof, an amine compound, a growth factor, an agent to control osmolarity and/or ionic strength and/or maintain membrane potential, a flavin, a compound that participates in or is a product of the glycolytic pathway, an alcoholamine, a cyclic alcohol, a phospholipid or portion thereof, a salt of selenious acid, a divalent or trivalent cation, a pteridine derivative, a valeric acid, and/or a coenzyme.  
     
     
         24 . The cell culture medium of  claim 1 , wherein said at least one cell is selected from the group consisting of a plant cell and an animal cell.  
     
     
         25 . The cell culture medium of  claim 24 , wherein said at least one cell is an animal cell.  
     
     
         26 . The cell culture medium of  claim 25 , wherein said animal cell is selected from the group consisting of a mammalian cell, a fish cell, an insect cell, an amphibian cell, and an avian cell.  
     
     
         27 . The cell culture medium of  claim 26 , wherein said animal cell is a mammalian cell.  
     
     
         28 . The cell culture medium of  claim 25 , wherein the at least one cell is an epithelial cell.  
     
     
         29 . The cell culture medium of  claim 26 , wherein said mammalian cell is selected from the group consisting of a 293 cell, a PER-C6 cell, a CHO cell, a COS cell, a BHK cell, a HeLa cell, and a Sp2/0 cell.  
     
     
         30 . The cell culture medium of  claim 29 , wherein said mammalian cell is a 293 cell.  
     
     
         31 . The cell culture medium of  claim 24 , wherein said at least one cell is in suspension culture.  
     
     
         32 . The cell culture medium of  claim 1 , wherein said medium substantially lacks or does not contain transferrin.  
     
     
         33 . The cell culture medium of  claim 1 , wherein said medium substantially lacks or does not contain insulin.  
     
     
         34 . The cell culture medium of  claim 1 , wherein said medium substantially lacks or does not contain any polyanionic compounds.  
     
     
         35 . The cell culture medium of  claim 1 , wherein said medium substantially lacks or does not contain dextran sulfate.  
     
     
         36 . The medium of  claim 1 , wherein LIPOFECTAMINE 2000 is admixed to said medium for said introduction.  
     
     
         37 . A method of making a medium comprising admixing water and at least one ingredient selected from the group consisting of an amino acid, a sugar, a fatty acid, a vitamin, a pH buffer, a surfactant, a trace metal or salt or hydrate thereof, an amine compound, a growth factor, an agent to control osmolarity and/or ionic strength and/or maintain membrane potential, a flavin, a compound that participates in or is a product of the glycolytic pathway, an alcoholamine, a cyclic alcohol, a phospholipid or portion thereof, a salt of selenious acid, a divalent or trivalent cation, a pteridine derivative, a valeric acid, and/or a coenzyme, 
 wherein said medium supports introduction of one or more macromolecules into at least one eukaryotic cell in culture and supports cultivation of said at least one cell subsequent to said introduction,    wherein it is not necessary to remove medium used during said introduction from the presence of said at least one cell to support growth of said at least one cell, and/or    wherein after said introduction growth is accomplished in cultivation in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which said introduction occurred.    
     
     
         38 . A method of cultivating eukaryotic cells comprising: 
 (a) contacting said cells with the cell culture medium of  claim 1;  and    (b) maintaining said cells under conditions suitable to support cultivation of said cells in culture.    
     
     
         39 . The method of  claim 38 , wherein said culture is suspension culture.  
     
     
         40 . The method of  claim 38 , further comprising introducing at least one or more compounds or macromolecules into at least one of said cells in the presence of said medium.  
     
     
         41 . The method of  claim 40 , wherein said one or more compounds or macromolecules include at least one nucleic acid molecule.  
     
     
         42 . The method of  claim 41 , wherein said at least one nucleic acid molecule is deoxyribonucleic acid (DNA).  
     
     
         43 . The method of  claim 38 , wherein said medium is a serum-free medium.  
     
     
         44 . The method of  claim 38 , wherein said medium is a chemically defined medium.  
     
     
         45 . The method of  claim 38 , wherein the medium is a low protein or protein-free medium.  
     
     
         46 . The method of  claim 38 , wherein said medium substantially lacks or does not contain materials obtained from an animal or substantially lacks or does not contain animal derived materials.  
     
     
         47 . The method of  claim 38 , said medium comprising at least one compound selected from the group consisting of a polyol, a hydroxypyridine derivative, 1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene, ethylenedi-amine-N,N′-tetramethylenephosphonic acid, trisuccin, an acidic saccharide (e.g. ferrous gluconate), a glycosaminoglycan, diethylenetriaminepentaacetic acid, nitrilotriacetic acid mono-, bis-, or tris-substituted 2,2′-bipyridine, a hydroxamate derivative (e.g. acetohydroxamic acid), an amino acid, deferoxamine, ferrioxamine, iron basic porphine, porphyrin and derivatives thereof, DOTA-lysine, a texaphyrin, a sapphyrin, a polyaminocarboxylic acid, an α-hydroxycarboxylic acid, a polyethylenecarbamate, picolinic acid, 4-pyridoxic acid, 3-hydroxy-2-pyridinemaltol, ethyl maltol, Ustilago ferrichrome, nicotinic acid-N-oxide and IRC011.  
     
     
         48 . The method of  claim 47 , wherein said medium comprises a hydroxypyridine derivative.  
     
     
         49 . The method of  claim 48 , wherein said hydroxypyridine derivative is selected from the group consisting of 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one, 1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4-one, 1-methyl-3-hydroxypyrid-2-one, 3-hydroxy-2(1H)-pyridinone, pyridoxal isonicotinyl hydrazone, nicotinic acid-N-oxide, and 2-hydroxy-nicotinic acid.  
     
     
         50 . The method of  claim 49 , wherein said hydroxypyridine derivative is 2-hydroxypyridine-N-oxide.  
     
     
         51 . The method of  claim 38 , said medium comprising one or more ingredients selected from the group of an amino acid, a sugar, a fatty acid, a vitamin, a pH buffer, a surfactant, a trace metal or salt or hydrate thereof, an amine compound, a growth factor, an agent to control osmolarity and/or ionic strength and/or maintain membrane potential, a flavin, a compound that participates in or is a product of the glycolytic pathway, an alcoholamine, a cyclic alcohol, a phospholipid or portion thereof, a salt of selenious acid, a divalent or trivalent cation, a pteridine derivative, a valeric acid, and/or a coenzyme.  
     
     
         52 . The method of  claim 38 , wherein said eukaryotic cells are selected from the group consisting of plant cells and animal cells.  
     
     
         53 . The method of  claim 52 , wherein said eukaryotic cells are animal cells.  
     
     
         54 . The method of  claim 53 , wherein said animal cells are selected from the group consisting of mammalian cells, fish cells, insect cells, amphibian cells, and avian cells.  
     
     
         55 . The method of  claim 52 , wherein said animal cells are epithelial cells.  
     
     
         56 . The method of  claim 54 , wherein said animal cells are mammalian cells.  
     
     
         57 . The method of  claim 56 , wherein said mammalian cells are selected from the group consisting of 293 cells, PER-C6 cells, CHO cells, COS cells, BHK cells, HeLa cells, and Sp2/0 cells.  
     
     
         58 . The method of  claim 57 , wherein said mammalian cells are 293 cells.  
     
     
         59 . The method of  claim 38 , wherein said medium substantially lacks or does not contain transferrin.  
     
     
         60 . The method of  claim 38 , wherein said medium substantially lacks or does not contain insulin.  
     
     
         61 . The method of  claim 38 , wherein said medium substantially lacks or does not contain any polyanionic compound.  
     
     
         62 . The method of  claim 38 , wherein said medium substantially lacks or does not contain dextran sulfate.  
     
     
         63 . The method of  claim 40 , wherein LIPOFECTAMINE 2000 is admixed to said medium for said introducing.  
     
     
         64 . A method for introducing one or more macromolecules into at least one eukaryotic cell in culture, said method comprising: 
 (a) culturing at least one eukaryotic cell in said medium of  claim 1  in culture;    (b) introducing at least one macromolecule into said culture under conditions sufficient to cause one or more of said at least one macromolecule to be introduced in said at least one cell; and    (c) cultivating said at least one cell in said medium to produce a product whose production is controlled by said at least one molecule,    wherein growth of said at least one cell continues in said medium in the absence of said medium being with fresh medium,    wherein it is not necessary to remove medium used during said introducing from the presence of said at least one cell to support growth of said at least one cell, and/or    wherein after said introducing growth is accomplished in cultivation in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which said introducing occurred.    
     
     
         65 . The method of  claim 64 , wherein said medium also supports cultivation of said at least one cell prior to said introducing.  
     
     
         66 . The method of  claim 64 , wherein replenishing, replacing or supplementing said medium with fresh medium after said introducing does not improve cultivation, relative to cultivation in said medium without said replenishing, replacing or supplementing.  
     
     
         67 . The method of  claim 64 , wherein replenishing, replacing or supplementing said medium with fresh medium does not improve the protein yield of said at least one cell, relative to the protein yield in medium without said replenishing, replacing or supplementing.  
     
     
         68 . The method of  claim 64 , wherein said culture is suspension culture.  
     
     
         69 . The method of  claim 64 , wherein said medium is not replenished, replaced or supplemented during said production for at least four hours subsequent to said introducing.  
     
     
         70 . The method of  claim 64 , wherein said medium is not during said production for at least twelve hours subsequent to said introducing.  
     
     
         71 . The method of  claim 64 , wherein said medium is not during said production for at least twenty-four hours subsequent to said introducing.  
     
     
         72 . The method of  claim 64 , wherein said medium is not during said production for at least thirty-six hours subsequent to said introducing.  
     
     
         73 . The method of  claim 64 , wherein said medium is not during said production for at least forty-eight hours subsequent to said introducing.  
     
     
         74 . The method of  claim 64 , wherein said medium is not during said production for at least sixty hours subsequent to said introducing.  
     
     
         75 . The method of  claim 64 , wherein said medium is not replenished, replaced or supplemented during said production for at least seventy-two hours subsequent to said introducing.  
     
     
         76 . The method of  claim 64 , wherein said medium is not replenished, replaced or supplemented during said production for at least ninety hours subsequent to said introducing.  
     
     
         77 . The method of  claim 64 , wherein said medium supports the introduction of one or more macromolecules into said at least one cell, and supports cultivation of said cell subsequent to said introduction.  
     
     
         78 . The method of  claim 64 , wherein said medium supports growth of said at least one cell prior to said introducing, said medium supporting growth not being prior to said introducing.  
     
     
         79 . The method of  claim 64 , wherein introduction of said at least one macromolecule into said at least one cell is accomplished in the same volume of medium in which said cell is cultivated after introduction of said macromolecules.  
     
     
         80 . The method of  claim 64 , wherein said at least one macromolecule includes at least one nucleic acid molecule.  
     
     
         81 . The method of  claim 80 , wherein said at least one nucleic acid molecule is deoxyribonucleic acid (DNA).  
     
     
         82 . The method of  claim 80 , wherein said at least one nucleic acid molecule includes a vector.  
     
     
         83 . The method of  claim 64 , wherein said contacting comprises the use of one or more transfection agents.  
     
     
         84 . The method of  claim 64 , wherein said medium is a serum-free medium.  
     
     
         85 . The method of  claim 64 , wherein said medium is a chemically defined medium.  
     
     
         86 . The method of  claim 64 , wherein said medium is a low protein or protein-free medium.  
     
     
         87 . The method of  claim 64 , wherein said medium substantially lacks or does not contain animal derived materials.  
     
     
         88 . The method of  claim 64 , said medium comprising at least one compound selected from the group consisting of a polyol, a hydroxypyridine derivative, 1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene, ethylenedi-amine-N,N′-tetramethylenephosphonic acid, trisuccin, an acidic saccharide (e.g. ferrous gluconate), a glycosaminoglycan, diethylenetriaminepentaacetic acid, nitrilotriacetic acid mono-, bis-, or tris-substituted 2,2′-bipyridine, a hydroxamate derivative (e.g. acetohydroxamic acid), an amino acid, deferoxamine, ferrioxamine, iron basic porphine, porphyrin and derivatives thereof, DOTA-lysine, a texaphyrin, a sapphyrin, a polyaminocarboxylic acid, an α-hydroxycarboxylic acid, a polyethylenecarbamate, picolinic acid, 4-pyridoxic acid, 3-hydroxy-2-pyridinemaltol, ethyl maltol, Ustilago ferrichrome, nicotinic acid-N-oxide and IRC011.  
     
     
         89 . The method of  claim 64 , wherein said medium comprises a hydroxypyridine derivative.  
     
     
         90 . The method of  claim 89 , wherein said hydroxypyridine derivative is selected from the group consisting of 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one, 1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4-one, 1-methyl-3-hydroxypyrid-2-one, 3-hydroxy-2(1H)-pyridinone, pyridoxal isonicotinyl hydrazone, nicotinic acid-N-oxide, and 2-hydroxy-nicotinic acid.  
     
     
         91 . The method of  claim 90 , wherein said hydroxypyridine derivative is 2-hydroxypyridine-N-oxide.  
     
     
         92 . The method of  claim 64 , said medium comprising one or more ingredients selected from the group consisting of an amino acid, a sugar, a fatty acid, a vitamin, a pH buffer, a surfactant, a trace metal or salt or hydrate thereof, an amine compound, a growth factor, an agent to control osmolarity and/or ionic strength and/or maintain membrane potential, a flavin, a compound that participates in or is a product of the glycolytic pathway, an alcoholamine, a cyclic alcohol, a phospholipid or portion thereof, a salt of selenious acid, a divalent or trivalent cation, a pteridine derivative, a valeric acid, and/or a coenzyme.  
     
     
         93 . The method of  claim 64 , wherein said at least one cell is selected from the group consisting of a plant cell and an animal cell.  
     
     
         94 . The method of  claim 93 , wherein said cell is an animal cell.  
     
     
         95 . The method of  claim 94 , wherein said animal cell is selected from the group consisting of a mammalian cell, a fish cell, an insect cell, an amphibian cell, and an avian cell.  
     
     
         96 . The method of  claim 94 , wherein the animal cell is an epithelial cell.  
     
     
         97 . The method of  claim 95 , wherein said animal cell is a mammalian cell.  
     
     
         98 . The method of  claim 97 , wherein said mammalian cell is selected from the group consisting of a 293 cell, a PER-C6 cell, a CHO cell, a COS cell, a BHK cell, a HeLa cell, and a Sp2/0 cell.  
     
     
         99 . The method of  claim 98 , wherein said mammalian cell is a 293 cell.  
     
     
         100 . The method of  claim 64 , wherein said medium substantially lacks or does not contain transferrin.  
     
     
         101 . The method of  claim 64 , wherein said medium substantially lacks or does not contain insulin.  
     
     
         102 . The method of  claim 64 , wherein said medium substantially lacks or does not contain any polyanionic compound.  
     
     
         103 . The method of  claim 64 , wherein said medium substantially lacks or does not contain dextran sulfate.  
     
     
         104 . The method of  claim 64 , wherein said medium comprises butyrate.  
     
     
         105 . The method of  claim 64 , wherein said one or more macromolecules are introduced into at least about 20 percent of cells in said culture.  
     
     
         106 . The method of  claim 64 , wherein said one or more macromolecules are introduced into from about 20 percent to about 100 percent of cells in said culture.  
     
     
         107 . The method of  claim 64 , further comprising culturing said at least one cell in said medium after said introducing of said at least one macromolecule, without removing medium containing said macromolecules from said culture after said introducing and prior to said cultivation.  
     
     
         108 . The method of  claim 64 , further comprising determining, quantitating or measuring the effect of said introducing of said macromolecules on cells in said culture.  
     
     
         109 . The method of  claim 64 , further comprising determining, quantitating or measuring the effect of said introducing of said at least one nucleic acid molecule into said at least one cell.  
     
     
         110 . The method of  claim 109 , wherein the level or presence or absence of at least one polypeptide encoded by said at least one nucleic acid molecule in said at least one cell is measured, quantitated or determined.  
     
     
         111 . The method of  claim 64 , wherein LIPOFECTAMINE 2000 is admixed to said medium for said introducing.  
     
     
         112 . A kit for the cultivation and transfection of cells in vitro, said kit comprising the cell culture medium of  claim 1 .  
     
     
         113 . The kit of  claim 112 , further comprising one or more agents for the introduction of at least one nucleic acid molecule into at least one cell.  
     
     
         114 . The kit of  claim 112 , further comprising one or more nucleic acid molecules.  
     
     
         115 . The kit of  claim 114 , further comprising at least one cell.  
     
     
         116 . The kit of  claim 115 , further comprising instructions for culturing said at least one cell in culture and/or for introducing at least one nucleic acid molecule into at least one cell in culture.  
     
     
         117 . The kit of  claim 116 , wherein said culture is suspension culture.  
     
     
         118 . A composition comprising the cell culture medium of  claim 1  and at least one component selected from the group consisting of at least one eukaryotic cell, one or more agents for the introduction of at least one nucleic acid molecule into at least one cell, and one or more nucleic acid molecules.  
     
     
         119 . The composition of  claim 118 , wherein said at least one nucleic acid molecule includes deoxyribonucleic acid (DNA).  
     
     
         120 . The composition of  claim 118 , wherein each of said at least one nucleic acid molecule includes a vector.  
     
     
         121 . The composition of  claim 118 , wherein said medium is a serum-free medium.  
     
     
         122 . The composition of  claim 118 , wherein said medium is a chemically defined medium.  
     
     
         123 . The composition of  claim 118 , wherein the medium is a low protein or protein-free medium.  
     
     
         124 . The composition of  claim 118 , wherein said medium substantially lacks or does not contain material from an animal source or substantially lacks or does not contain animal derived materials.  
     
     
         125 . The composition of  claim 126 , wherein said medium comprises at least one compound is selected from the group consisting of a polyol, a hydroxypyridine derivative, 1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene, ethylenedi-amine-N,N′-tetramethylenephosphonic acid, trisuccin, an acidic saccharide (e.g. ferrous gluconate), a glycosaminoglycan, diethylenetriaminepentaacetic acid, nitrilotriacetic acid mono-, bis-, or tris-substituted 2,2′-bipyridine, a hydroxamate derivative (e.g. acetohydroxamic acid), an amino acid, deferoxamine, ferrioxamine, iron basic porphine, porphyrin and derivatives thereof, DOTA-lysine, a texaphyrin, a sapphyrin, a polyaminocarboxylic acid, an α-hydroxycarboxylic acid, a polyethylenecarbamate, picolinic acid, 4-pyridoxic acid, 3-hydroxy-2-pyridinemaltol, ethyl maltol, Ustilago ferrichrome, nicotinic acid-N-oxide and IRC011.  
     
     
         126 . The composition of  claim 126 , wherein said medium comprises a hydroxypyridine derivative.  
     
     
         127 . The composition of  claim 118 , wherein said hydroxypyridine derivative is selected from the group consisting of 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one, 1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4-one, 1-methyl-3-hydroxypyrid-2-one, 3-hydroxy-2(1H)-pyridinone, pylidoxal isonicotinyl hydrazone, nicotinic acid-N-oxide, and 2-hydroxy-nicotinic acid.  
     
     
         128 . The composition of  claim 126 , wherein said hydroxypyridine derivative is 2-hydroxypyridine-N-oxide.  
     
     
         129 . The composition of  claim 118 , said medium further comprising one or more ingredients selected from the group of ingredients consisting of at least one amino acid, at least one vitamin, at least one inorganic salt, at least one organic salt, at least one trace metal, at least one nucleotide, at least one buffering salt, at least one sugar, at least one lipid and at least one hormone.  
     
     
         130 . The composition of  claim 118 , wherein said at least one eukaryotic cell is selected from the group consisting of a plant cell and an animal cell.  
     
     
         131 . The composition of  claim 119 , wherein said cell is an animal cell.  
     
     
         132 . The composition of  claim 131 , wherein said animal cell is selected from the group consisting of a mammalian cell, a fish cell, an insect cell, an amphibian cell, and an avian cell.  
     
     
         133 . The composition of  claim 131  wherein said animal cell is an epithelial cell.  
     
     
         134 . The composition of  claim 132 , wherein said animal cell is a mammalian cell.  
     
     
         135 . The composition of  claim 134 , wherein said mammalian cell is selected from the group consisting of a 293 cell, a PER-C6 cell, a CHO cell, a COS cell, a BHK cell, a HeLa cell, and a Sp2/0 cell.  
     
     
         136 . The composition of  claim 135 , wherein said mammalian cell is a 293 cell.  
     
     
         137 . The composition of  claim 118 , wherein said medium substantially lacks or does not contain transferrin.  
     
     
         138 . The composition of  claim 118 , wherein said medium substantially lacks or does not contain insulin.  
     
     
         139 . The composition of  claim 118 , wherein said medium substantially lacks or does not contain any polyanionic compound.  
     
     
         140 . The composition of  claim 118 , wherein said medium substantially lacks or does not contain dextran sulfate.