Defined systems for epithelial cell culture and use thereof

Abstract

The present invention provides cell culture media formulations which support the in vitro cultivation of animal epithelial cells. The media comprise at least one fibroblast growth factor (FGF) and at least one agent that induces increased intracellular cAMP levels, and optionally comprise ascorbic acid. The present invention also provides methods of cultivating animal epithelial cells in vitro using these cell culture media formulations, kits comprising the media, cell culture compositions comprising the culture media and an animal epithelial cell, and compositions that may be used as replacements for organ or gland extracts in animal cell culture media.

1 . A serum-free cell culture medium comprising a fibroblast growth factor (FGF) and an agent causing an increase in intracellular levels of cyclic adenosine monophosphate (cAMP), wherein said medium is capable of supporting the cultivation of an animal epithelial cell in vitro.  
     
     
         2 . The medium of  claim 1 , wherein said FGF is selected from the group consisting of FGF-1 (aFGF), FGF-2 (bFGF) and FGF-7 (KGF).  
     
     
         3 - 6 . (canceled)  
     
     
         7 . The medium of  claim 1 , wherein said agent causing an increase in intracellular levels of cAMP is a β-adrenergic receptor agonist.  
     
     
         8 . The medium of  claim 1 , wherein said agent is cholera toxin or forskolin.  
     
     
         9 - 10 . (canceled)  
     
     
         11 . The medium of  claim 7 , wherein said agent is isoproterenol.  
     
     
         12 . The medium of  claim 1 , said medium further comprising ascorbic acid.  
     
     
         13 . (canceled)  
     
     
         14 . The cell culture medium of  claim 1 , wherein said medium formulation is a 10× concentrated medium formulation.  
     
     
         15 . The cell culture medium of  claim 1 , said medium further comprising one or more ingredients selected from the group of ingredients consisting of an amino acid, a vitamin, an inorganic salt, adenine, ethanolamine, D-glucose, epidermal growth factor (EGF), heparin, N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES), hydrocortisone, insulin, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, T3, thymidine and transferrin.  
     
     
         16 . (canceled)  
     
     
         17 . The cell culture medium of  claim 15 , wherein said amino acid ingredient comprises one or more amino acids selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, 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.  
     
     
         18 . The cell culture medium of  claim 15 , wherein said vitamin ingredient comprises one or more vitamins selected from the group consisting of biotin, choline chloride, D-Ca ++ -pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine and vitamin B 12 .  
     
     
         19 . The cell culture medium of  claim 15 , wherein said inorganic salt ingredient comprises one or more inorganic salts selected from the group consisting of a calcium salt, CuSO 4 , FeSO 4 , KCl, a magnesium salt, a manganese salt, sodium acetate, NaCl, NaHCO 3 , Na 2 HPO 4 , Na 2 SO 4 , a selenium salt, a silicon salt, a molybdenum salt, a vanadium salt, a nickel salt, a tin salt and a zinc salt.  
     
     
         20 . A cell culture medium comprising the ingredients adenine, ethanolamine, D-glucose, N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES), hydrocortisone, insulin, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, T3, thymidine, transferrin, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, biotin, choline chloride, D-Ca ++ -pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine, vitamin B 12 , a calcium salt, CuSO 4 , FeSO 4 , KCl, a magnesium salt, a manganese salt, sodium acetate, NaCl, NaHCO 3 , Na 2 HPO 4 , Na 2 SO 4 , a selenium salt, a silicon salt, a molybdenum salt, a vanadium salt, a nickel salt, a tin salt, and a zinc salt, 
 wherein each ingredient is present in an amount which supports the cultivation of an animal epithelial cell in vitro.    
     
     
         21 . The medium of  claim 20 , said medium further comprising heparin, epidermal growth factor (EGF), a fibroblast growth factor (FGF) and an agent causing an increase in intracellular levels of cyclic adenosine monophosphate (cAMP).  
     
     
         22 . (canceled)  
     
     
         23 . A cell culture medium obtained by combining a basal medium with heparin, EGF, a fibroblast growth factor (FGF) and an agent causing an increase in intracellular levels of cyclic adenosine monophosphate (cAMP), wherein said medium is capable of supporting the cultivation of an animal epithelial cell in vitro.  
     
     
         24 - 29 . (canceled)  
     
     
         30 . The medium of  claim 23 , wherein said agent is cholera toxin or forskolin.  
     
     
         31 - 32 . (canceled)  
     
     
         33 . The medium of  claim 23 , wherein said agent is isoproterenol.  
     
     
         34 . The animal cell culture medium obtained according to  claim 23 , wherein said basal medium is obtained by combining one or more additional ingredients selected from the group consisting of adenine, ethanolamine, D-glucose, epidermal growth factor (EGF), heparin, N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES), hydrocortisone, insulin, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, T3, thymidine, transferrin, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, biotin, choline chloride, D-Ca ++ -pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine, vitamin B 12 , a calcium salt, CuSO 4 , FeSO 4 , KCl, a magnesium salt, a manganese salt, sodium acetate, NaCl, NaHCO 3 , Na 2 HPO 4 , Na 2 SO 4 , a selenium salt, a silicon salt, a molybdenum salt, a vanadium salt, a nickel salt, a tin salt and a zinc salt, 
 wherein each ingredient is added in an amount which supports the cultivation of an animal epithelial cell in vitro.    
     
     
         35 - 40 . (canceled)  
     
     
         41 . A method of cultivating an animal epithelial cell comprising the steps of 
 (a) contacting said cell with the cell culture medium of  claim 1;  and    (b) cultivating said cell under conditions suitable to support cultivation of said cell.    
     
     
         42 - 46 . (canceled)  
     
     
         47 . A kit for the culture of an animal epithelial cell, said kit comprising a carrier means having in close confinement therein one or more container means, wherein a first container means contains the culture medium of  claim 1 .  
     
     
         48 . A kit for the culture of an animal epithelial cell, said kit comprising a carrier means having in close confinement therein one or more container means, wherein a first container means contains the culture medium of  claim 23 .  
     
     
         49 . A kit for the culture of an animal epithelial cell, said kit comprising a carrier means having in close confinement therein one or more container means, wherein a first container means contains the culture medium of  claim 20  and a second carrier means contains at least one component selected from the group consisting of heparin, epidermal growth factor (EGF), a fibroblast growth factor (FGF), an agent causing an increase in intracellular levels of cyclic adenosine monophosphate (cAMP), and ascorbic acid.  
     
     
         50 . A composition comprising the culture medium of  claim 1  and an animal epithelial cell.  
     
     
         51 - 55 . (canceled)  
     
     
         56 . A composition comprising heparin, EGF, a fibroblast growth factor (FGF), and an agent causing an increase in intracellular levels of cyclic adenosine monophosphate (cAMP), wherein said composition replaces an organ or gland extract in an animal cell culture medium.  
     
     
         57 . The composition of  claim 56 , further comprising ascorbic acid.  
     
     
         58 - 63 . (canceled)  
     
     
         64 . The composition of  claim 56 , wherein said agent is cholera toxin or forskolin.  
     
     
         65 - 66 . (canceled)  
     
     
         67 . The composition of  claim 56 , wherein said agent is isoproterenol.  
     
     
         68 . The composition of  claim 56 , wherein said composition is a 1×-1000× concentrated formulation.  
     
     
         69 - 72 . (canceled)