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 . The medium of  claim 2 , wherein said FGF is aFGF. 
     
     
         4 . The medium of  claim 1 , wherein said agent causing an increase in intracellular levels of cAMP functions through interaction with a cellular G-protein. 
     
     
         5 . The medium of  claim 1 , wherein said agent causing an increase in intracellular levels of cAMP functions by directly increasing intracellular cAMP levels. 
     
     
         6 . The medium of  claim 1 , wherein said agent causing an increase in intracellular levels of cAMP functions by inhibiting a cAMP phosphodiesterase. 
     
     
         7 . The medium of  claim 1 , wherein said agent causing an increase in intracellular levels of cAMP is a .beta.-adrenergic receptor agonist. 
     
     
         8 . The medium of  claim 4 , wherein said agent is cholera toxin or forskolin. 
     
     
         9 . The medium of  claim 5 , wherein said agent is dibutyryl cAMP. 
     
     
         10 . The medium of  claim 6 , wherein said agent is isobutylmethylxanthine or theophylline. 
     
     
         11 . The medium of  claim 7 , wherein said agent is isoproterenol. 
     
     
         12 . The medium of  claim 1 , said medium further comprising ascorbic acid. 
     
     
         13 . The medium of  claim 1 , wherein said medium is a 1.times. medium formulation. 
     
     
         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 . The medium of  claim 15 , said medium further comprising ascorbic acid. 
     
     
         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 , NaHPO 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 - 72 . (canceled)