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 - 72 . (canceled) 
     
     
         73 . A method of cultivating a keratinocyte cell comprising the steps of:
 (a) contacting said cell with a defined, 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 the medium is capable of supporting the cultivation of an animal epithelial cell in vitro; and   (b) cultivating said cell under conditions suitable to support cultivation of said cell.   
     
     
         74 . The method of  claim 73 , where said FGF in said medium is selected from the group consisting of FGF-1 (aFGF), FGF-2 (bFGF) and FGF-7 (KGF). 
     
     
         75 . The method of  claim 73 , wherein said FGF in said medium is aFGF. 
     
     
         76 . The method of  claim 75 , wherein the concentration of said aFGF in said medium is selected from the group consisting of about 0.0001 to about 10 mg/L, about 0.001 to about 0.1 mg/L, and about 0.005 mg/L. 
     
     
         77 . The method of  claim 75 , wherein the concentration of said aFGF in said medium is about 0.005 mg/L. 
     
     
         78 . The method of  claim 73 , wherein said agent in said medium causing an increase in intracellular levels of cAMP is a β-adrenergic receptor agonist. 
     
     
         79 . The method of  claim 73 , wherein said agent in said medium is isoproterenol. 
     
     
         80 . The method of  claim 79 , wherein the concentration of said isoproterenol in said medium is selected from the group consisting of about 0.01 to about 10 mg/L, about 0.1 to about 5 mg/L, and about 0.25 mg/L. 
     
     
         81 . The method of  claim 79 , wherein said isoproterenol in said medium is at a concentration of about 0.25 mg/L. 
     
     
         82 . The method of  claim 79 , wherein said medium further comprises 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, triiodothyronine (T3), thymidine and transferrin. 
     
     
         83 . The method of  claim 82 , wherein said medium comprises EGF at a concentration which is selected from the group consisting of about 0.00001 to about 10 mg/L, about 0.0001 to about 0.1 mg/L, and about 0.0002 mg/L. 
     
     
         84 . The method of  claim 82 , wherein said EGF in said medium is at a concentration of about 0.0002 mg/L. 
     
     
         85 . The method of  claim 82 , wherein said medium comprises heparin and the said heparin in said medium is at a concentration which is selected from the group consisting of about 1 to about 500 U.S.P. units/liter, about 5 to about 50 U.S.P. units/liter, and about 50 U.S.P. units/liter. 
     
     
         86 . The method of  claim 85 , wherein said heparin in said medium is at a concentration of about 50 U.S.P. units/liter. 
     
     
         87 . The method of  claim 82 , wherein said medium comprises an amino acid and the said amino acid is 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. 
     
     
         88 . The method of  claim 82 , wherein said medium comprises a vitamin and the said vitamin is selected from the group consisting of biotin, choline chloride, D-Ca ++ -pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, riboflavin, thiamine and vitamin B 12 . 
     
     
         89 . The method of  claim 82 , wherein said medium comprises an inorganic and the said inorganic salt is 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. 
     
     
         90 . The method of  claim 82 , wherein said medium further comprises ascorbic acid. 
     
     
         91 . The method of  claim 90 , wherein said ascorbic acid in said medium is L-ascorbic acid phosphate, magnesium salt. 
     
     
         92 . The method of  claim 91 , wherein the concentration of said L-ascorbic acid phosphate, magnesium salt, in said medium is selected from the group consisting of about 0.001 to about 10 mg/L, about 0.01 to about 5 mg/L, and about 0.1 mg/L.