Human liver epithelial cell line and culture media therefor

A method for transforming normal adult human liver epithelial cells to create a continuous human liver epithelial cell line, which comprises:

• culturing normal adult human liver epithelial cells in a cell culturing medium consisting essentially of an amount of the following ingredients effective to sustain proliferation of normal adult human liver epithelial cells:
• an effective amount of ornithine to allow proliferation of normal adult human liver epithelial cells;
• growth factor inactivated serum;
• retinoic acid in an amount of 1 to 300 nM;
• an effective amount of calcium ions high enough to promote cell proliferation but low enough to avoid cellular differentiation between 0.2 and 1 mM;
• an effective cell proliferation promoting amount of glucose;
• an effective amount of insulin to aid cells in glucose uptake;
• an effective cell proliferation promoting amount of hydrocortisone;
• an effective amount of epidermal growth factor to bind epidermal growth factor receptors on cells;
• an effective amount of transferrin to increase DNA synthesis in cells;
• an effective amount of cholera toxin to increase DNA synthesis in cells;
• an effective amount triiodothyronine to increase DNA synthesis in cells;
• an effective proliferation promoting amount of mammalian hormones;
• an effective cell proliferation promoting amount of medium conditioned by HepG2 hepatoblastoma cells;
• said medium containing no arginine;
• and
• contacting said cells with a vector containing an SV40-T antigen gene to effect transformation and to produce a continuous human liver epithelial cell line.

A continuous human liver epithelial cell line isolated from normal adult human liver epithelial cells wherein cells of said continuous cell line have the following properties:

• i) non-tumorigenic;
• ii) express SV40 T antigen;
• iii) 20% of the cells express albumin when grown in a serum-free medium;
• and
• iv) 30 to 40% of the cells express albumin when grown in a medium containing serum;
• that is the cell line deposited as ATCC CRL 10149.

A method for proliferative culture of normal adult human liver epithelial cells, which comprises:

• culturing normal adult human liver epithelial cells in a cell culturing medium, wherein said culturing medium sustains proliferation of normal adult human liver epithelial cells and consists essentially of the following ingredients in about the amounts indicated:
• ______________________________________ INGREDIENT mg/liter ______________________________________ L-cystine.2HCl 47.0 L-histidine 41.9 L-isoleucine 7.9 L-leucine 26.2 L-lysine.HCl 73.0 L-methionine 9.0 L-phenylalanine 9.9 L-threonine 23.8 L-tryptophan 4.1 L-tyrosine.2Na.2H.sub.2 O 15.7 L-valine 23.4 Nonessential Amino Acids L-alanine 17.8 L-asparagine.H.sub.2 O 30.0 aspartic acid 26.6 glutamic acid 29.4 L-glycine 15.0 L-proline 69.1 L-serine 21.0 Amino Acid Derivatives putrescine.2HCl 0.32 Water Soluble Vitamins and Coenzymes d-biotin 0.07 folic acid 1.32 DL-A-lipoic acid (thioctic) 0.21 nicotinamide 0.04 D-pantothenic acid 0.24 pyridoxine.HCl 0.06 riboflavin 0.04 thiamine.HCl 0.34 vitamin B12 (Cyanocobalamine) 1.36 Carbohydrates/Derivatives pyruvic acid 174.0 sodium acetate 295.6 Nucleic Acid Derivatives hypoxanthine 4.1 thymidine 0.7 Lipids/Derivative choline chloride 14.0 inositol 18.0 Bulk Inorganic Ions (Salts) NaCl 5844.0 KCl 283.3 Na.sub.2 HPO.sub.4 126.4 KH.sub.2 PO.sub.4 58.5 MgSO.sub.4 19.3 MgCl.sub.2.6H.sub.2 O 105.7 Inorganic Trace Elements CuSO.sub.4.5H.sub.2 O 0.002 Buffers and Indicators HaHCO.sub.3 1176.0 HEPES buffer 7149.0 phenol red 1.1 L-glutamine 2 mM Growth factors insulin 10 .mu.g/ml hydrocortisone 0.2 .mu.M epidermal growth factor 5.0 ng/ml transferrin 10 .mu.g/ml Other ingredients phosphoethanolamine 0.5 .mu.M cholera toxin 25 .mu.g/ml triiodothyronine 10 nM retinoic acid 10 nM ornithine 2 mM CaCl.sub.2 0.4 mM glucose 2.0 mg/ml bovine pituitary extract 7.5 .mu.g/ml "Ex-cyte .RTM." 312 .mu.g/ml FeSO.sub.4.7H.sub.2 O 2.7 .mu.M ZnSO.sub.4.7H.sub.2 O 0.5 .mu.M Chemically denatured Fetal Bovine Serum 10% Na.sub.2 SeO.sub.3 3.0 .times. 10.sup.-8 M MnCl.sub.2.4H.sub.2 O 1.0 nM Na.sub.2 SiO.sub.3.9H.sub.2 O 5.0 .times. 10.sup.-7 M (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O 1.0 nM NH.sub.4 VO.sub.3 5.0 nM NiSO.sub.4.6H.sub.2 O 0.5 nM SnCl.sub.2.2H.sub.2 O 0.4 nM Gentamicin 50 .mu.g/ml. ______________________________________

A continuous human liver epithelial cell line isolated from normal adult human liver eptithelial cells, wherein cells of said continuous cell line have the following properties:

• i) non-tumorigenic;
• ii) life span in vitro of at least 40 population doublings;
• iii) express SV40 T antigen;
• iv) 20% of the cells express albumin when grown in a serum-free medium;
• and
• v) 30 to 40% of the cells express albumin when grown in a medium containing serum.