DEVELOPMENT OF NOVEL HUMAN HEPATOCYTE CELL LINES.

A.Svanberg¹, ², J.O'Dwyer1, ², P.Goldfarb², J.Sinden¹

¹ReNeuron Ltd, 10 Nugent road, Surrey Research Park, Guildford, Surrey GU2 7AF, UK
²School of Biomedical & Molecular Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK

Human hepatocyte cell lines expressing the full range of normal hepatocyte functions including the appropriate xenobiotic-metabolising enzymes would be of great value in the study of drug metabolism and toxicity. Primary human liver tissue from a 15 week fetus was obtained according to UK ethical compliance, dissociated with collagenase and expanded on collagen coated tissue culture plastic ware. Arginine-free William's E media supplemented with 10% dialysed fetal bovine serum (dFBS), 0.4mM ornithine, 5.5µM hydrocortisone, 100nM insulin, 2mM glutamine, 20ng/ml epidermal growth factor (EGF), 100 IU/ml Penicillin and Streptomycin was used to discourage the growth of fibroblasts and other non-parenchymal liver cell types. Actively dividing primary cells were immortalized by retroviral infection with a human c-myc gene fused to a mutated estradiol receptor, which can be activated by 4-hydroxytamoxifen (4-OHT). In this system, the presence of 4-OHT drives the hepatocytes to undergo proliferation. Following infection, the cells were placed in selective medium and surviving clones picked. Analysis of these proliferating cell lines showed that they are positive for the immortalizing transgene and for hepatocyte markers including albumin and cytokeratin-18In depth analysis of further hepatocyte markers using QRT-PCR based assays is currently in progress to confirm that these cell lines behave as mature human hepatocytes in the absence of 4-OHT and growth factors.

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