Serum-free PFMR-4 medium vas found to support the long-term multiplication of normal human liver epithelial cells. Human liver epithelial cells from three separate autopsy cases were induced to divide for up to 12 divisions (4 passages). At passage 4 these cells were positive for cytokeratin 18 and a small proportion was also positive for albumin. Additionally, human hepatocytes in primary culture were transfected with SV40 large T antigen gene. The life span of these cells was extended to 40 population doublings at which time they became quiescent. These transfected cells were also positive for cytokeratin 18, T antigen and a small number were positive for albumin. Infection with a zip-neo virus containing the SV40 large T antigen gene has yielded a cell line that has also shown an extended doubling potential of greater than 40 population doublings and has to date shown no indications of senescence. These cells are positive for cytokeratin 18 and T antigen. Differentiation studies with the normal and SV40 transformed human liver epithelial cells using intrasplenic nude mouse injections and in vitro 3-- dimensional collagen matrices are being employed in order to induce the liver epithelial cells to re-express a more differentiated phenotype.
