Background: Liver cell lines closely resembling primary hepatocyte are essential for research on hepatitis viruses and hepatocyte function. Currently used cell lines are derived from hepatic tumours and have altered gene expression.

Aims: The generation and characterisation of novel human hepatocyte lines (HHLs) derived from healthy human liver, retaining the primary hepatocyte phenotype.

Results: Primary hepatocytes were immortalised with Moloney's mouse leukaemia virus expressing E6 and E7 proteins of human papillomavirus, and cultures propagated long‐term. All HHLs contained markers of hepatocyte and biliary phenotype (cytokeratins 7, 8, 18 and 19), Cytochrome P450 and albumin. The HHLs did not express high levels of p53 or α‐fetoprotein. When grown in a collagen sandwich culture, or at the air–liquid interface, HHLs were maintained as monolayer whereas Huh‐7 and HepG2 formed thick layers. All HHLs showed increased capacity to bind recombinant hepatitis C virus‐like particles in comparison with Huh‐7 and HepG2. We also demonstrate that HHLs contained active gap junctions, and that the cells respond to stimulation with IFN‐α by upregulation of major histocompatibility complex (MHC)‐I and ‐II.

Conclusions: These HHLs retain primary hepatocyte phenotype and should be useful for investigating mechanisms of entry and replication of hepatotropic viruses, and should also be valuable in the study of hepatocyte biology and pathology.