Abstract
Within a few days of being isolated, primary human hepatocytes undergo a rapid dedifferentiation process and lose susceptibility to hepatitis B virus (HBV) infection in vitro. This fact has limited their further application. In this study, a convenient and feasible method of preventing this dedifferentiation was established, by co-culturing human fetal hepatocytes with hepatic non-parenchymal cells to maintain the differentiation features of human fetal hepatocytes. Isolated hepatic cells were seeded at a low density, and cultured in dimethyl sulfoxide-free medium for a month to allow rapid proliferation of non-parenchymal cells. Subsequently, 2% dimethyl sulfoxide was added to induce formation of typical hepatic islands, in which hepatocytic features could be further maintained for up to an additional 3 months. These hepatic islands, formed of piled-up hepatocytes, were surrounded and invaded by non-parenchymal cells. Protein expression profiles showed that the human fetal hepatocytes underwent a rapid maturation process, and the hepatocytic features were well preserved. Most importantly, these human fetal hepatocytes still exhibited susceptibility to HBV infection after long-term maintenance, for as long as 10 weeks. This co-culture method has overcome the pre-existing disadvantages of primary human hepatocytes for virological studies, and provides a valuable approach to long-term maintenance of primary human hepatocytes for studies of HBV infection for prolonged periods.