title: Long-term maintenance of human fetal hepatocytes and prolonged susceptibility to HBV infection by co-culture with non-parenchymal cells.
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.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Zhou
authors.forename: Fei
authors.initials: F
authors.lastname: Zhao
authors.forename: Jiafu
authors.initials: J
authors.lastname: Li
authors.forename: Zhikui
authors.initials: Z
authors.lastname: Cheng
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Tian
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Zhi
authors.forename: Yayun
authors.initials: Y
authors.lastname: Huang
authors.forename: Kanghong
authors.initials: K
authors.lastname: Hu
mesh_terms: D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D006515:Hepatitis B virus / Q000254:growth & development*; D022781:Hepatocytes / Q000502:physiology* / Q000821:virology*; D006801:Humans; D007231:Infant, Newborn; D014776:Virus Cultivation / Q000379:methods
chemical_list: D003470:Culture Media
keywords: Co-culture; Hepatitis B virus; Human fetal hepatocyte; Infection; Non-parenchymal cell