Crucial Factors for de novo Establishment of Long-term Primary Culture of Tree Shrew Hepatocytes

Tree shrews（Tupaia Belangeri）are phenogenetically close to humans and primates and generally recognized as a prominent small animal model for studies of heptitis virus type B (HBV). Isolating and culturing tree shrew hepatocytes is the first key step toward the cellular model of HBV infection in vitro. Due to the lack of details in previous reports, establishing the model has been a matter of arbitary experiences. In this study, we validated the superiority of perfusion over mechanic dispersion for hepatocyte separation and isolation. Subsequent cultures showed that dimethyl sulphoxide (DMSO) could suppress the growth of fibroblast-like cells and maintain the hepatocytes in the differentiated status. Furthermore, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) could sustain growth and survival of the hepatocytes in the long-term culture. The combination of DMSO and HGF/EGF could maintain the hepatocytes in a longer and more stable differentiated status with clear trend to form liver sinus-like structures. Following this detailed method as a laboratory routine would permit plentiful starting material for study of HBV infection in vitro and drug-screening, as well as studies on hepatitis viruses type C and D, and herpes simplex virus.