title: Revival, characterization, and hepatitis B virus infection of cryopreserved human fetal hepatocytes.
abstract: Primary human hepatocytes are considered the ideal cellular model for in-vitro studies of liver-specific pathology, such as hepatitis B virus (HBV) infection. However, poor accessibility, limited cell numbers, and lot-to-lot variation of primary human hepatocytes limit their broad application. Human fetal hepatocytes were isolated from postmortem embryonic liver tissues by two-step collagenase perfusion and cryopreserved. A monolayer of cryopreserved human fetal hepatocytes was established by optimizing such conditions as cell density and viability and purification of viable cells by Percoll. Finally, revived human fetal hepatocytes were characterized and infected with HBV. A large number of viable human fetal hepatocytes could be isolated and cryopreserved, with seeding density and viability being critical for the establishment of a compact monolayer culture. Using low-viability cryopreserved human fetal hepatocytes, a typical monolayer was established by purification with Percoll. The revived cells were actively proliferative, showed identical morphologic characteristics to non-cryopreserved cells, and had a typical hepatic gene expression profile. Moreover, this optimized model was susceptible to HBV infection and could be used to screen entry inhibitors against HBV infection. In conclusion, these methods can be used on human fetal hepatocytes to provide a cell bank for studies of the early stages of HBV infection.
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors: Ming
authors: M
authors: Zhou
authors: Biomedical Center, Hubei University of Technology, Wuhan, Hubei, China.
authors: Yayun
authors: Y
authors: Huang
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors: Zhikui
authors: Z
authors: Cheng
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors: Fei
authors: F
authors: Zhao
authors: Department of Gynecology and Obstetrics, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China.
authors: Jiafu
authors: J
authors: Li
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors: Xiaoguang
authors: X
authors: Zhi
authors: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors: Xiaohui
authors: X
authors: Tian
authors: Biomedical Center, Hubei University of Technology, Wuhan, Hubei, China.
authors: Weihua
authors: W
authors: Sun
authors: Biomedical Center, Hubei University of Technology, Wuhan, Hubei, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China. Electronic address: hukgh@wh.iov.cn.
authors: Kanghong
authors: K
authors: Hu
mesh_terms: D002452:Cell Count; D018929:Cell Culture Techniques; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation; D006515:Hepatitis B virus / Q000254:growth & development*; D022781:Hepatocytes / Q000502:physiology* / Q000528:radiation effects / Q000821:virology*; D006801:Humans; D007231:Infant, Newborn
keywords: Cryopreservation; HBV infection; Human fetal hepatocyte; Revival