Korean J Gastroenterol.  2008 Sep;52(3):150-160.

In vitro Culture of Hepatitis C Virus (HCV) Using Immortalized Hepatocyte

• Choi JE
• Hur W
• Shin JY
• Piao LS
• Yoon SK

Abstract

BACKGROUND/AIMS: It is essential to develop an in vitro culture model of primary hepatocytes for the study of hepatocellular function and the pathogenesis of hepatitis C virus (HCV) infection. In this study, we have established the immortalized primary human hepatocyte (IPHH) and performed in vitro culture of HCV derived from human patient.
METHODS
Primary human hepatocytes were isolated from surgically resected liver tissue and then were immortalized by transfection with the SV40 large T antigen. The characterization of the IPHH during culture was analyzed by immunocytochemistry, RT-PCR, Western blot, ELISA, and soft agar assay. Next, sera and/or liver tissue homogenates from surgically resected liver tissues of patients with HCV infection were inoculated for the culture of HCV in IPHH. After HCV RNA extraction from IPHH and culture media, positive or negative stranded HCV RNA was examined by specific nest RT-PCR.
RESULTS
IPHH expressed liver-associated proteins but did not express alpha-fetoprotein. Also IPHH showed ammonia removal activity. With regard to its malignant potential, colony formation in soft agar assay was not observed. Next, positive and negative stranded HCV RNAs in IPHH infected with patient's sera plus liver tissue homogenates were clearly detected whereas those in IPHH infected with only patient's sera were not detected.
CONCLUSIONS
These results demonstrated the phenotypic characteristics of IPHH and the feasibility in vitro culture system of HCV infected human samples. This system might be useful for study of pathogenesis of HCV infection or hepatocyte-based applications.

Keyword

Primary hepatocyte; Immortalization; Hepatitis C virus; Culture

MeSH Terms

Antigens, Viral, Tumor/genetics
Base Sequence
Carcinogenicity Tests
Cell Culture Techniques
Cells, Cultured
Cells, Immobilized
Hepacivirus/isolation & purification/*physiology
Hepatocytes/metabolism/physiology/*virology
Humans
Liver Function Tests
Models, Biological
RNA Probes
RNA, Viral/analysis
Reverse Transcriptase Polymerase Chain Reaction