[In vitro culture of hepatitis C virus (HCV) using immortalized hepatocyte].
OBJECTIVE
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.
Choi
Jung Eun
JE
WHO Collaborating Center of Viral Hepatitis, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Hur
Wonhee
W
Shin
Ju Yeop
JY
Piao
Lian Shu
LS
Yoon
Seung Kew
SK
D000957:Antigens, Viral, Tumor / Q000235:genetics; D001483:Base Sequence; D015197:Carcinogenicity Tests; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D018914:Cells, Immobilized; D016174:Hepacivirus / Q000302:isolation & purification / Q000502:physiology*; D022781:Hepatocytes / Q000378:metabolism / Q000502:physiology / Q000821:virology*; D006801:Humans; D008111:Liver Function Tests; D008954:Models, Biological; D015347:RNA Probes; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000957:Antigens, Viral, Tumor; D015347:RNA Probes; D012367:RNA, Viral