title: [In vitro culture of hepatitis C virus (HCV) using immortalized hepatocyte].
abstract: 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.
authors.affiliation: WHO Collaborating Center of Viral Hepatitis, College of Medicine, The Catholic University of Korea, Seoul, Korea.
authors.forename: Jung Eun
authors.initials: JE
authors.lastname: Choi
authors.forename: Wonhee
authors.initials: W
authors.lastname: Hur
authors.forename: Ju Yeop
authors.initials: JY
authors.lastname: Shin
authors.forename: Lian Shu
authors.initials: LS
authors.lastname: Piao
authors.forename: Seung Kew
authors.initials: SK
authors.lastname: Yoon
mesh_terms: 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
chemical_list: D000957:Antigens, Viral, Tumor; D015347:RNA Probes; D012367:RNA, Viral