Primary hepatocyte culture supports hepatitis C virus replication: A model for infection-associated hepatocarcinogenesis

Abstract

Analysis of progressive changes in hepatic gene expression that underlie hepatocarcinogenesis following hepatitis C virus (HCV) infection require examination of long-term cultures of normally differentiating primary human hepatocytes. We report a culture system of primary hepatocytes that support productive replication of infectious HCV. Hepatic functions were analyzed by reverse-transcription polymerase chain reaction amplification of total cell RNA from cultures maintained in serum-free defined medium for up to 190 days. Sustained hepatic function was assessed by expression of albumin, alpha-fetoprotein, cytochrome P4502E1, cytokeratin-18, type-1 collagen, transforming growth factor-beta 1, matrix metalloproteinase-2 (MMP-2), MMP-13, and interferon alpha-receptors 1 and 2. Normally differentiated human primary hepatocytes supported productive replication of infectious clones of HCV genotypes 1a, 1b, and 2a; virus infection was inhibited by antibodies against CD81 virus entry factor. Virus released into the culture media of HCVinfected primary hepatocytes repeatedly passage to naïve hepatocytes. Replication of the three HCV genotypes shows interferon sensitivity observed in natural infections. Conclusion: Sustained cultures of physiologic host cells for the propagation of infectious HCV strains should accelerate studies of host response to HCV infection and progressive liver disease.

Mechanism of Hepatitis C Virus (HCV)-induced Osteopontin and Its Role in Epithelial to Mesenchymal Transition of Hepatocytes

“…Briefly, the hepatic stellate cell line (CFSC-8B) was used as a feeder cell layer, and a freshly isolated human hepatocyte suspension (Cambrex, Walkersville, MD) was seeded over the feeder cell line in a hepatocytedefined medium, as described previously (30). Primary hepatocyte cultures form spherical masses after 30 days in co-culture.…”

“…The in vitro transcribed J6/JFH-1 plasmid was transfected into primary human hepatocytes (PHH) as described previously (30). To verify if HCV particles were released in the culture supernatant of transfected PHH, conditioned media were collected and used to infect naive PHH as described previously (30). Total cellular RNA was extracted using TRIzol (Invitrogen), and HCV copy number was analyzed using quantitative RT-PCR (data not shown).…”

“…The hepatocyte cultures containing spherical masses were harvested with 0.05% trypsin in hepatocyte-defined medium (supplemented with 1% FBS), reseeded in 6-well plates, and propagated in hepatocyte-defined medium. The in vitro transcribed J6/JFH-1 plasmid was transfected into primary human hepatocytes (PHH) as described previously (30). To verify if HCV particles were released in the culture supernatant of transfected PHH, conditioned media were collected and used to infect naive PHH as described previously (30).…”

MicroRNA silencing of tumor suppressor DLC-1 promotes efficient hepatitis C virus replication in primary human hepatocytes

“…Long-term cultures of primary human hepatocytes were maintained in a defined medium that supported productive replication of HCV as described. 12 RNA Isolation, Reverse-Transcription, and Quantitative PCR. Total cell RNA was extracted using TRIzol LS Reagent (Invitrogen) according to the manufacturer's instructions and processed for viral RNA quantitation by way of nested reverse-transcription polymerase chain reaction (RT-PCR).…”

“…Primary human hepatocytes were transfected with HCV genotypes 1a, 1b, and 2a (1 lg/ 10 6 cells) as described. 12 Virus released in the culture medium was filtered through 0.25-lm filters from infected cells.…”