Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocytelike morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies.H epatitis B, one of the major infectious diseases worldwide, is caused by a small enveloped DNA virus, the hepatitis B virus (HBV). HBV exhibits a very narrow host range and shows a strong tropism for liver parenchymal cells. It has therefore been assumed that susceptibility to HBV infection is restricted to differentiated cells. Accordingly, it was found that only human hepatocyte primary cultures were susceptible to HBV infection (1-4). However, the use of this model is hampered by the limited availability and the inherent variability of human liver material. Even though several human hepatoma-derived cell lines support HBV replication after HBV DNA transfection (5-9), none of them are susceptible to HBV infection.We describe here a hepatoma-derived cell line that expresses a representative panel of liver-specific genes and is susceptible to HBV infection. This goal was achieved by combining an original selection procedure applied early after the cell line establishment in culture and the use of appropriate culture conditions, allowing the commitment of these cells to an optimal differentiation status. MethodsIsolation of the Cell Line and Culture Conditions. Cells were isolated from a liver tumor of a female patient suffering from hepatocarcinoma and hepatitis C infection. All experimental procedures were conducted in conformity with French laws and regulations and were approved by the National Ethics Committee. The samples were minced into small pieces, washed with Hepes buffer (pH 7.7; 140 mM NaCl͞2.68 mM KCl͞0.2 mM Na 2 HPO 4 ͞10 mM Hepes), and digested with 0.025% collagenase D (Boehringer Mannheim) diluted in the same buffer supplemented with 0.075% CaCl 2 under gentle stirring at 37°C. The cell suspension was washed twice in Hepes buffer and resuspended in a William's E medium supplemented with 10% FCS, 100 units͞ml penicillin, 100 g͞ml streptomycin, 5 g͞ml insulin, and 5 ϫ 10 Ϫ7 M hydrocortisone hemisuccinate. Cell suspension was distributed in several dishes without any coating feeder layer. After several weeks, cell growth was sufficient to fulfill the culture dishes. Cells appeared well differentiated, with a hepatocyte-like ...

ABSTRACT:Most human hepatocyte cell lines lack a substantial set of liverspecific functions, especially major cytochrome P450 (P450)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the HepaRG cells, derived from a human hepatocellular carcinoma, which exhibit a high differentiation pattern after 2 weeks at confluency to determine whether they could mimic human hepatocytes for drug metabolism and toxicity studies. We show that when passaged at low density, these cells reversed to an undifferentiated morphology, actively divided, and, after having reached confluency, formed typical hepatocyte-like colonies surrounded by biliary epithelial-like cells. By contrast, when seeded at high density, hepatocyte-like clusters retained their typical differentiated morphology. Transcripts of various nuclear receptors (aryl hydrocarbon receptor, pregnane X receptor, constitutive androstane receptor, peroxisome proliferator-activated receptor ␣), P450s (CYP1A2, 2C9, 2D6, 2E1, 3A4), phase 2 enzymes (UGT1A1, GSTA1, GSTA4, GSTM1), and other liver-specific functions were estimated by reverse transcriptase-quantitative polymerase chain reaction and were found to be expressed, for most of them, at comparable levels in both confluent differentiated and high-density differentiated HepaRG cells and in cultured primary human hepatocytes. For several transcripts, the levels were strongly increased in the presence of 2% dimethyl sulfoxide. Measurement of basal activities of several P450s and their response to prototypical inducers as well as analysis of metabolic profiles and cytotoxicity of several compounds confirmed the functional resemblance of HepaRG cells to primary cultured human hepatocytes. In conclusion, HepaRG cells constitute the first human hepatoma cell line expressing high levels of the major P450s involved in xenobiotic metabolism and represent a reliable surrogate to human hepatocytes for drug metabolism and toxicity studies.

No abstract