Immortalized Hepatocyte-like Cell Lines for HBV Studies

An immortalized hepatocyte-like cell line (imHC) [39 (link),40 (link)], HepG2 cell line (ATCC, Manassas, VA, USA) and HepaRG (Thermo Fisher Scientific, Waltham, MA, USA) were used. All cell lines were cultured in DMEM/F12 (Hyclone), 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin at 37 °C, 5% CO₂. Prior to hepatitis B virus (HBV) infection, 1 × 10⁶ cells were seeded onto each well of a 6-well plate overnight. The quiescent stage was achieved in differentiated medium [42 (link)] (Williams’ E medium, 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 50 µM hydrocotisone, 2 mM L-glutamine (GlutaMax, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 2% DMSO (Sigma Corporation of American, Ronkonkoma, NY, USA) for 2 weeks.