Isolation and Culture of Liver Cells

HSCs, liver sinusoidal endothelial cells (LSEC) and hepatocytes (HEP) were isolated for mouse liver and cultured according to validated protocols20 (link)21 (link). Kupffer cells (KC) were isolated by fluorescence-activated cell sorting after collagenase/pronase perfusion, using an F4/80 antibody (Invitrogen, USA). Cell purity and functionality were confirmed on morphology and by quantitative polymerase chain reaction (qPCR) for following marker genes (Supplementary Methods S2): Cyp3a11 (HEP), CD32b (LSEC), desmin and Acta2 (HSC)22 (link). 2 Hours after isolation (4 hours for HEP), cells were washed and either solvent or OCA was added to the medium at concentrations of 0.1, 1 and 10 μM, together with vehicle, 1 mg/mL TGF-β1 (R&D Systems, Wiesbaden-Nordenstadt, Germany), TNF-α or LPS. All cells were collected for molecular analysis 24 h after incubation, except for culture-activated HSC that were further stimulated for 7 days. LX2 cells were provided by Vijay H. Shah (Mayo Clinic, Rochester, NY), originally established by Scott Friedman23 (link).