Type 1 collagen coated dishes

HSDCs were enriched as previously described [17 (link)] with some modifications. Briefly, fetal liver tissues were removed from pregnant C57BL/6 mice at embryonic day 12.5. Dissociated liver cells were centrifuged at 500 rpm for 3 min in cold phosphate-buffered saline (PBS) and then cultured on six-well ultralow attachment plates (Corning, Corning, NY, USA) at a density of 5 × 10⁵ cells per milliliter in standard Dulbecco’s modified Eagle’s medium/F12 (Sigma, St Louis, MO, USA) supplemented with B27 (Gibco, Grand Island, NY, USA), insulin-transferrin-selenium X (ITS-X, Gibco), 10 mmol/L HEPES (Gibco), antibiotics, 20 ng/mL epidermal growth factor (EGF; Sigma), 20 ng/mL basic fibroblast growth factor (bFGF; R&D Systems, Minneapolis, MN, USA), and 20 ng/mL hepatocyte growth factor (HGF; Sigma). Hepatic spheroids were collected at day 6 by centrifugation at 300 rpm for 2 min and plated on type I collagen-coated dishes (Becton Dickinson, San Jose, CA, USA). After growing to subconfluency, the HSDCs were passaged using Accutase (Gibco), and the HGF concentration was reduced to 10 ng/mL after plating on collagen-coated dishes.

LPCs were isolated as described previously [24 (link)]. Briefly, liver tissues were removed from CDE diet-fed mice after in-situ perfusion using a two-step collagenase perfusion method and then were incubated with 1 mg/ml collagenase D (Roche) and 1 mg/ml pronase (Roche) at 37 °C for 30 min. Non-parenchymal cells (NPCs) were separated from hepatocytes by repeated low-speed centrifugation. Next, NPCs were collected and suspended in a discontinuous gradient of 20% and 50% Percoll (GE Healthcare, Pittsburgh, PA, USA) and centrifuged continuously at 1400 × g for 20 min to enrich LPCs. The enriched LPCs were labelled with APC-conjugated anti-EpCAM and FITC-conjugated anti-CD45 (eBioscience, San Diego, CA, USA) antibodies, and EpCAM⁺CD45⁻ cells were isolated by fluorescence-activated cell sorting. These LPCs were cultured in type I collagen-coated dishes (BD Biosciences, San Jose, CA, USA). The standard culture medium for LPCs was DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum, 1 μg/ml insulin (Sigma-Aldrich), 1 × 10^− 7 mol/L dexamethasone (Sigma-Aldrich), 1% penicillin/streptomycin (Invitrogen), 50 ng/ml hepatocyte growth factor (PeproTech, Rocky Hill, NJ, USA), 20 ng/ml epidermal growth factor (PeproTech), 20 ng/ml FGF (PeproTech) and 1× insulin–Transferrin–Selenium–Ethanolamine (Invitrogen).