Iscove basal medium

1000 cells of phHSC were well mixed into reduced growth factor BME2 (Basement Membrane Extract, Type 2; Pathclear) and seeded on 24-well plates. After polymerization of BME2, classical culture medium was added to the cells with or without TGF-β (10 ng/ml). Cells were passaged by mechanical dissociation into small fragments by trituration using a plastic pipet with splitting medium of Iscove basal medium (Merck Biochrom, Germany) containing 1% HEPES (1M; Thermo Fisher Scientific, USA), 1% GlutaMAX Supplement (Thermo Fisher Scientific, USA), and 0.2% Primocin (InvivoGen, France), and transferred to fresh BME2 or plastic plates where indicated.

phHSC were isolated by the Biobank of the Department of General, Visceral and Transplant Surgery in LMU using a modified two-step collagenase perfusion procedure followed by low-speed centrifugation. The supernatant from the first centrifugation at 72× g, which contained the non-parenchymal cell (NPC) fraction, was then used for the isolation of phHSC using a three-layer density gradient [26 (link)]. phHSC were cultured in Iscove basal medium (Merck Biochrom, Germany) containing 1% sodium pyruvat (Merck Biochrom, Germany), 1% NEAA (non essential amino acids; Merck Biochrom, Germany), 1% L-glutamin (Sigma-Aldrich, Germany), 1% antibiotics (Sigma-Aldrich, Germany), and 10% fetal bovine serum (PAN-Biotech, Germany) in a humidified atmosphere with 5% CO₂ at 37 ^°C as described previously [27 ].

Cells were kept in Iscove Basal Medium (Merck Millipore; phHSCs) or in Dulbecco’s modified Eagle medium (Sigma-Aldrich, Darmstadt, Germany; pmHSCs and LX-2 cells) containing 2% (LX-2) or 10% (phHSCs and pmHSCs) fetal bovine serum (Merck Millipore) and antibiotics (Sigma-Aldrich). All cells were cultivated in a humidified atmosphere with 5% CO₂ and 21% O₂ at 37°C. LX-2 cells were stimulated with 10 ng/mL TGF-β (PeproTech, Hamburg, Germany) or 30 ng/mL PDGF-BB (Biomol, Hamburg, Germany) and co-incubated with 1000 nM CEP-1347 (Tocris, Bristol, United Kingdom) for 1 hour, 24 hours, or 48 hours, where indicated. phHSCs and pmHSCs were cultured on uncovered plastic dishes for up to 13 days to induce a spontaneous activation. For these long-term experiments, culture media in phHSCs were replaced with fresh media and CEP-1347 3 times a week.