PHH were obtained from Lonza (Walkersville, MD, USA), and HSCs, KCs and LECs were provided by Samsara (San Diego, CA, USA). PHH, HSCs and LECs were seeded in red phenolfree William E medium (A12176; Gibco, Invitrogen Corp., Waltham, MA, USA) supplemented with 5% fetal bovine serum, primary hepatocyte thawing and plating supplements solution (CM3000; ThermoFisher Scientific, Waltham, MA, USA) and 1% of a non-essential amino acids solution (11140050; Gibco).
PHHs, LECs and hepatic stellate primary cells were embedded at 0.5×106, 0.1×106 and  0.1×106 cells/mL, respectively, in a half RAFTTM 3D collagen hydrogel (016-0R92; Lonza) in 96-well plates, as recommended by the provider. Cells were cocultured in DMEM (low-glucose, pyruvate, no glutamine, no red phenol; 11054; Gibco) supplemented with bovine serum/ free fatty acid-free 0.125% solution (A7030; Sigma-Aldrich, St Louis, MO, USA), 50 U/mL penicillin/streptomycin (15140122; Gibco), dexamethasone 0.1 mM, ITS-G 1X (41400045; Gibco), GlutaMAXTM 1X (35050061; Gibco), HEPES 15 mM (15630080; Gibco), non-essential amino acids solution 1X (11140050; Gibco), acid L-ascorbic 2.5 mg/mL (A4403; Sigma-Aldrich) and glucagon 0.1 mg/mL (G2044; Sigma-Aldrich). This liver coculture medium is named “healthy media”. After 3 days of culture, the cocultures were incubated either in healthy media or in a media mimicking the NASH environment and supplemented with glucose 25 mM (Sigma-Aldrich), oleate acid 40 mM (SigmaAldrich), palmitate acid at 60 mM (Cayman Chemical, Ann Arbor, MI, USA) and tumor necrosis factor-a 5 ng/mL (PeproTech, Cranbury, NJ, USA). Healthy and NASH medium were changed every 2-3 days. At day 6, KCs were added to the coculture at 0.2×106 cells/mL. Supernatants and embedded cells were sampled on days 3, 6, 8, 10, 13 and 15 for analysis.