Spheroid cultures

Cryopreserved primary human hepatocytes (PHH) and crude non-parenchymal cells (NPC) were obtained from KaLy-Cell (KLC; Plobsheim, France) and Lonza (Basel, Switzerland). NPC were passaged as previously described22. Donor characteristics of PHH and NPC are listed in Table 1. PHH were seeded in 96-well Corning® Costar® Ultra-Low Attachment Plates (Merck, Kenilworth, NY) or 96-well Nunclon™ Sphera™ U-Shaped-Bottom Microplate (Thermo Fisher Scientific, Waltham, MA) with NPC at a cell ratio of 4:1 (1500:375) as previously described18,22. Spheroids were cultured in William's E medium (Thermo Fisher Scientific) supplemented with 2 mM L-glutamine (Sigma-Aldrich, Saint Louis, MO), 100 units/mL penicillin (Sigma-Aldrich), 100 µg/mL streptomycin (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), ITS X-100 (Thermo Fisher Scientific) and 10% fetal bovine serum (FBS; Thermo Fisher Scientific). The medium was changed 5 days after seeding and every 2–3 days thereafter using the medium described above without adding FBS. Induction of liver fibrosis and treatment of spheroids with various substances was initiated 7 days after seeding. The spheroids were harvested on day 14. Spheroids were cultured in 100 µl medium under standard cell culture conditions at 37 °C in a humidified incubator at 5% CO2. Unless otherwise stated, the combination of PHH from donor 1 and NPC from donor 6 was used.