Figure 1

Overview of spheroid generation and study design. Spheroids were generated by incubating 2000 primary human hepatocytes for 7 days in 384- or 96-well ultra-low attachment plates. Initiation of spontaneous cell aggregation was observed within one day after seeding. Hepatocytes from five individual donors were used in two independent experiments for WT-1, WT-2 and TM6SF2 E67K-2, and in one independent experiment for WT-3 and TM6SF2 E167K-1, respectively (data from repeat experiments shown in Supplementary Fig. S7). For each experiment, an individual vial of cryopreserved primary hepatocytes was used for spheroid preparation. Bright-field images representatively show spheroid formation by spontaneous aggregation of hepatocytes obtained from donor WT-1. Hepatocytes from all other donors showed similar spheroid formation behavior, yielding fully formed spheroids after 7 days. Arrows in the timeline depict medium changes for incubation with FA and samplings for bright field (Figs 2A–C and S3) and confocal (Figs 5B and S4) imaging, intracellular ATP content (Fig. 3), intracellular and secreted APOB (Figs 4 and 7A,B), intracellular total TG content (Fig. 5A) and gene expression profiling (Figs 6 and 7C), respectively. Bars = 200 µm. Abbreviations: ATP, adenosine triphosphate; APOB, APOLIPOPROTEINB100; FA, fatty acid; TG, triglyceride, WT, wild type.