Organoid generation and culture

Human iPS-HLCs were dissociated into single cells with TrypLE Select Enzyme and passed through a strainer to remove the cell aggregates. The cells were suspended in Matrigel at 5 × 10⁴ cells/40 μL droplet, seeded in each well of the 24-well plates, and cultured in the organoid medium containing Y-27632 (Fujifilm Wako Pure Chemical, Osaka, Japan). The medium was replaced with the organoid medium without Y-27632 every 2 days. Table S1 shows the composition of the organoid medium, which was previously used by Huch et al.³⁷ to generate liver organoids. The passage interval was about 8 days. The organoids were washed with cold 0.5 mM EDTA (Thermo Fisher Scientific)/PBS and treated with TrypLE Select. They were resuspended in fresh Matrigel and seeded at 40 to 60 μL each in the center of a well of a 24-well plate. The medium was replaced every 3 days.