“…2 A. BG01-derived HEs, ECs, and HscLCs were dissociated into single-cell suspensions by treatment with TrypLE (12604-021, Gibco) for 3 min and collected using DMEM (12100-046, Gibco) containing 10% FBS (16000-044, Gibco). 1.5 × 10 4 HE, 3 × 10 4 ECs, and 5 × 10 3 HscLCs were seeded together in ultra-low attachment 96-well plates (ULA PrimeSurface® 96U, MS-9096UZ, SIMADZU) in cold multilineage liver organoid differentiation medium [ 37 – 40 ] [DM, advanced DMEM/F12 media supplemented with 1 × HEPES (15630-080, Gibco), 1 × GlutaMAX (35050-061, Gibco), 1 × penicillin/streptomycin (15140-122, Gibco), 0.1 mg/ml BSA (A7096, Sigma), 1 × B27 supplement minus vitamin A (12587-010, Gibco), 25 ng/ml BMP7 (120-03p, Peprotech), 100 ng/ml FGF19 (100-32, Peprotech), 25 ng/ml HGF (CYT-244, Prospec), 10 nM (Leu15)-Gastrin I (G9145, Sigma), 1.25 mM N-acetyl-L-cysteine (A9165, Sigma), 0.5 μM A83-01 (2939, Tocris Bioscience), 10 μM DAPT (ab120633, Abcam), 3 μM dexamethasone (D4902, Sigma), 0.566 mg/ml growth factor-reduced Matrigel (M354230, Corning), 100 ng/ml VEGF-165 (100-20, Peprotech), 50 ng/ml bFGF (100-18b, Peprotech), and 10 μM ROCK inhibitor Y27632 (1254, Tocris Bioscience)]. On days 1 and 3, cold DM was added to the organoid culture.…”