A83 01

Three lots of PHHs (lots DOO; Celsis, HC10-10; XENOTECH, HC4-24; XENOTECH) were used to generate organoids (Table S1). PHHs were washed with cold Advanced DMEM/F12 (Thermo Fisher Scientific) and spun at 400 g for 5 min. The cell pellet was mixed with Matrigel (growth factor reduced, Corning) and 1 × 10⁴ cells were seeded per well in a 24-well plate. After the Matrigel had solidified, 500 μl of organoid expansion medium was added to each well. The organoid expansion medium was prepared as described in a previous report [13 (link)]. Briefly, Advanced DMEM/F12 was supplemented with 1% Antibiotic Antimycotic Solution and 1 × GlutaMAX (GIBCO), 10 mM HEPES (Nacalai Tesque), 2% B27 supplement (GIBCO), 1.25 mM N-Acetylcysteine (Sigma), 10 mM Nicotinamide (Sigma), 10 nM recombinant gastrin (Merk), 50 ng/ml EGF (R&D), 10% R-Spondin1 conditioned medium (homemade), 100 ng/ml recombinant human FGF10 (peprotech), 25 ng/ml recombinant human HGF (R&D), 5 μM A83-01 (Wako), and 10 μM Forskolin (Wako). During cultivation, the medium was refreshed every 3 days. For the establishment of the organoids, the medium was supplemented with 25 ng/ml Recombinant Noggin (R&D), 7.5 ng/mL Recombinant Wnt3a (R&D) and 10 μM Y27632 (Wako) for the first 3–4 days. Passage was performed in a 1:3 split ratio once every 10–14 days. Organoids passaged 5–10 times were used in all experiments.

Hepatocytes harvested from 5- to 8-week-old rats were sorted into 96-well collagen-coated plates filled with 100 μL/well of Dulbecco’s modified Eagle medium/F12 (Life Technologies) containing 2.4 g/L NaHCO₃ and l-glutamine, which was supplemented with 5 mmol/L HEPES (Sigma), 30 mg/L L-proline (Sigma), 0.05% bovine serum albumin (Sigma), 10 ng/mL epidermal growth factor (Sigma), insulin-transferrin-serine-X (Life Technologies), 10^-7 mol/L dexamethasone (Sigma), 10 mmol/L nicotinamide (Sigma), 1 mmol/L ascorbic acid-2 phosphate (Wako), an antibiotic/antimycotic solution (Life Technologies), and 3 small molecules: 10 µmol/L Y-27632 (Wako), 0.5 µmol/L A-83-01 (Wako), and 3 µmol/L CHIR99021 (Axon Medchem, Reston, VA). On day 1, the cell and nuclei number for each well was determined, and wells that contained dead cells and, in the 8c plates, cells greater than doublets were excluded from further analysis. Ten days after seeding, cells from each of the formed colonies were counted manually.

The differentiation protocol was as described previously [[14] , [15] (link), [16] , [17] (link)]. In short, approximately 6 × 10⁵ freshly isolated rat hepatocytes were seeded on 100 mm collagen-coated dishes (Asahi Techno Glass, Tokyo, Japan) in differentiation medium composed of DMEM/F12 consisting of 2.4 g/L NaHCO3 and l-glutamine added with 10 mM Y-27632 (AdooQ BioScience), 0.5 mM A-83-01 (Wako Pure Chemical), and 3 mM CHIR99021 (AdooQ BioScience) (YAC medium). Furthermore, the medium included 5 mM HEPES, 30 mg/mL l-proline, 0.5 mg/mL BSA, 10 ng/mL epidermal growth factor, insulin-transferrin-serine-X, 0.1 mM dexamethasone (Dex), 10 mM nicotinamide, 1 mM ascorbic acid-2 phosphate, 100 U/mL penicillin, and 100 mg/mL streptomycin. The medium was altered 1 day after seeding and every other day thereafter. The cells achieved 90% confluence within 2 weeks. The features of CLiPs were evaluated by immunofluorescent staining (CK19, EpCAM). To confirm the ability of rCLiP to differentiate into mature hepatocytes, Oncostatin M and dexamethasone were added to rCLiP. The respective mature and undifferentiated hepatocyte components were examined using PCR. In this study, we evaluated SOX9 and EpCAM as characteristics of progenitor cells, CYP1A1 and Tryptophan 2,3-dioxygenase as characteristics of mature hepatocytes.