Cc 3199

Cell culture plates were coated with 0.5 mg/ml rat tail tendon collagen type Ⅰ (Solarbio, C8062, Beijing, China) overnight at 4°C and washed with PBS for three times before cell seeding. Murine hepatocytes were isolated from C57BL/6 N mice by a retrograde perfusion of liver with 30–40 ml HBSS (Gibco, 14175–095) containing 0.5 mM EGTA (Sigma, 03777), followed with about 40 ml low glucose DMEM containing 100U/mL collagenase type IV (Gibco, 17104–019). Hepatocytes were cultured in DMEM with 10% fetal bovine serum for 4 h for attachment. Then the medium was replaced with hepatocyte culture medium (LONZA, CC-3199 and CC-4182). The hepatocytes were treated with APAP (5 and 10 mM) in the absence or presence of CSA (50 μM) or chloroquine (20 μM) for 24 h. Cells were double stained with calcein and propidium iodide (Beyotime Biotech, C2015). After incubation at 37°C for 30 min, the fluorescence intensity was detected by Tecan’s Spark multimode reader to assess the percentage of cell death. For representative images, hepatocytes were stained with propidium iodide and Hoechst 33258 (10 μg/ml) for 30 min followed by fluorescence microscopy.

HCC tissue was cut into 3-mm³ pieces and washed with 4 °C Hank’s balanced salt solution (Lonza, Basel, Switzerland) supplemented with 1× antibiotic-antimycotic (A/A) solution (Sigma-Aldrich, St Louis, MO, USA) and 1× penicillin-streptomycin (P/S) (Lonza) in 100-mm Petri dishes. After three washes with DMEM/nutrient mixture F-12 (DMEM/F12; Gibco) supplemented with 10% FBS, 1× A/A solution, and 1× P/S, the cells were resuspended in 10 ml of the same solution and incubated at 4 °C for 16 h. Next, tissue was washed with fresh DMEM/F12 and incubated with 2 ml of 2× collagenase II (BD Biosciences, Franklin Lakes, NJ, USA) at 37 °C in a shaking chamber for 90 min. After incubation, the tissues were washed with DMEM/F12 several times until the supernatant was clear. The pellet was resuspended in hepatocyte basal medium (Lonza, CC3199) containing 1× A/A solution, 10% FBS, and 5 μg/ml hepatocyte growth factor (R&D systems, Minneapolis, MN, USA) and plated on collagen type I-coated T-25 flasks (BD Biosciences) with 5×10⁵cells.

Cryopreserved PHHs (454543; Corning) were thawed with cryopreserved hepatocyte plating medium and cryopreserved hepatocyte recovery medium (Thermo Fisher Scientific) according to manufacturer instructions in a Matrigel-coated dish at 2 × 10⁵ cells/cm². The medium was replaced with fresh hepatocyte culture medium (cc-3199; Lonza Group AG, Basel, Switzerland) at 4 to 6 h after thawing. HepG2 cells (ATCC) were cultured in Dulbecco Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Corning) and 100 U/mL penicillin–streptomycin (Thermo Fisher Scientific). To evaluate CYP3A4-mediated hepatotoxicity, DM-differentiated organoids were treated with troglitazone (50 μM) and propionate (1 μM), an SCFA mixture (acetate:propionate:butyrate = 1:1:1), or CYP3A4 inhibitor ketoconazole (1 μM) 3 days after differentiation for another 6 days. PHHs and HepG2 cells were seeded in 96-well plates, and the following day, a troglitazone and SCFA mixture was treated daily for 3 days. Toxicity was evaluated using EZ-cytox (Dogenbio, Seoul, Korea) following the manufacturer’s instructions. The absorbance of the medium was measured using a Spectra Max M3 microplate reader (Molecular Devices, San Jose, CA, USA).