4 2 hydroxyethyl 1 piperazineethanesulfonic acid

Primary human hepatocytes (PHHs) were collected from humanized liver chimeric NOG‐TKm30 mice^[19] using the two‐step collagenase–pronase liver perfusion method as previously reported.^[20] The collected PHHs were seeded in 12‐well or 24‐well collagen type I‐coated microplates (AGC Techno Glass Co., Ltd.). The PHH culture medium was Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque, Inc.) supplemented with 5 μg/ml L‐proline (Sigma‐Aldrich), 32.2 μg/ml 2‐phospho‐L‐ascorbic acid trisodium (Sigma‐Aldrich), 0.02 μg/ml dexamethasone (Sigma‐Aldrich), 0.005 μg/ml recombinant human epidermal growth factor (Sigma‐Aldrich), 25 μl/L human insulin solution (Sigma‐Aldrich), 20 mM 4‐(2‐hydroxyethyl)‐1‐piperazine ethanesulfonic acid (Nacalai Tesque, Inc.), 0.37% NaHCO3 (Nacalai Tesque, Inc.), 2% dimethyl sulfoxide (DMSO) (Nacalai Tesque, Inc.), 10% fetal bovine serum (FBS; Life Technologies), 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Life Technologies). The culture medium was changed on the indicated days. HepG2‐hNTCP‐C4‐iCas9 cells were maintained in DMEM/F‐12 (Life Technologies) containing G418 Disulfate Aqueous Solution and 10% FBS at 37°C under 5% CO2. Doxycycline (DOX) was administered at 1 μg/ml to induce Cas9.