We prepared mouse embryonic fibroblasts (MEFs) (KBL9284600; Kitayama Labes, Nagano, Japan) inactivated by mitomycin C treatment on 0.1% gelatin-coated dishes (1.2 × 106 cells/10 cm dish). Mice were not directly involved in the study. Primed hiPSCs frozen in liquid nitrogen were thawed as quickly as possible in warmed maintenance medium, comprising DMEM/F12 (D6421; Sigma, St. Louis, Missouri, USA) supplemented with 20% (v/v) KSR (lot No. 1517496; Invitrogen, Waltham, Massachusetts, USA), 0.1 mM non-essential amino acids (11140-050; Gibco), 2 mM l -glutamine (25030-081; Gibco), 5 ng/mL recombinant human basic FGF (068-04544; Wako, Osaka, Japan), and 0.1 mM 2-mercaptoethanol (131-14572; Wako. Cells suspended in maintenance medium were distributed on MEF-coated dishes and maintained in a CO2 incubator under 2% CO2 at 37 °C. The medium was changed daily.
For passaging, primed hiPSC colonies were harvested by incubation in 0.25% (w/v) trypsin and 0.1 mg/mL collagenase IV in PBS containing 20% (v/v) KSR and 1 mM CaCl2 for 6–8 min at 37 °C. The harvested clumps were broken into smaller pieces by gentle pipetting. The passages were performed at a split ratio of 1:4–6.
For passaging, primed hiPSC colonies were harvested by incubation in 0.25% (w/v) trypsin and 0.1 mg/mL collagenase IV in PBS containing 20% (v/v) KSR and 1 mM CaCl2 for 6–8 min at 37 °C. The harvested clumps were broken into smaller pieces by gentle pipetting. The passages were performed at a split ratio of 1:4–6.