Single-Cell, Feeder-Free Culture of hiPSCs

Human iPSCs were cultured in the single-cell and feeder-free (SFF) culture system as previously described (Ono et al., 2014 (link)). Briefly, cells were grown at 37°C and 5% CO₂ in MT-fCFA medium, a modified chemically defined medium (MT-CDM) supplemented with activin A (338-AC; R&D Systems, Minneapolis, MN, USA) and FGF2 (100-18B; Peprotech, Rocky Hill, NJ, USA). For passaging, the cells were treated with 0.005% trypsin/0.002% EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 3 min, mixed with 250 μg/ml of trypsin inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), centrifuged at 180× g for 5 min, and resuspended in MT-fCFA medium containing 2.4 μM thiazovivin (Wako, Osaka, Japan) and 4.7 μg/ml human fibronectin (FN; 356008; Corning, NY, USA). The cells were plated on collagen type I (Col I)-coated dishes (IWAKI, Tokyo, Japan) at a dilution ratio of 1:5. Cells routinely received fresh medium every day and were passaged when 70%–80% confluence was reached, which normally occurred every 2–3 days. For cryopreservation, cells were suspended in STEM-CELLBANKER (BLC-3S; ZENOAQ, Fukushima, Japan) and frozen at −80°C, following the manufacturer’s instructions.