Feeder-free hiPSC Maintenance and Passaging

The hiPSC cells, 454E2 line (HPS0077, RIKEN BRC, female)³² (link) and 1383D6 line (HPS1006, RIKEN BRC, male),³³ (link) were obtained from RIKEN BioResource Research Center (BRC), Cell Engineering Division (RIKEN cell bank). To culture hiPSCs under feeder-free conditions, StemFit AK02N medium (Ak02N, Ajinomoto, Japan) and polystyrene tissue culture plates coated with Laminin-511 fragment (iMatrix-511 silk; 892021, Matrixome, Japan) were used. On the first day after passage, AK02N medium supplemented with 10 μM Y-27632 (HY-10071; Wako, Japan) was used. The medium was changed every other day from the day after the passage. The iPSCs were passaged every 6–8 days using 0.5 × CTS TrypLE Select (A12859-01, Thermo Fisher Scientific, USA) supplemented with 0.5 mM EDTA in PBS(−) or only 0.5 mM EDTA in PBS(−).