Rh-iPSC maintenance and passage

Rh-iPSC medium was replaced with 1 mL per 60-mm dish of dissociation solution consisting of 20 mL of 2.5% trypsin (Invitrogen), 40 mL of KO serum replacement (Invitrogen), and 2 mL of 100 mM CaCl₂ to 138 mL of Dulbecco’s PBS (D-PBS)(−) (Nacalai, Japan), and the cells were then incubated at 37°C in a CO₂ incubator for 5 min. After the incubation, MEFs and dissociation solution were removed, and 1 mL of Rh-iPSC culture medium was added. The colonies were broken up into small cell clumps by pipetting. About one-fifth of the cell suspension was transferred to a new MEF dish, although the split ratio may require adjustment depending on the iPSC line. The medium was replaced with fresh medium every day.