Splitting hESC/hiPSC lines on MEF using Accutase

1. Coat culture dish with sterile 0.1% gelatin solution for about 5 min at room temperature. 2. Thaw frozen MEF vial at 37◦C water bath quickly. 3. Transfer cells into 15 ml conical tube and add fibroblast media (DMEM + 10% FBS). 4. Wash cells via spinning at 1500 rpm, 5 min. 5. Plate MEF at a density of 1.5–2.0 × 104/cm2. 6. Let MEF settle down at least 8 hours before use. (Overnight is best and MEF can be used within 2 days) • Splitting hPSCs with Accutase