How to culture pluripotent stem cells in suspension: Passaging of PSC cultures in suspension

Set up the desired number of suspension culture vessels with stem scale medium before beginning the passage procedure.

Swirl culture vessels slowly to gather spheroids to the center, then pipette or pour the spheroid-containing medium into a 50 mL conical tube. Wash the empty vessel walls with stem scale medium to collect any remaining spheroids.

Centrifuge collected spheroids at 200× gravity for 4 minutes, then aspirate the spent medium and add pre-warmed stem pro accutase cell dissociation reagent.

Allow spheroids to dissociate in a 37°C water bath for 10-15 minutes, periodically mixing by flicking or gently shaking the tube until the suspension becomes cloudy.

Triturate the cell suspension 5-7 times using a P1000 micropipette to break up remaining spheroids into single cells or small clusters.

Add stem scale medium (3 mL per 1 mL accutase) to inactivate the dissociation reagent, then centrifuge and resuspend cells in fresh stem scale medium supplemented with 10 µM Y27632.

Count and seed 100,000-150,000 cells per mL in fresh non-tissue culture treated vessels, then place on the CO₂ resistant orbital shaker in the incubator.