Figure 1.

hPSC Passaging

(A) For routine passaging, hPSC colonies are treated with Gentle Cell Dissociation Reagent for 6–8 min at room temperature (15°C–25°C; RT) until the cells start to round up within the colonies.

(B) Extended exposure to the reagent (shown here is 15 min at RT) leads to detachment of the colonies (red arrows) and increased cell death.

(C) Optimal size of aggregates (50–200 μm) when performing “aggregate-passaging” with Gentle Cell Dissociation Reagent.

(D−F) Colony densities on the day after splitting, showing the outcome of optimal (D), too sparse (E) and too dense (F) splitting.

(G) Optimal Accutase digestion (5 min at 37˚C) is achieved when cells start to round up within the colonies.

(H) Overdigestion with Accutase (shown here is 10 min at 37˚C) leads to detachment of the colonies (red arrows) and increased cell death.

Scale bars, 400 μm.