Organoid Passaging Protocol: A Step-by-Step Guide with Modifications

The organoids were passaged every 7-10 days using a previously described method1 with slight modifications. In brief, the medium was removed, and 250ul of mild trypsin (TrypLE Express, Gibco, Cat# 12605-010) was added to each well. All wells were then collected into a 15ml centrifuge tube. The organoids were broken into small fragments by repeatedly pipetting using a 1ml pipette gun. The centrifuge tube was placed in an incubator at 37°C for 10 minutes to digest the organoids. Afterward, the tube was repeatedly pipetted 10-20 times to further promote the separation of the organoids. Finally, the tube was centrifuged at 600g for 5 minutes. The cell precipitates were resuspended with Advanced DMEM/F12 (Thermo Fisher Scientific, 12634010) and transferred to a 1.5 ml microcentrifuge tube. The tube was centrifuged again under the same conditions. After carefully aspirating and discarding the supernatant, the cell precipitates were placed on ice and reseeded in 48-well plates at a splitting ratio of 1:2 or 1:3, depending on cell density. The culture medium was changed every 2-3 days.