Mix 500µL DNAse with 5mL Papain.

Transfer single or pooled organoid to 60 mm dish.

Aspirate excess media, add 2.5mL Papain + DNAse solution.

With a razor blade mince organoid (<1 mm).

Transfer plate to an orbital shaker 70rpm (inside incubator).

With 1-mL pipette dissociate pieces (Mix up-down 30 times).

Put in orbital shaker 0h 20m 0s.

In the meantime, add 5mL Earle´s medium + 3mL Inhibitor to a 15-mL conical tube.

Remove samples from the orbital shaker. With a 1-mL tip, mix up-down 30 times.

Take 2mL (upper part) into new tube using a 40 µm cell strainer. Wait 1-3 min to debris to settle.

Transfer cell suspension to the inhibitor tube. Invert to mix 5 times.

Centrifuge 300rpm.

Aspirate supernatant, resuspend in 500µL to 1mL 0.5% BSA-PBS (Up-down 30 times).

Filter the resuspended cells (900µL) with a 30 µm cell strainer.

Count the cells for the final suspension and dilute. Resuspend at 1000 cells/μl in 0.04% BSA-PBS.