Peter van Mourik

97 Forskolin-induced swelling protocol 7. Aspirate and discard the supernatant and add the required amount of medium and matrigel to the organoid pellet. Note: For freezing, organoids must be seeded in a 1:1 ratio after trypsinization. Thus, 1 well of organoids from a 24-wells plate can be seeded into 1 new well of a 6-well tissue culture plate. 8. Mix the organoid suspension by resuspending without creating bubbles. o Tip: Avoid bubbles by not fully aspirating and dispensing the pipet, but always keep a small amount of liquid in the tube. 9. Seed 250 μL in a single well of a pre-warmed 6-well tissue culture plate by seeding 25 * 10 μL matrigel drops . 10. Place the plate in the incubator at 37 °C and leave the matrigel to solidify for 20-30 min. 11. Add 2.5 mL of fresh CM +/+ + ROCKi in each 6-well plate well and transfer the plate to the incubator. Note: 1-2 day old organoid cultures are ready to be frozen. This increases the efficiency of survival after thawing. 12. Detach the matrigel and organoids by aspirating 1 mL Ad-DF+++ and forcefully dispensing the medium directly onto the matrigel drops in the well several times with a p1000. If necessary gently scrape well with end of pipet tip to detach everything. 13. Transfer the organoid suspension to a sterile 15 mL tube. 14. Wash the wells with another 1 mL of Ad-DF+++ and transfer to the same 15 mL tube. 15. Fill up the 15 mL tube with with 12 mL of cold Ad-DF+++ and pipet up and down with a 5 mL pipet. 16. Centrifuge the suspension for 3 min at 450 g at 4 °C, remove the supernatant and keep the pellet. 17. Dissolve the organoid pellet with cold Recovery™ Cell Culture Freezing Medium and pipet up and down to properly resuspend all the organoid structures. Note: one full well from a 6-well plate is frozen in 1 mL of Recovery™ Cell Culture Freezing Medium and is divided over 2 cryovials. Each vial should contain enough cells to be thawed into ≥ 4 wells of 24-well plate. 18. Transfer 0.5 mL of organoids suspension in Recovery™ Cell Culture Freezing Medium to sterile cryovials. 19. Place the cryovials at -80°C in a cell container that will freeze the organoids 1°C per minute (e.g. mr Frosty). 4