Peter van Mourik

98 Chapter 4 20. After 24 h, transfer the cryovials for storage in liquid nitrogen. B. Freezing protocol B 1. Perform step 1-4 and 7,8 of ‘General handling and passaging of human colon organoid cultures’, part B. However, shear/ disrupt the colon organoids 30 – 35 times to create even smaller organoid structures. 2. Add cold Recovery™ Cell Culture Freezing Medium Use 1 mL of Recovery™ Cell Culture Freezing Medium in 1 cryovial per isolated well. 1 frozen well in 1 cryovial can be thawed into 3- 4 wells of a 24 - well plate > pellet of 1 full well + 1 mL Recovery™ Cell Culture Freezing Medium or FM > add to 1 cryovial > pellet of 3 full wells + 3 mL Recovery™ Cell Culture Freezing Medium or FM > divide over 3 cryovials 3. Place the cryovials at -80°C in a cell container that will freeze the organoids 1°C per minute (e.g. mr Frosty). 4. After 24 h, transfer the cryovials for storage in liquid nitrogen.