Peter van Mourik

96 Chapter 4 Freezing human colon organoid cultures Timing ~2 h Two different methods of organoids freezing are described here: A. Freezing trypsinized human colon organoids after 1-3 days replating (Freezing protocol A). This is an efficient method to store source colon organoid samples in a master cell bank. B. Freezing small organoid structures directly after disruption (Freezing protocol B); This is a fast, timesaving and efficient procedure appropriate for storing a local working cell bank. 1. Disrupt and freeze organoids when well is full (7 - 10 days cultured organoids) 2. Freeze one full well (of a 24-wells plate) per cryovial which should be sufficient to plate into 3-4 new wells after thawing. 3. Prepare cold Recovery™ Cell Culture Freezing Medium. 4. Prepare cold Ad-DF+++ 5. Prepare ROCKi 10 mM stock. 6. Prepare warm CM +/+ with additional ROCKi 10 µM. 7. For freezing protocol A: thaw (and keep) matrigel on ice and dilute 1:1 with cold CM +/+ (= 50 % matrigel) 8. Prepare 0.05% Trypsin EDTA to 18-23 ° C A. Freezing protocol A 1. Perform step 1-4 and 7,8 of ‘General handling and passaging of human colon organoid cultures’, part B. 2. Aspirate and discard the supernatant and add 4 mL of 0.05% Trypsin EDTA, and vortex for 30 s. 3. Put the tube in a warm water bath at 37 °C for 1 min and vortex vigorously for 30 s. 4. Inspect the solution in the tube by horizontally placing the tube on the light microscopy stage (4x objective), adjust focus so that organoids in solution are in visible. Observe the size of the organoid structures. If intact organoids are still visible put the tube in a warm water bath at 37 °C for an additional 1 min and vortex vigorously for 30 sec. 5. When the organoids are sufficiently disrupted (~100 x smaller), add 8 mL of Ad-DF+++ to neutralize the trypsin and resuspend 10 times the organoid suspension. 6. Centrifuge the tube for 3 min at 450 g at 4 °C.