Peter van Mourik

99 Forskolin-induced swelling protocol Thawing human colon organoids from frozen nitrogen stock vials Timing ~3 h CRITICAL: Pre-warm 24- and 96-wells plates used for organoid culturing to 37 °C for a minimum of >1 day. This is essential to ensure that after the colon organoids are plated, the matrigel polymerizes in the well efficiently and forms stable drops. Note: Human colon organoids cultures can be thawed from two different types of nitrogen stock vials: Thawing cultures that have been collected after 2-3 days in culture after trypsinization (freezing protocol A) or thawing cultures from vials with small organoid structures frozen directly after disruption (freezing protocol B). 1. Prepare warm Ad-DF+++ (37 °C). 2. Thaw (and keep) matrigel on ice or at 4 °C and dilute 1:1 with cold CM +/+ (= 50 % matrigel) 3. Prepare ROCKi 10 mM stock. 4. Prepare warm CM +/+ with additional ROCKi 10 µM. Note: Addition of ROCKi is only necessary during the first week after thawing A. Colon organoid thawing procedure 1. Add 13 mL pre-warmed Ad-DF+++ in a 15 mL tube 2. Rinse the outside of the cryovial with warm water or place in 37 °C water bath until the cell suspension in the vial is thawed. Clean the outside of the cryovial with 70 % EtOH. Alternative: first clean the outside of the cryovial with 70 % EtOH and then add warm Ad-DF+++ to the organoid suspension to thaw it. 3. Rapidly transfer organoids to 15 mL tube with 13 mL Ad-DF+++ (37 °C). When thawing colon organoids frozen according to freezing protocol A: 1. Gently mix the suspension and centrifuge 450 g for 5 min at 4 °C. 2. Discard supernatant and resuspend pellet in 100 µL of 50 % matrigel. 3. Check the organoid density under the microscope by seeding a 7.5 µL matrigel test drop. The matrigel drop should contain >100 structures of very small colon organoid structures. Organoids frozen according to freezing protocol A should be thawed and seeded with higher density due to the smaller sized structures compared to protocol B. To ensure optimal outgrowth and budding of the colon organoid structures it is suggested to seed multiple densities to cover the most optimal density for recovery and proliferation. 4