Peter van Mourik

100 Chapter 4 4. Plate 4* 7.5 µL matrigel drops without bubbles to a single well of a (pre-warmed) 24-wells tissue culture plate. Usually one cryovial should contain enough organoids to seed in 3-4 wells. 5. Dilute the remaining colon organoid suspension 1:1 with matrigel and plate two additional wells. 6. Again, dilute the remaining colon organoid suspension 1:1 with matrigel and plate the fourth well. When thawing colon organoids frozen according to freezing protocol B: 1. Gently mix the organoid suspension and centrifuge 130 g for 5 min at 4 °C. 2. Discard the supernatant and resuspend the organoid pellet in 100 µL of 50 % matrigel. 3. Check the organoid density under the microscope: seed the organoid structures with a 10-20% higher density compared to organoids already in culture, aiming for 50-70 structures per drop (10-20% of the structures are usually not viable). Organoids frozen according to freezing protocol B should be thawed and seeded with lower density due to the larger sized structures compared to protocol A. 4. Plate 4* 7.5 µL matrigel drops without bubbles per single well of a (pre-warmed) 24-wells tissue culture plate. Usually one cryovial should contain enough organoids to seed in 3-4 wells. Colon organoids will settle to the bottom quickly, so resuspend the organoids in the matrigel suspension frequently while plating. 5. Incubate a maximum of 10 min at 37 °C. Thawed human intestinal organoids are sensitive to being left without warm CM +/+ 6. Add 500 µL of (pre-warmed) CM +/+ with additional ROCKi inhibitor (10 µM)to each well. Refresh the medium with 500 µL CM +/+, every 2 - 3 days (Monday, Wednesday, Friday). 7. Incubate at 37 °C, 5% CO 2 for 1 week. CRITICAL: Monitor the progress of the thawed organoid structures daily.