Peter van Mourik

107 Forskolin-induced swelling protocol C. Human colon organoid culturing & workflow (also see table 7) 1. When received, store cryovials with organoid reference lines in liquid nitrogen until ready for the FIS experiments. 2. When ready, thaw a cryovial per reference organoid line as described in thawing protocol A 3. Seed 3 - 4 wells of a pre- warmed 24- well plate for each organoid reference line and add CM +/+ with additional ROCKI. 4. Check the organoid structures by light microscopy after 3 days and ensure there is enough space for the structures to expand. If not consider reseeding the cells with decreased organoid density to allow more space to proliferate and expand. 5. After 7 days of culture check the growth and quality of the organoid structures (compare to structures in Figure 10) : a. When the organoids are not yet budding, but appear small and round, densely proliferating organoids: take up and wash the organoids, centrifuge at 130 g for 5 min at 4 °C without disruption/shearing and reseed 1 well into 3 new wells to create more space and allow for continuous proliferation of the structures. b. When the organoids appear as already “budding” and healthy, large structures: disrupt 1 well according to ‘General handling and passaging of human colon organoid cultures’ (without the cleaning step) and seed into 3-4 new wells. 6. After week 2 and a further 7 days in culture check the appearance of the organoid structures. a. When grown into appropriate budding, proliferated organoid structures (see Figure 10): Freeze two full wells into two cryovials according to freezing protocol B . Take the 3 rd full well, disrupt and wash the organoids and seed into 3-4 new wells (without the cleaning step). b. When not yet grown/proliferated into efficient budding organoid structures and the organoid structures still appear dense and small: repeat the washing and reseeding step without disruption/shearing and seed in 3 wells. 7. After week 3, all organoid lines should be of good culture quality, meaning they should appear as efficiently budding, proliferating structures and sufficient growth. Colon organoids are then ready to be frozen and to start experimental cultures to use in the FIS assay. Make sure a working cell bank is stored for every reference organoid cell line before any FIS experiments are started (see table 7). 4