Peter van Mourik

91 Forskolin-induced swelling protocol General handling and passaging of human colon organoid cultures 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. A. Preparations 1. Prepare cold Ad-DF+++ and warm CM +/+. 2. Thaw (and keep) matrigel on ice or at 4 ºC and dilute 1:1 with cold CM +/+ (= 50 % matrigel). Note: the splitting ratio should be usually 1:4 - 1:8 24-wells. If seven day old organoid cultures cannot be split according to this ratio because they have not proliferated sufficiently, check the medium quality or reseed organoids in 1:1 wells without disruption to allow more time to recover or proliferate until disruption. B. Passaging colon organoids 1. Discard the CM +/+ from the wells containing the colon organoids. 2. Detach the matrigel with the organoids by aspirating 1 mL Ad-DF+++ and forcefully dispensing the medium directly onto the matrigel drops in the well several times with a p1000 filter tip. If necessary gently scrape the well with the end of the pipet tip to detach residue. 3. Transfer the colon organoid suspension to a sterile 15 mL tube on ice. Several wells (up to 12) can be combined in the 1 mL. 4. Put a p200 tip (without filter) on top of a p1000 filter tip (see figure 7) and place the tip in the 1 mL organoid suspension on the bottom of the tube. Mechanically shear/disrupt the organoid structures by pipetting the 1 mL organoid suspension up and down 15-20 times through the p1000/ 200 tip combination. The organoids will be broken up into smaller parts. Resuspend with firm speed and fully take up and dispense the 1 mL suspension. Slow, mechanic shearing does not create efficient disruption. Minor foam formation is normal but try to prevent it by avoiding foam aspiration or dispension. 4