Peter van Mourik

87 Forskolin-induced swelling protocol Organoid crypt isolation from human colon (rectal) biopsies Timing ~3 h CRITICAL: All laboratory procedures related to colon organoid or cell cultures should be performed in a laminar flow to avoid contaminations and to protect the operator from biological risks. CRITICAL: Always keep matrigel at 4°C or on ice to prevent polymerization above 10 °C. 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 cold PBS0 (4 °C). 2. Thaw (and keep) matrigel on ice or at 4 °C and dilute 1:1 with cold CM +/+ (= 50 % matrigel). 3. Prepare CM +/+ with additional gentamicin (50 µg/mL) and vancomycin (50 µg/ mL) to reduce the risk of infection. B. EDTA incubation CRITICAL: In every step of this procedure, when handling the biopsies with pipettes, it is necessary to pre-coat the pipettes with Ad-DF+++ by aspirating media into the pipettes and discard the media. This covers the inside of the pipets with liquid and prevents biopsies from adhering to the plastic. 1. Wash the colon biopsies several times in cold PBS0 until the supernatant is clear using the following procedure: a. Pipet the biopsies up and down ~10 times in 10 mL with a 10 or 25 mL pipet. b. Let the biopsies settle at the bottom for 30 seconds. c. Remove the supernatant and add 10 mL of clean cold PBS0. d. Repeat these steps 2-4 times until the biopsies and supernatant are clear of debris. 2. Discard the supernatant and add 10 mL of clean PBS0 to the biopsies and supplement it with 200 μL ultrapure EDTA pH 8, 0.5 M (final concentration 10 mM). 4