Colon Organoid Differentiation Protocol

Colon organoids were collected at days 7 to 9 after passaging. Cell Recovery Solution was used for 40 min at 4°C to dissolve Matrigel, followed by incubation of organoids in Trypsin/EDTA (Thermo Fisher Scientific, catalog no. 12605036) at 37°C for 5 min. Next, organoids were mechanically dissociated into single cells, resuspended in EM without nicotinamide, and seeded onto type I collagen (50 μg/ml)–coated 12-well 0.4-μm pore polyester Transwell inserts (Corning, 3493) at a density of 3 × 10⁵ cells per Transwell. After 3 to 5 days of incubation, monolayers were confluent and we initiated differentiation as described previously (8 (link)). For differentiation, apical medium was replaced with Advanced DMEM/F12 plus Hepes, Glutamax, and Pen/Strep and basolateral media with differentiation medium, which is EM without L-WRN conditioned medium, nicotinamide, prostaglandin E2, SB202190, and thiazovivin, but supplemented with human recombinant noggin (100 ng ml⁻¹; PeproTech, catalog no. 120-10C) and 20% R-spondin conditioned medium (Sigma-Aldrich, catalog no. SCC111). Monolayer integrity was monitored with transepithelial electrical resistance (TEER) measurements, which were performed using the EndOhm-12 chamber with an EVOM2 meter (World Precision Instruments). Monolayers were used for further experimentation at days 7 to 9 after seeding.