Organoid-based 2D monolayer culture

Generation of an organoid-based 2D monolayer culture was performed by removing supernatant from wells containing 3D organoids and subsequent addition of 1 ml ice cold PBS. Matrigel was dissected by pipetting and organoids were collected in a tube pre-filled with 10 ml ice cold PBS. After centrifugation at 4°C and 600 x g for 10 min, supernatant was discarded. Pellet was resuspended in 0.05% Trypsin/EDTA and incubated for 5 min at 37°C before resuspending the solution 20 times with a 1,000 μl tip and another 15 times with a 200 μl tip mounted on a 1,000 μl tip. 10% (v/v) ice cold fetal bovine serum in DMEM was added and the tube was centrifuged at 3,000 x g, 4°C for 10 min. Supernatant was discarded and the pellet resuspended in monolayer medium (S3 Table). Cells were counted and 2 * 10⁵ cells seeded on precoated (Matrigel 1:40 in PBS) Snapwells^® (Corning, Kaiserslautern, Germany; diameter: 12 mm; pore size: 0.4 μm). Basolateral chamber was filled with 3 ml monolayer medium, apical chamber with 0.5 ml monolayer medium, respectively. After 16 days of cultivation, medium was changed to differentiation medium for another 2 days (S4 Table) until experiments were conducted after a total incubation time of 18 days.