Passaging and Stimulation of Colonoids

For experiments, colonoids were passaged using TrypLE techniques as already described but with modifications^{3 (link), 27 (link), 29 (link)}. First, Matrigel domes were disrupted by adding ice-cold PBS containing 5 mM EDTA and incubating on ice for 15 min. Colonoids were washed twice with base media, resuspended in TrypLE express (Gibco) and incubated at 37 °C with 5% CO₂ for 2 × 5 min. The colonoids were then rapidly disrupted into single cell suspensions with repeated pipetting through a p1000 tip, and an equal volume of organoid media was added. Cells were centrifuged, then resuspended in base media. The cells were counted using an automated cell counter and the appropriate number of cells (15,000 cells/well for human; 10,000 cells/well for mouse) were seeded using Matrigel as described in the colonoid isolation section in a 24-well plate. Colonoids were stimulated (days 5–7 after seeding for mice, day 10–14 for human) with base media with FliC (100 ng/ml; InvivoGen) or human IL-1β (10 ng/mL; Sigma) or murine IL-1β (10 ng/mL; PeproTech) or LPS O55:B5 (1000 ng/ml; InvivoGen) with or without recombinant IL-37 (100 to 0,1 ng/ml; R&D) or corresponding volume of base media for 4 h.