Derivation and Culture of Colonic Organoids

Colonic organoids were derived as previously described from Muc2^+/+ and Muc2^-/- littermates (23 (link), 24 (link)). Briefly, the colons were excised, and crypts digested using a gentle cell dissociation buffer (StemCell Technologies) and approximately 100 crypts were embedded into GFR-matrigel (BD) domes. Organoids were fed every two days with advanced DMEM supplemented with L-WRN (ATCC CRL-3276) as well as N2, B27, GlutaMax, SB202190, Nicotinamide, N-acetylcysteine, A83-01, and mEGF. Colonoids were passaged once every 7-10 days with TrypeLE. For experiments, colonoids were grown for 7 days in complete media and then fed with L-WRN-conditioned media containing only N2, B27, Nicotinamide, and Glutamax for 3 days. Five µM DAPT was administered to the experimental media 24 hours prior to harvesting for experiments using colonoids skewed to a goblet cell phenotype.