Intestinal organoid culture

The clonal wild-type human intestinal organoid line ASC-5a from donor STE0072 (female) was derived in a previous study.⁶ Intestinal organoids were cultured as previously described⁵⁸ in 10 μL domes of Cultrex Pathclear Reduced Growth Factor Basement Membrane Extract (BME) (3533–001, Amsbio) in growth medium consisting of Advanced DMEM/F12 (Gibco), 1× B27, 1× glutamax, 10 mmol/L HEPES, 100 U/ml penicillin-streptomycin (all Thermo Fisher), 1.25 mM N-acetylcysteine, 10 μM nicotinamide, 10 μM p38 inhibitor SB202190 (all Sigma-Aldrich) and the following growth factors: 0.5 nM Wnt surrogate-Fc fusion protein, 2% noggin conditioned medium (both U-Protein Express), 20% Rspo1 conditioned medium (in-house), 50 ng/mL EGF (Peprotech), 0.5 μM A83-01, and 1 μM PGE2 (both Tocris). For the last two passages, organoids were cultured in medium without antibiotics for 4 days. They were exposed to 0.05% (w/v) FastGreen dye (Sigma) apically, and 5 μg/mL of gentamicin (Sigma) for three days. Primocin (1X, InvivoGen) was added for three days prior to passage or single cell isolation. Single cells were isolated for PTA by dissociating organoids with TrypLE express (Gibco) followed by fluorescence-activated cell sorting (FACS) on an SH800S Cell Sorter (Sony).