Healthy human colonoids (from FARMM study) were generated and propagated in Matrigel (Corning, 356231) on 24-well polystyrene plates as previously described (48) with full growth medium comprising basal medium (advanced DMEM supplemented with 10% FBS, 1× penicillin/streptomycin, 1× GlutaMAX, 10 mM HEPES, 1× N2 supplement, 1× B27 supplement, all from Invitrogen), 1 mM N-acetyl-cysteine, 10 nM leu-gastrin (all from MilliporeSigma) and WRN cell conditioned medium (WRN cells from ATCC; basal medium and WRN conditioned medium mixed at 1:1 ratio), and supplements (500 nM A83-01 [Tocris], 10 μM SB-20210, 10 mM nicotinamide, 50 ng/mL human EGF [all from MilliporeSigma], and 2 μL/mL 1× Primocin [InvivoGen]). For the first 48 hours of colonoid culture, growth medium was supplemented with 10 μM Y27632, 2.5 μM CHIR99021 (both from MilliporeSigma), and 2.5 μM thiazovivin (Reprocell). Media was replaced every 48 hours.

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