Culturing Healthy Human Colon Organoids

All experimentation using human organoids described herein was approved by the ethical committee at University Medical Center Utrecht (UMCU; TcBio #12-093). Informed consent for tissue collection, generation, storage and use of the organoids was obtained from the patients at UMCU. Human healthy colon organoids were cultured as described previously (van de Wetering et al., 2015 (link)). Organoid culture medium contained advanced DMEM/F12 medium (Invitrogen) supplemented with penicillin/streptomycin (Sigma-Aldrich), 10 mM HEPES (Life Technologies), 1x Glutamax (Life Technologies) (defined as AdvDMEM^+/+/+) including 1X B27 (Invitrogen), 10 mM nicotinamide (Sigma-Aldrich), 1.25 mM N-acetylcysteine (Sigma-Aldrich), Noggin-conditioned medium (10% v/v), R-spondin1-conditioned medium (20% final volume if not indicated otherwise), 50 ng/ml EGF (Peprotech), WNT3a conditioned medium (50% final volume if not indicated otherwise, produced using stably transfected L cells), 500 nM TGF-β type I receptor inhibitor A83-01 (Tocris) and 10 µM P38 inhibitor SB202190 (Sigma-Aldrich). IWP-2 (R&D systems) was used at 2.5 μM for 2 days.