Leu15 gastrin

HCCOs were passaged after dissociation with 0.25% Trypsin-EDTA (Gibco) for 5 minutes. Cells were seeded into reduced growth factor BME2 (Basement Membrane Extract Type 2; R&D Systems) mixed 1:1 with an expansion medium (EM). After polymerization of the BME2, an EM (based on Huch et al^{24 (link)}) was added to the cells. The composition is advanced DMEM/F-12 (Gibco) supplemented with 1:50 B-27 (Gibco), 1:100 N-2 (Gibco), 10 mM nicotinamide (Sigma), 1.25 mM N-acetyl-l-cysteine (Sigma), 10 nM [Leu¹⁵]-gastrin (Sigma), 10 µM forskolin (Tocris), 5 µM A83-01 (Sigma), 50 ng/mL EGF (PeproTech), 100 ng/mL FGF10 (PeproTech), 25 ng/mL HGF (PeproTech), and 10% RSPO1-conditioned medium (homemade). Cryovials were prepared by resuspending dissociated organoids in a Recovery Cell Culture Freezing Medium (Gibco) before freezing. Expansion of HCCOs was exclusively performed under 20% O₂ (normoxia).

Colonic glands were isolated, treated with EDTA, and then resuspended in 30 to 50 μl of Matrigel (BD Bioscience) and plated in 24-well plates. WRN containing DMEM/F12 with Hepes (Sigma-Aldrich) containing 20% FBS, 1% penicillin-streptomycin, and recombinant mouse epidermal growth factor (50 ng/ml; Life Technologies) was used for culturing Apc^KO colon organoids. For the first 2 to 3 days after seeding, the medium was also supplemented with 10 mM ROCK inhibitor Y-27632 (Sigma-Aldrich) and 10 mM SB431542 (Sigma-Aldrich), an inhibitor for the transforming growth factor–β (TGF-β) type I receptor to avoid anoikis. For passage, colon organoids were dispersed by trypsin-EDTA and transferred to fresh Matrigel. Passage was performed every 3 to 4 days with a 1:3 to 1:5 split ratio. For human colon organoid culture, the previous medium was supplemented with antibiotics Primocin (100 μg/ml; InvivoGen) and Normocin (100 μg/ml; InvivoGen), serum-free supplements 1× B27 [Thermo Fisher Scientific (Gibco)] and 1× N2 [Thermo Fisher Scientific (Gibco)], chemical supplements 10 mM nicotinamide (Sigma-Aldrich) and 500 mM N-acetyl cysteine (Sigma-Aldrich), hormone 50 mM [Leu15]-Gastrin (Sigma-Aldrich), growth factor FGF10 (100 μg/ml; recombinant human; Thermo Fisher Scientific), and 500 nM A-83-01 (Sigma-Aldrich), which is an inhibitor of the TGF-β receptors ALK4, ALK5, and ALK7.