Crypt Isolation and Organoid Culture: Crypt isolation and organoid culture from human intestinal samples were carried out as previously described (Fujii et al., 2015). For normal tissues, the stroma was removed using fine scissors and the remaining epithelium was further shredded into 1-mm3 pieces. Epithelial fragments were washed vigorously at least five times with ice cold PBS until the supernatant was free of debris and were treated with 2.5 mM EDTA (Thermo Fisher Scientific) for 1 hr at 4°C with gentle rocking to release crypts. Liberated crypts were suspended in Matrigel and dispensed onto 48-well plates as 25 μL droplets. After Matrigel polymerization, crypts were overlaid with the medium as described below. Advanced Dulbecco’s Modified Eagle’s Medium/F12 (Thermo Fisher Scientific) was supplemented with penicillin/streptomycin (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), 2 mM GlutaMAX (Thermo Fisher Scientific), 1 × B-27 Supplement (Thermo Fisher Scientific) to prepare a basal medium. An expansion medium was made by supplementing the basal medium with 10 nM gastrin I (Sigma-Aldrich), 1 mM N-acetylcysteine (Sigma-Aldrich), 100 ng/ml recombinant mouse Noggin (PeproTech), 50 ng/ml recombinant mouse EGF (Thermo Fisher Scientific), 100 ng/ml recombinant human IGF-1 (BioLegend), 50 ng/ml recombinant human FGF-basic (FGF-2) (Peprotech), 1 μg/ml recombinant human R-spondin1 (R&D), 500 nM A83-01 (Tocris) and 50% Afamin-Wnt-3A serum-free conditioned medium (Mihara et al., 2016). The organoids were passaged once a week by physical dissociation using fire-polished Pasteur pipettes with a split ratio of 1:5. EGF was removed from the first passage onward to generate differentiated cells. For specific experiments, 10 μM SB202190 (Sigma-Aldrich) was used. Organoid establishment from an ulcerative colitis-related dysplasia was performed as previously described (Fujii et al., 2016). The tumor tissue was washed with ice cold PBS and digested in Liberase TH Research Grade (Roche) for 1 hr at 37°C. The remaining fragments were further dissociated in TrypLE Express (Thermo Fisher Scientific) for 20 min at 37°C. Digested tissue was embedded in Matrigel and maintained in the culture medium described above. The medium was supplemented with 10 μM Y-27632 (FUJIFILM Wako Pure Chemical Corporation) for the first two days of culture. Sequencing analysis identified KRASG12V mutation in this organoid line and confirmed its tumor origin. Full paper
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