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Long-term 3D PureCol EZ Gel (Fujii medium) Ileum (N39) organoids were cultured in expansion medium consisting of DMEM/F12 supplemented with 100 U ml −1 P/S, 10 mM HEPES, 1× Glutamax, 2% B-27 with VitA, 3% (v/v) recombinant R-spondin-3, 2% (v/v) recombinant noggin, 1 mM N -acetylcysteine, 0.5 nM Wnt surrogate, 0.1 µg ml −1 hIGF-1 (Peprotech), 0.05 µg ml −1 hFGF2 (Peprotech), 10 nM gastrin, 0.5 µM A83-01, 0.05 µg ml −1 hEGF, 3 µM CHIR 99021 and 10 µM forskolin (Tocris) 42