6–8 week-old BALB/C mice were euthanized by decapitation. 10 cm of terminal ileum was taken immediately and washed with pre-cooled (4°C) PBS. Adipose tissue and mesentery in the intestine were removed. Intestinal contents were washed away with 4°C pre-chilled PBS. After the ileum was cut into 2 mm pieces, pipetting was repeated 6 times with PBS. The supernatant was removed after the tissue was precipitated. EDTA-containing PBS was added to precipitates and incubated for 30 min at 4°C on a shaker. After incubation, pipetting was repeated 6 times with PBS until the crypts are dispersed. 200–500 crypts per well were suspended in Matrigel (Corning). Complete ENR medium (all components from Thermo Fisher Scientific) consisted of advanced DMEM/F12 (Gibco), antibiotic and antifungal (×100), 1 mM N-acetylcysteine (Sigma-Aldrich), B27 Supplement, N2 Supplement, EGF, Noggin (R&D Systems), R-spondin-1-conditioned medium was added for the culture. The ENR medium was replaced every 2 to 3 days. The surface area of intestinal organoids was measured from non-overlapping photographs of the organoids randomly taken in a well using an inverted microscope (Carl Zeiss). Each photo was analyzed using ImageJ software (NIH) and the Zen image program (Carl Zeiss)50 (link).
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