Irradiation and Metformin Effect on Intestinal Organoids

The crypts were isolated from the small intestines of mice, and isolated crypts were cultured as previously reported [45 (link)]. The organoids were cultured onto four-well plates at the same ratio, and then exposed to 137Cs γ-rays from a Gamma Cell-3000 irradiator (MDS Nordion International, Kanata, Ontario, Canada). Metformin (20 μM; Sigma-Aldrich, St. Louis, MO, USA) was administered after irradiation on day 0, and the medium was changed every three days. For examination of intestinal organoid viability after irradiation, we performed methylthiazolyl tertrazolium (MTT) assays, as previously reported [46 (link)]. Briefly, organoids were incubated with 10% MTT for 2–3 h, and the insoluble formazan crystal were dissolved in solubilization solution. MTT-stained organoids indicated surviving cells.