Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor

Figure 1 Establishment of small intestinal organoid culture under epidermal growth factor/noggin/r-spondin1 and epidermal growth factor/noggin/r-spondin1-/CHIR99021/VPA conditions. Crypts were isolated from the small intestines of C57/B6 mice at ages 9-12 wk and were resuspended in growth factor-reduced Matrigel. A: Time course of the growth of isolated crypts at passage 0 (P0) under two different culture media. Enterospheres formed on day 1, budding appeared on day 3, and robust budding was observed on days 5-10. Scale bars: 50 μm. B: Organoids were incubated with the thymidine analog EdU (green) for 1 h, and freshly isolated crypts were cultured for 6 d. Images were analyzed by fluorescence microscopy, and nuclei were double stained with Hoechst (blue). Scale bars: 50 μm. C: Numbers of organoids grown in two different media for 7 d. Organoids exhibiting at least two budding structures in each group were counted. The data are shown as means ± SDs of triplicate experiments (^bP < 0.01, Student’s t-tests).