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

Figure 2 Phenotypic differences of intestinal organoids cultured under epidermal growth factor/noggin/r-spondin1-/CHIR99021/VPA or epidermal growth factor/noggin/r-spondin1 conditions upon continual passage. Organoids cultured for 7 d from freshly isolated crypt were split (1:4) and were cultured. Passage was performed once per week. A: Representative morphology of organoids cultured on day 7 under ENR or ENR-CV conditions upon continual passage. Scale bars; 50 μm. B: Quantitative real-time polymerase chain reaction analysis of relative mRNA expression levels of markers for intestinal stem cells (Lgr5) in organoids at early passage (P0-4) or late passage (P8-12) after culture for 6 d under ENR or ENR-CV conditions. GAPDH was used as an internal control. The data are shown as means ± SEMs of two independent experiments (^bP < 0.01, two-way analysis of variance with Dunnett’s T3 tests) and normalized to the value for the ENR condition. Note that the mean of the sum from each passage with triplicate experiments in the indicated early and late passages was used. C: Organoids cultured on day 6 at late passage (P10) were incubated with the thymidine analog EdU (green) for 1 h. Images were analyzed by fluorescence microscopy. Nuclei were double stained with Hoechst (blue). Scale bars: 50 μm. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; ENR: Epidermal growth factor/Noggin/R-spondin1; ENR-CV: ENR/CHIR99021/VPA.