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

Figure 3 Comparison of the compositions of differentiated intestinal epithelial cells in long-term cultured intestinal organoids under two different media. A: Quantitative real-time polymerase chain reaction analysis of relative mRNA expression of markers for intestinal epithelial cells (Muc2 for goblet cells, ChgA for enteroendocrine cells, Alp for enterocytes, and Lyz1 for paneth cells) in organoids at early passage (P0-4) or late passage (P8-12) cultured 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 test) and normalized to the ENR value. Note that the mean of the sum from each passage with triplicate experiments in the indicated early and late passages was used. Raw crypts: freshly isolated crypts from mice. B: Representative images show lysozyme (paneth cells), Muc2 (goblet cells), and ChgA (enteroendocrine cells) staining of organoids cultured for 6 d at passages 3 and 10 under ENR-CV conditions. Three-dimensional reconstructed confocal images are shown. Nuclei were double stained with DAPI (blue). Scale bars: 50 μm. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; ENR: Epidermal growth factor/Noggin/R-spondin1; ENR-CV: ENR/CHIR99021/VPA.