Generation of apical-out intestinal organoids after treatment with 1 µM CHIR. (A) Schematic illustration of the experimental procedures regarding the generation of apical-out intestinal organoids after treatment with 1 µM CHIR. Apical-out intestinal organoids through suspension culture without Matrigel matrix after treatment with 1 µM CHIR began to emerge at Day 7. Scale bar: 50 µm. (B) The paracellular permeability characteristics of the epithelial layer in apical-out intestinal organoids for comparison of treatment with 1 µM CHIR at Day 3 and spontaneous differentiation at Day 6. (C) Comparison of fluorescent intensity regarding apical-out intestinal organoids generated after treatment of 1 µM CHIR at Day 3 and spontaneous differentiation at Day 6. In addition, we generated apical-out intestinal organoids after spontaneous differentiation at Day 6 as a control (Lee et al., 2021). The fluorescence intensity was calculated using ImageJ software. There was no significant difference in the fluorescence intensity levels. Significant differences between groups were analyzed by Student’s t test. A p value less than or equal to 0.05 indicated statistical significance (*p value ≤ 0.05).