Figure 2.

The presence of endothelium enhances the establishment of the epithelial barrier. (A) Representative phase-contrast images of the colonic epithelial cells on day 5 of the Colon Intestine-Chip culture in the presence or absence of endothelial cells. (B) The P_app to 3 kilodaltons dextran Cascade blue, of endothelial or epithelial cells, where stretch (10% strain, 0.15 Hz) was applied in the presence or absence of endothelium, over the course of an 8-day culture. Data shown correspond to 1 representative of 3 independent experiments. n = 3–11 chips/condition, means ± 95% CI, 2-way analysis of variance, Tukey post hoc test. (C) Representative confocal fluorescent images showing the establishment of strong epithelial TJs on day 5 of culture in the presence of endothelium. TJs were stained with anti–ZO-1 and anti–E-cadherin and cytoskeleton with phalloidin (gray). Cell nuclei are shown in blue. (D) Representative confocal immunofluorescence images against the basolateral transporter Na⁺K⁺ adenosine triphosphatase (ATPase) (magenta) and the apical transporter SLC26A3 (green), indicating the establishment of a polarized epithelial monolayer on day 5 of culture only during the co-culture of colonocytes with endothelium. Cell nuclei are shown in blue. The plots indicate the mean fluorescent intensity distribution for each channel along the basal–apical axis of the epithelial cells. The quantification was performed across 3 different fields of view in 3 individual chips for each condition. (E) Representative scanning electron microscopy (SEM) images of the colonic epithelial cells on day 5 of the Colon Intestine-Chip culture, indicating maturation of the epithelial brush border in the presence of endothelial cells. (F) Box plots showing the density of microvilli per μm², according to quantification of SEM images on day 5 of culture. The presence of endothelium significantly increases the density of microvilli per μm². The quantification was performed across 6–11 different fields of view for each condition. Each individual point represents a single field of view. Unpaired t test. ∗∗P < .01 and ∗∗∗∗P < .0001. DAPI, 4′,6-diamidino-2-phenylindole.