Figure 4.

Endothelium supports the establishment of a tight epithelial barrier over the course of the culture. (A) Representative confocal fluorescent images showing the establishment of strong epithelial TJs on day 8 of the 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. (B) 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 only during the co-culture of colonocytes with endothelium, on day 8 of culture. 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. (C) The gene expression of the 2 epithelial junction proteins, Cdh1 and Tjp1, was confirmed in epithelial cells on days 5 and 8 of culture of the Colon Intestine-Chip. The on-chip culture enhances the expression of both proteins. The graph represents fragments per kilobase of transcript per million mapped reads (FPKM) values generated from bulk RNA-seq analysis of the epithelial cells and plotted on a box plot, where each individual point represents a single chip. n = 3–6 chips/condition, means ± 95% CI, 1-way analysis of variance, Tukey post hoc test. (D) The gene expression of the 2 ion transporters, Atp1a1 and Slc26a3, was confirmed in epithelial cells on days 5 and 8 of culture of the Colon Intestine-Chip. The on-chip culture enhances the expression of both transporters. The graph represents FPKM values generated from bulk RNA-seq analysis of the epithelial cells and plotted on a box plot, where each individual point represents a single chip. n = 3–6 chips/condition, means ± 95% CI, 1-way analysis of variance, Tukey post hoc test. (E) Representative scanning electron microscopy (SEM) images of the colonic epithelial on day 8 of the Colon Intestine-Chip culture, where stretch (10% strain, 0.15 Hz) is applied in the presence or not of endothelium. Endothelium does not significantly contribute on the establishment of a dense microvilli network at a later stage of the culture. (F) Box plots showing the density of microvilli per μm², according to quantification of SEM images on day 8 of culture. The quantification was performed across 8–9 different fields of view for each condition. Each individual point represents a single field of view. Unpaired t test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. DAPI, 4′,6-diamidino-2-phenylindole.