Fig 1: 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 Papp 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.
Fig 2: The presence of stretch does not significantly contribute in the establishment of a tight epithelial barrier. (A) Representative phase-contrast images of the colonic epithelial on day 5 of the Colon Intestine-Chip culture in the presence or not of endothelium and/or stretch. (B) The Papp of the colonic epithelial cells to 3 kilodaltons of dextran Cascade blue, over the course of an 8-day culture, in the presence or absence of stretch (10% strain, 0.15 Hz) and/or endothelium. Stretch does not contribute significantly to the establishment of the epithelial barrier. 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 widefield fluorescent tile images showing the uniform expression of the TJ proteins, ZO-1 (green) and anti–E-cadherin (magenta), along the Colon Intestine-Chip (day 5) when endothelium is present. Cell nuclei are shown in blue. (D) Representative confocal immunofluorescence images indicating the basolateral localization of the transporter Na+K+ adenosine triphosphatase (ATPase) (magenta) and the apical localization of the transporter SLC26A3 (green) on day 5 of culture. Cell nuclei are shown in blue. The application of stretch does not affect the establishment of the polarity of the epithelial cells. The plots indicate the mean fluorescent intensity distribution for each channel, SLC26A3 (green) or Na+K+ATPase (magenta), 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 on day 5 of the Colon Intestine-Chip culture in the presence or not of endothelium and/or stretch, show the significant contribution of stretch on the maturation of the epithelial brush border only in the absence of endothelial cells. (F) Box plots showing the density of microvilli per μm2, according to quantification of SEM images on day 5 of culture, in the presence (top graph) and absence (bottom graph) of endothelium and/or stretch. The quantification was performed across 3–11 different fields of view for each condition. Each individual point represents a single field of view. Unpaired t test. ∗P < .05 and ∗∗P < .01. DAPI, 4′,6-diamidino-2-phenylindole.
Fig 3: 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.
Supplier Page from Abcam for Anti-SLC26A3 antibody