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. 2019;8(4):653-655.e5.
doi: 10.1016/j.jcmgh.2019.07.009. Epub 2019 Jul 26.

Building a Thick Mucus Hydrogel Layer to Improve the Physiological Relevance of In Vitro Primary Colonic Epithelial Models

Y Wang  1 , R Kim  2 , C E Sims  1 , N L Allbritton  3
Affiliations

Building a Thick Mucus Hydrogel Layer to Improve the Physiological Relevance of In Vitro Primary Colonic Epithelial Models

Y Wang et al. Cell Mol Gastroenterol Hepatol. 2019.
No abstract available

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Figures

Figure 1
Figure 1
VIP-assisted ALI culture generates physiologic mucus layer above intestinal epithelial cells. (A) Culture schematic. (B) Dose-dependent water secretion of VIP at 24 hours. (C) Immunofluorescence of monolayers differentiated under submerged and ALI conditions in the presence of VIP. Green: anti-Muc2; blue: Hoechst 33342. (D) Removal of hydrated mucus with forceps. (E) Side-view confocal micrograph showing tissues with mucus separating bacteria from epithelial cells at 0 and 6 days, respectively. Green: green fluorescent protein (GFP)–expressing Escherichia coli; blue: Hoechst 33342. (F) Plot of mucus thickness vs duration of ALI. (G) Representative side-view confocal micrograph showing 1 μm red fluorescent beads separated from epithelial cells by mucus. (H) Immunofluorescence staining of paraffin-embedded, sectioned monolayers. DM, differentiation medium.
Figure 2
Figure 2
Effect of Clostridium difficile toxin A and Escherichia coli (EC) on human colonic epithelium in the absence or presence of the VIP-enhanced mucus layer. (A) Cell-culture schematic for panels B and C. (B) Permeability and (C) IL-8 secretion of the epithelium after 4-hour exposure to toxin A. (D) Cell culture schematic for panels E, F, and G. (E–G) Production of inflammatory cytokines after a 24-hour co-culture of green fluorescent protein (GFP)–expressing EC, epithelium, and peripheral blood mononuclear cells (PBMC): (E) tumor necrosis factor alpha (TNF-α), (F) IL-6, and (G) IL-1β. Unpaired t test: **P < .005; #not statistically significant. n = 3 samples per condition. N.S., not significant.
Supplemental Figure 1
Supplemental Figure 1
Air-liquid interface (ALI) culture generates a compact, clumped-appearing mucus layer. (A) Schematic of culture formats. (B) Immunofluorescence staining of sectioned monolayers. Green: Muc2; blue: Hoechst 33342. White arrows indicate goblet cells. (C) Scanning electron microscopy images. Top panel: submerged culture. Bottom panel: ALI culture. (D) The mucus layer was overlaid with 1-μm red fluorescent beads (top, red) or GFP-expressing EC (bottom, green). The nuclei of intestinal cells were stained with Hoechst 33342 (blue/aqua). The yellow dashed line shows the boundary between the mucus and microbeads or EC. A, apical; B, basal.
Supplemental Figure 2
Supplemental Figure 2
(A, B) Effect of culture method and VIP (0 and 330 ng/mL) on the number of goblet cells. (A) Immunofluorescence images of monolayers stained for Muc2 (green). (B) The percentage of the monolayer surface area positive for Muc 2 fluorescence. *P < .05; **P < .005; #not statistically significant. n = 3. (C, D) The hydrated mucus layer generated by VIP-assisted air-liquid interface (ALI) culture. (C) Side-view confocal micrograph showing tissues with bacteria-separating mucus accumulation at 2 and 4 days, respectively. Green: GFP-expressing EC; blue: Hoechst 33342. (D) Scanning electron microscopy of hydrated mucus layer (overlaid with GFP-expressing EC). The mucus layer was partially removed using tweezers to reveal the epithelium (lower arrow) below the mucus. The upper right panel shows bacteria (rod-shaped structures) on the surface of the mucus layer. (E–G) Characterizations of the hydrated mucus layer generated by VIP-assisted ALI culture. (E) Visualization of the mucus layer by overlaying 1-μm red fluorescent beads. Top: adding beads to the mucus layer without rinsing. Bottom: adding beads to the mucus layer after gently rinsing. (F) The mucus layer was overlaid with a mixture of 0.02-μm red fluorescent beads (#F8786; ThermoFisher) and 5_μm green fluorescent beads (#G0500; ThermoFisher) for 4 hours. (G) Immunofluorescence of sectioned monolayers. Red: AGR2 or Meprin β; blue: Hoechst 33342. There was minimal Meprin β expression, as was expected because the tissue is derived from the large and not small intestine and the β subunit is not predominant in the large intestine.
Supplemental Figure 3
Supplemental Figure 3
Effect of Clostridium difficile toxin A on human colonic epithelium in the absence or presence of the VIP-enhanced mucus layer. (A) Confocal microscopic and Scanning electron microscopy images of F-actin (top panel) and zonula occludens-1 tight junction (middle panel). (B) Permeability and (C) IL-8 secretion of epithelium after 8-hour exposure to toxin A. Unpaired t test: *P < .05; **P < .005; #not statistically significant. n = 3. Scale bar = 20 μm.
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