Novel chicken two-dimensional intestinal model comprising all key epithelial cell types and a mesenchymal sub-layer

Abstract

The intestinal epithelium plays a variety of roles including providing an effective physical barrier and innate immune protection against infection. Two-dimensional models of the intestinal epithelium, 2D enteroids, are a valuable resource to investigate intestinal cell biology and innate immune functions and are suitable for high throughput studies of paracellular transport and epithelial integrity. We have developed a chicken 2D enteroid model that recapitulates all major differentiated cell lineages, including enterocytes, Paneth cells, Goblet cells, enteroendocrine cells and leukocytes, and self-organises into an epithelial and mesenchymal sub-layer. Functional studies demonstrated the 2D enteroids formed a tight cell layer with minimal paracellular flux and a robust epithelial integrity, which was maintained or rescued following damage. The 2D enteroids were also able to demonstrate appropriate innate immune responses following exposure to bacterial endotoxins, from Salmonella enterica serotype Typhimurium and Bacillus subtilis. Frozen 2D enteroids cells when thawed were comparable to freshly isolated cells. The chicken 2D enteroids provide a useful ex vivo model to study intestinal cell biology and innate immune function, and have potential uses in screening of nutritional supplements, pharmaceuticals, and bioactive compounds.

Keywords: 2D enteroid; Bacteria; Barrier integrity; Chicken; Gut model; Immune response; Intestinal epithelium; Organoid.

Figure 1

Growth and formation of 2D enteroids. 2D enteroid grown on Matrigel-coated cell culture plastic. A Bright field image of epithelial cells forming islands on day 1 of culture. B Fibroblast-like cells seen around the edge of the epithelial cell islands (arrows). C A confluent polygonal epithelial cell layer on day 3 and on D day 6 of cell culture. E Higher magnification of 2D enteroid on day 6 of culture. F Bright field image of 2D enteroid grown on an uncoated cell culture plastic. Representative images of 5–6 independent experiments. Scale bars: 100 µm.

Figure 2

Cellular characterisation of the apical layer of 2D enteroids. Immunolocalisation of (A–F) apical epithelial cell types in 2D enteroids grown on Matrigel coated cell culture plastic at day 6 of culture. All cells are counterstained with DAPI (blue). The cells are stained for A E-cadherin (epithelial cells with adherens junctions, green) and vimentin (red), but vimentin expression is not evident in the sub-epithelial layer after mild cell permeabilisation with saponin, B villin (enterocytes, green), C Lysozyme (Paneth cells, red), D Chromogranin A (enteroendocrine cells, red), E Muc5AC (Goblet cells, green), and F CD45⁺ (leukocytes, green). Representative images from 3–4 independent experiments. Scale bars: 50 µm. G mRNA expression of the stem cell marker Lgr5 in freshly isolated villi, at day 2 and 5 of culture, grown on uncoated cell culture plastic. Data represents the median and 95% CI of three independent experiments.

Figure 3

Cellular characterisation of the basal layer of 2D enteroids. mRNA expression of A α-smooth muscle actin and B desmin in freshly isolated villi and in 2D enteroids grown on uncoated and Matrigel coated cell culture plastic at day 5 of culture. Data is represented as the mean of three independent experiments and 95% CI. Immunolocalisation of basal mesenchymal cell types in 2D enteroids grown on Matrigel coated cell culture plastic (C–F) or transwells (G–I). After removal of the apical cells, the cells are stained for C, D vimentin (mesenchyme, green). E, F To demonstrate the subepithelial layer, the cells are permeabilised with Triton X100 on day 2 and double stained for E-cadherin (epithelial cells with adherens junctions, green) and vimentin (mesenchyme, red). Vimentin expression is evident in the sub-epithelial layer. Images from 3 independent experiments. All cells are counterstained with DAPI (nuclei, blue). Scale bars: 50 µm. (G-J) Immunolocalisation of F-actin (red) and DAPI (nuclei, blue) in 2D enteroids grown on Matrigel coated transwells at day 4 of culture. Z- stack of G Apical, H Middle and I, J Basal slices. I, J DAPI stained nuclei in the basal slices demonstrate the presence of nuclei in the subepithelial layer (white arrowheads), whereas long F-actin filaments from mesenchymal cells (I) cross the basal layer slice (white arrows). The lines on the main photo indicate the image area represented in the transverse slices at the bottom and side of each photo. Representative images of 1 experiment. Scale bars: 15 µm.

Figure 4

Tight junction formation of 2D enteroids. 2D enteroids grown on Matrigel coated A cell culture plastic or B, C transwells. Cells are stained after 3 days in culture for ZO1 (red) expression with a low power image (A) and inset magnified. Nuclei are stained with DAPI (blue). Representative images of four independent experiments. Scale bar: 30 µM. B Paracellular transport measured by diffusion of 4 kDa FITC-dextran across the 2D enteroids from the apical to basal compartment in Matrigel coated transwells. Measurements were taken at 0.5, 1, 2, 3, 4 h post-application of FITC-dextran to the apical chamber. To allow for normalisation across all time points, fluorescence measurements included a sample of cell culture media only. Data is converted to percentage maximum diffusion, measured in wells with Matrigel coated inserts and no cells. ***Represents statistically significant difference between Matrigel coated wells with cells and with no cells. Data is the median of four independent experiments and 95% CI. (C) Measurement of trans-epithelial electrical resistance (TEER) reaching maximum TEER at day 3 and maintained until day 7 of culture. Data is represented as median of four independent experiments and 95% CI.

Figure 5

Enhancement of intestinal epithelial integrity. 2D enteroids grown on Matrigel coated transwells. The duration of the maximum TEER of 2D enteroids grown can be extended by A addition of CHIR99021 (10 mM) in the culture media from initial seeding for longer than 24 h (control, 24 h), B increasing Ca²⁺ concentration from control concentration 1.05 mM to 2 mM by addition of calcium chloride (duplicate wells) or by C addition of sodium butyrate (NaB; 3, 5, 7 mM; duplicate wells). D NaB and Ca²⁺ rescue epithelial integrity of a leaky gut model of 2D enteroids grown in low calcium media (LCM with 0.26 mM Ca²⁺). The barrier integrity could be rescued by restoring Ca²⁺ concentration to the control concentration (1.05 mM Ca²⁺) or by adding NaB (7 mM) from day 3 of culture. All additions were made every second day, from day 3 of culture, to the apical and basal compartments of the transwell. Statistical significance compared to control wells are *P < 0.05, **0.05 < P > 0.001, ***P < 0.001. Data is presented as the median of 3–5 independent experiments and 95% CI.

Figure 6

Bacterial components have no effect on intestinal epithelial integrity. 2D enteroids grown on Matrigel coated transwells. LPS from A S. enterica serotype Typhimurium, B E. coli O55:B5, C S. enterica serotype Enteritidis (0, 0.1, 1 and 10 μg/mL) and D Heat inactivated Avian Pathogenic E. coli (MOI 0, 10, 10, 100) were added from day 3 of culture to the apical and basal compartments. TEER measurements were analysed from day 3 to day 7 or 8 of culture. Data is represented as the mean of 3 independent experiments and 95% CI.

Figure 7

Bacterial components induce inflammatory responses in 2D enteroids. Confluent 2D enteroids cultured on uncoated plastic wells were treated on day 5 for 6 h with LTA from Bacillus subtilis or LPS from S. enterica serotype Typhimurium (1 and 10 μg/mL). Inflammatory responses are assessed by qRT-PCR A IL-6 and B IL-8 (CXCLi2) mRNA expression shown as fold change relative to control 2D enteroids. Statistical significance calculated in comparison to control cells and shown as *P < 0.05. Data is the median of four independent experiments and 95% CI.

Figure 8

Resurrection and cellular characterisation of 2D enteroids after cryopreservation. Immunolocalisation of 2D enteroids grown from cryopreserved intestine cells on Matrigel-coated transwells, on day 4 of culture. Z stack of co-immunolocalisation of F-actin (apical brush border, red) with intestinal epithelial cells (green); A ZO1 (Tight Junctions), B Villin (enterocytes), C Lysozyme (Paneth cells), D Muc5AC (Goblet cells) and E Chromogranin A (enteroendocrine cells). F Magnification of Z-stack of Muc5AC showing DAPI stained nuclei in basal cells (white arrow heads). Scale bars: 15 µm. G Vimentin expression (green) in basal mesenchymal cells in the absence of epithelial cells. Scale bar: 50 µm. Nuclei are stained with DAPI. The data represents three independent experiments. H, I Cells were thawed and grown as 2D enteroids in transwells; CHIR99021 was added to Maintenance Media for H 24 h and I 72 h. Data is the median of 3–4 independent experiments and 95% CI.