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.
Highlights
The small intestine is a highly complex structure with several roles; in addition to nutrient and water absorption from ingested food and secretion of digestive enzymes it acts as physiological barrier, prevents invasion of commensal and pathogenic bacteria in the lumen and regulates the intestinal immune system
Establishment of 2 Dimensional (2D) enteroids culture system To establish a chicken 2D enteroid culture system, we plated dissociated single cells from villi isolated from the intestine of ED18 embryos in a cell culture medium containing the growth factors, Epidermal growth factor (EGF), Noggin, R-spondin, CHIR99021 and ROCK inhibitor
Recent advances in the generation of 3 Dimensional (3D) chicken enteroids have improved the complexity of the cultured organoids to closer resemble native tissue [25], but 2D chicken intestinal models are still relatively primitive with partial characterisation of epithelial cell types, lacking the multi-cellular interactions found in vivo, and with limited scope for functional studies [17,18,19,20,21,22,23,24, 27]
Summary
The small intestine is a highly complex structure with several roles; in addition to nutrient and water absorption from ingested food and secretion of digestive enzymes it acts as physiological barrier, prevents invasion of commensal and pathogenic bacteria in the lumen and regulates the intestinal immune system. To study the intestine ex vivo, three-dimensional (3D) intestinal organoids have been developed in several mammalian species from adult stem cells or isolated crypts and villi including human [3, 4] mouse [4, 5], pig [6], cow [7], and horse [8]. Successful intestinal monolayers have subsequently been developed in many of these species; human [9, 10], mouse [9, 11, 12], pig [13], cow [14], and cat [15]. There are currently no chicken intestinal monolayer models that demonstrate expression of all epithelial cell types and form a confluent cell layer with robust epithelial integrity, that can be used to study paracellular permeability and barrier function
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