Abstract
Cattle are an economically important domestic animal species. In vitro 2D cultures of intestinal epithelial cells or epithelial cell lines have been widely used to study cell function and host–pathogen interactions in the bovine intestine. However, these cultures lack the cellular diversity encountered in the intestinal epithelium, and the physiological relevance of monocultures of transformed cell lines is uncertain. Little is also known of the factors that influence cell differentiation and homeostasis in the bovine intestinal epithelium, and few cell-specific markers that can distinguish the different intestinal epithelial cell lineages have been reported. Here we describe a simple and reliable procedure to establish in vitro 3D enteroid, or “mini gut”, cultures from bovine small intestinal (ileal) crypts. These enteroids contained a continuous central lumen lined with a single layer of polarized enterocytes, bound by tight junctions with abundant microvilli on their apical surfaces. Histological and transcriptional analyses suggested that the enteroids comprised a mixed population of intestinal epithelial cell lineages including intestinal stem cells, enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We show that bovine enteroids can be successfully maintained long-term through multiple serial passages without observable changes to their growth characteristics, morphology or transcriptome. Furthermore, the bovine enteroids can be cryopreserved and viable cultures recovered from frozen stocks. Our data suggest that these 3D bovine enteroid cultures represent a novel, physiologically-relevant and tractable in vitro system in which epithelial cell differentiation and function, and host–pathogen interactions in the bovine small intestine can be studied.
Highlights
The mucosal surface that lines the mammalian gastrointestinal tract is continuously exposed to commensal and pathogenic microorganisms
Paneth cells are absent in the large intestine, where regenerating islet-derived family member 4 (REG4)-expressing deep secretory cells play a similar role in the maintenance of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)+ intestinal stem cells in colonic crypts [7]
In the current study we describe the development of a 3D in vitro culture system that is more representative of the cellular diversity within the bovine intestinal epithelium
Summary
The mucosal surface that lines the mammalian gastrointestinal tract is continuously exposed to commensal and pathogenic microorganisms. 3D enteroids or “mini guts” are formed, comprising a single layer of intestinal epithelial cells surrounding a central closed lumen with many crypt bud domains [13] These enteroid cultures maintain much of the cellular diversity present within the intestinal epithelium in vivo [6, 13, 15,16,17,18]. Few physiologically-relevant in vitro systems are available to accurately study the interactions between enteric pathogens and the bovine intestinal epithelium, and most data have been derived from the analysis of 2D monolayers of epithelial cell lines or primary epithelial cells [27, 28]. Our data suggest that these bovine small intestinal crypt-derived enteroid cultures represent a useful physiologically-relevant in vitro system to study epithelial cell differentiation and function, and host–pathogen interactions in the bovine small intestine
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
