Adult Canine Intestinal Derived Organoids: A Novel In Vitro System for Translational Research in Comparative Gastroenterology

Abstract

1 Background: Large animal models, such as the dog, are increasingly being used over 2 rodent models for studying naturally occurring diseases including gastrointestinal (GI) 3 disorders. Dogs share similar environmental, genomic, anatomical, and intestinal 4 physiologic features with humans. To bridge the gap between currently used animal 5 models (e.g. mouse) and humans, and expand the translational potential of the dog 6 model, we developed a three dimensional (3D) canine GI organoid (enteroid and 7 colonoid) system. Organoids have recently gained interest in translational research as 8 this model system better recapitulates the physiological and molecular features of the 9 tissue environment in comparison with two-dimensional cultures.

Organoids are promising tools for species‐specific in vitro toxicological studies

“…The canine enteroids and colonoids have shown good colony-forming efficiency when cultured in human organoid defined media, including Wnt3a, R-spondin-1 and Noggin (Powell & Behnke, 2017). Two methods for canine enteroid culture are described in the literature: one method used collagenase digestion for crypt isolation (Powell & Behnke, 2017), while the other applied the EDTA chelation method (Chandra et al, 2018). The use of EDTA for crypt isolation significantly enhanced the purity of the crypt epithelium and decreased contamination with other cell types (Saxena et al, 2016), but its concentration and the time of incubation should be specific for each animal species because these parameters are related to length, numbers and size of villi, which are species-specific (Mahe et al, 2013).…”

“…The use of EDTA for crypt isolation significantly enhanced the purity of the crypt epithelium and decreased contamination with other cell types (Saxena et al, 2016), but its concentration and the time of incubation should be specific for each animal species because these parameters are related to length, numbers and size of villi, which are species-specific (Mahe et al, 2013). Canine jejunal enteroids are capable of reproducing the anatomical and physiological features of native jejunal tissue (Chandra et al, 2018). …”

“…Canine gastrointestinal organoids (enteroids and colonoids, obtained from small and large intestine, respectively) have been recently developed (Chandra et al, 2018). In a study performed by Chandra and colleagues, organoids were generated from intestinal ASCs isolated from whole jejunal tissue and from duodenal, ileal and colonic biopsy samples of dogs, demonstrating that the isolation of crypt epithelium can be performed from different canine intestinal regions (duodenum, jejunum, ileum and colon) (Chandra et al, 2018). The canine enteroids and colonoids have shown good colony-forming efficiency when cultured in human organoid defined media, including Wnt3a, R-spondin-1 and Noggin (Powell & Behnke, 2017).…”

“…Epithelial markers (keratin, chromogranin A and periodic acid-Schiff) expressed in whole jejunal tissue were found in canine enteroids, but mesenchymal (vimentin, actin) and immune cell (c-kit and CD3 T) markers were expressed only in the whole jejunal tissues, demonstrating that canine enteroids consist only of epithelial populations without other cell types(Chandra et al, 2018). Chandra et al (2018) used a canine-specific LGR5 probe to identify crypt base columnar SCs, as the presence of LGR5+ adult ISCs (namely the crypt base columnar SCs) are essential to develop the peculiar crypt-villus enteroid structure if a non-epithelial cell niche is not present.…”

“…Whole intestinal tissue contains epithelial, mesenchymal and immune cells, but enteroids and colonoids obtained from adult ISCs showed only epithelial cell populations. Epithelial markers (keratin, chromogranin A and periodic acid-Schiff) expressed in whole jejunal tissue were found in canine enteroids, but mesenchymal (vimentin, actin) and immune cell (c-kit and CD3 T) markers were expressed only in the whole jejunal tissues, demonstrating that canine enteroids consist only of epithelial populations without other cell types(Chandra et al, 2018). These findings proved that canine gastrointestinal organoids, being a good representation of the canine intestine, could be successfully used as innovative in vitro models in toxicology studies.…”