Derivation of adult canine intestinal organoids for translational research in gastroenterology

Abstract

Background Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures. Results Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research. Conclusions We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications. Electronic supplementary material The online version of this article (10.1186/s12915-019-0652-6) contains supplementary material, which is available to authorized users.

Recapitulation of the accessible interface of biopsy-derived canine intestinal organoids to study epithelial-luminal interactions

“…We have recently optimized the three-dimensional (3D) in vitro culture conditions of canine primary intestinal organoids and shown that isolated intestinal stem cells differentiate into organoids containing matured intestinal cell lineages within~8 days of culture [9]. The 3D organoid culture technology not only offers a more physiological platform compared with conventional 2D cell lines [10], but also provides a "personalized" modeling to investigate the effect of environmental stimuli or dietary interventions on intestinal epithelium [11].…”

“…We envision that our optimized protocol and the robust culture of canine-derived epithelium may enable to develop an advanced in vitro model to demonstrate complex hostgut microbiome crosstalk and pharmacological assessment under various disease milieus. of the primary intestinal epithelium [9]. A colonoid-derived monolayer was generated in a nanoporous insert of the Transwell pre-coated with the extracellular matrix (ECM) mix with Matrigel (100 μg/mL) and collagen I (30 μg/mL) by introducing the dissociated colonoid cells ( Fig 1B).…”

Dynamic Monitoring of Cross-Bred Dogs for Translational Research in Ulcerative Colitis

“…Many reports showed that mice or rats as experimental models of UC [28] [11,29]. But, dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans [30].In this paper, we suggest that cross-bred dogs represents a useful clinical model of UC.…”

Generation of Differentiating and Long-Living Intestinal Organoids Reflecting the Cellular Diversity of Canine Intestine

“…This absence of differentiated cells in expansion medium could not be overcome by prolonged cultivation: low-(≤5) and high-passage (≥10) canine intestinal organoids all tested negative for differentiation marker expression. Recently, Chandra et al published a well-written paper on the derivation of canine intestinal organoids and the presence of goblet and enteroendocrine cells [35]. The formulation of the expansion medium differs in a 5-fold higher concentration of p38-MAPK inhibitor and the addition of 8% fetal bovine serum FBS (Chandra) compared to the medium formulation used herein.…”