Isolation, culture and maintenance of rabbit intestinal organoids, and organoid-derived cell monolayers

Abstract

Abstract Organoids emulate many aspects of their parental tissue and have been used to study pathogen-host interactions, tissue development and regeneration, metabolic diseases, and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. We also report conditions that sustain an intestinal stem cell population in spheroid culture. Rabbit intestinal spheroids and monolayer cultures propagated and expanded most efficiently in L-WRN-conditioned medium that contained the signalling factors Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-β inhibitors. Organoid and monolayer differentiation was initiated by switching to a medium that contained less of the L-WRN-conditioned medium and was supplemented with ROCK and Notch signalling inhibitors. Using immunofluorescence staining and RT-qPCR, we demonstrate that organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. These findings demonstrate that our rabbit intestinal organoids have many of the multi-cellular characteristics of, and closely resemble, an intestinal epithelium. This newly established organoid culture system will provide a useful tool to study rabbit gastrointestinal physiology and disease. For example, organoids and organoid-derived cells may be used to propagate and study caliciviruses and other enterotropic pathogens that cannot be grown in conventional cell culture systems.