Robust Three-Dimensional (3D) Expansion of Bovine Intestinal Organoids: An In Vitro Model as a Potential Alternative to an In Vivo System

SIMPLE SUMMARY: The ability to recapitulate stem cells’ self-organising potential, creating three-dimensional (3D) structures of stem cells, has revolutionised various fields. Recently, in vitro 3D organoid systems are now used as alternative research tools because they serve the same purpose as in vivo systems. In the present study, we developed bovine intestinal organoids based on a 3D culture system and evaluated their characteristics. These bovine intestinal organoids, used in an in vitro model as a potential alternative to an in vivo system, hold great promise for further potential use in determining disease-modelling-based host–pathogen interactions and nutritional research for feed efficiency measurements. ABSTRACT: Intestinal organoids offer great promise for disease-modelling-based host–pathogen interactions and nutritional research for feed efficiency measurement in livestock and regenerative medicine for therapeutic purposes. However, very limited studies are available on the functional characterisation and three-dimensional (3D) expansion of adult stem cells in livestock species compared to other species. Intestinal crypts derived from intestinal organoids under a 3D culture system from the small intestine in adult bovine were successfully established and characterised for functionality testing, including the cellular potentials and genetic properties based on immunohistochemistry, immunocytochemistry, epithelial barrier permeability assay, QuantSeq 3′ mRNA-Seq. data and quantitative reverse transcription-polymerase chain reaction. Intestinal organoids were long-term cultivated over several passages of culture without loss of the recapitulating capacity of crypts, and they had the specific expression of several specific markers involved in intestinal stem cells, intestinal epithelium, and nutrient absorption. In addition, they showed the key functionality with regard to a high permeability for compounds of up to FITC-dextran 4 kDa, while FITC-dextran 40 kDa failed to enter the organoid lumen and revealed that the genetic properties of bovine intestinal organoids were highly similar to those of in vivo. Collectively, these results provide a reliable method for efficient isolation of intestinal crypts from the small intestine and robust 3D expansion of intestinal organoids in adult bovine and demonstrate the in vitro 3D organoids mimics the in vivo tissue topology and functionality. Finally, intestinal organoids are potential alternatives to in vivo systems and will be facilitated as the practical model to replace animal experiments for various purposes in the fields of animal biotechnology.