Robust Three-dimensional (3D) Expansion of Bovine Intestinal Stem Cells: A Model for Replacement of Animal Experiments

Abstract Background Intestinal organoids offer great promise for disease modelling based host-pathogen interactions and nutritional research for feed efficiency measurement in livestock as well as regenerative medicine for therapeutic purposes. However, very limited studies are available on the functional characterization and three-dimensional (3D) expansion of adult stem cells in livestock species compared to mammals. Therefore, we characterized intestinal stem cells derived from small intestine in adult bovine and cultivated intestinal organoids under in vitro 3D culture system.Results In this study, we successfully established intestinal organoids in bovine. 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. Furthermore, the genetic properties of intestinal organoids were highly similar to those of in vivo based on QuantSeq 3’ mRNA-Seq. data.Conclusions Collectively, these results provide a reliable method for efficient isolation of intestinal crypts from small intestine and robust 3D expansion of intestinal stem cells 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 system and will facilitate the practical use of a model to replace animal experiments in the fields of animal biotechnology for various purposes.