Establishment of intestinal organoids from small intestine of growing cattle (12 months old)

Recently, we reported the robust <italic>in vitro</italic> three-dimensional (3D) expansion of intestinal organoids derived from adult bovine (&gt; 24 months) samples. The present study aimed to establish an <italic>in vitro</italic> 3D system for the cultivation of intestinal organoids derived from growing cattle (12 months old) for practical use as a potential alternative to <italic>in vivo</italic> systems for various purposes. However, very few studies on the functional characterization and three-dimensional (3D) expansion of adult stem cells from livestock species compared to those from other species are available. In this study, intestinal crypts, including intestinal stem cells, from the small intestines (ileum and jejunum) of growing cattle were isolated and long-term 3D cultures were successfully established using a scaffold-based method. Furthermore, we generated an apical-out intestinal organoid derived from growing cattle. Interestingly, intestinal organoids derived from the ileum, but not the jejunum, could be expanded without losing the ability to recapitulate crypts, and these organoids specifically expressed several specific markers of intestinal stem cells and the intestinal epithelium. Furthermore, these organoids exhibited key functionality with regard to high permeability for compounds up to 4 kDa in size (e.g., FITC-dextran), indicating that apical-out intestinal organoids are better than other models. Collectively, these results indicate the establishment of growing cattle-derived intestinal organoids and subsequent generation of apical-out intestinal organoids. These organoids may be valuable tools and potential alternatives to <italic>in vivo</italic> systems for examining host-pathogen interactions involving epithelial cells, such as enteric virus infection and nutrient absorption, and may be used for various purposes.