Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces.

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

Adult Bovine-Derived Small and Large Intestinal Organoids: In Vitro Development and Maintenance.

Recent progress in bovine intestinal organoid research has expanded opportunities for creating improved in vitro models to study intestinal physiology and pathology. However, the establishment of a culture condition capable of generating organoids from all segments of the cattle intestine has remained elusive. Although previous research has described the development of bovine jejunal, ileal, and colonic organoids, this study marks the first report of successful bovine duodenal and rectal organoid development. Maintenance of these organoids through serial passages and cryopreservation was achieved, with higher success rates observed in large intestinal organoids compared to their small intestinal counterparts. A novel approach involving the use of biopsy forceps during initial tissue sampling streamlined the subsequent tissue processing, simplifying the procedure compared to previously established protocols in cattle. Additionally, our study introduced a more cost-effective culture medium based on Advanced DMEM/F12, diverging from frequently used commercially available organoid culture media. This enhancement improves accessibility to organoid technology by reducing culture costs. Crucially, the derived organoids from jejunum, ileum, colon and rectum faithfully preserved the structural, cellular, and genetic characteristics of in vivo intestinal tissue. This research underscores the significant potential of adult bovine intestinal organoids as a physiologically and morphologically relevant in vitro model. Such organoids provide a renewable and sustainable resource for a broad spectrum of studies, encompassing investigations into normal intestinal physiology in cattle and the intricate host-pathogen interactions of clinically and economically significant enteric pathogens.