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

Abstract

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. In addition, 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 the accessibility to organoid technology by reducing culture costs. Crucially, the derived organoids from the jejunum, ileum, colon, and rectum faithfully preserved the structural, cellular, and genetic characteristics of the 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.

Pathogen-epithelium interactions and inflammatory responses in Salmonella Dublin infections using ileal monolayer models derived from adult bovine organoids

“…The organoid-derived monolayer system is superior to the conventional cell-based monolayer models because it retains cellular heterogeneity derived from intestinal stem cells similar to the native intestinal tissue. Relatively abundant expression of the enteroendocrine cell marker gene and lower expressions of Paneth cell and goblet cell marker genes are in line with previous observations with bovine ileal organoids 19 , 30 . Additionally, the organoid-derived monolayer system offers significant advantages for in vitro studies of host–pathogen interactions compared to 3D organoid models.…”

“…The present study did not evaluate the direct gene expression of the donor tissue; thus, it is not possible to compare and discuss similarities or differences in genetic properties between the monolayers and their originating tissues. Compared with previous research 19 , there are considerable gene expression variations between 2D ileal monolayers and 3D organoids, demonstrating how culture methods influence cellular behavior. Nonetheless, the 2D monolayers are particularly valuable for studying specific aspects of host-pathogen interactions as well as intestinal physiology, providing key insights into these processes.…”

“…A stable intestinal epithelial monolayer was generated using adult bovine ileal organoids, as previously described 19 . The optimized culture conditions for the ileal monolayer were determined by varying the culture medium compositions and cell seeding densities (Supplementary Fig.…”

“…Ileal tissue samples were freshly obtained from four approximately 15- to 18 month-old cattle at a local slaughterhouse (Supplementary Table S2 ). The tissue was sampled and processed for crypt isolation and organoid development following the previously described protocol 19 . Briefly, approximately 4–8 mm sections of ileal tissue were sampled using biopsy forceps and rinsed with ice-cooled Dulbecco’s phosphate-buffered saline (PBS) containing 1 × penicillin/streptomycin and 25 μg/mL gentamicin.…”

“…Subsequently, each well received 300 μL of organoid culture medium once Matrigel was polymerized at 37 ℃ for 10–15 min. The organoid culture medium was prepared according to a previous study 19 (Supplementary Table S3 ). The medium was changed every other day for growth and maintenance of ileal organoids.…”