Background: The small intestine plays a crucial role in animals in maintaining homeostasis as well as a series of physiological events such as nutrient uptake and immune function to improve productivity. Research on intestinal organoids has recently garnered interest, aiming to study various functions of the intestinal epithelium as a potential alternative to an in vivo system. These technologies have created new possibilities and opportunities for substituting animals for testing with an in vitro model. Methods: Here, we report the establishment and characterisation of intestinal organoids derived from jejunum tissues of adult pigs. Intestinal crypts, including intestinal stem cells from the jejunum tissue of adult pigs (10 months old), were sequentially isolated and cultivated over several passages without losing their proliferation and differentiation using the scaffold-based and three-dimensional method, which indicated the recapitulating capacity. Results: Porcine jejunum-derived intestinal organoids showed the specific expression of several genes related to intestinal stem cells and the epithelium. Furthermore, they showed high permeability when exposed to FITC-dextran 4 kDa, representing a barrier function similar to that of in vivo tissues. Collectively, these results demonstrate the efficient cultivation and characteristics of porcine jejunum-derived intestinal organoids. Conclusions: In this study, using a 3D culture system, we successfully established porcine jejunum-derived intestinal organoids. They show potential for various applications, such as for nutrient absorption as an in vitro model of the intestinal epithelium fused with organ-on-a-chip technology to improve productivity in animal biotechnology in future studies.

Recent progress has been made to establish intestinal organoids for an in vitro model as a potential alternative to an in vivo system in animals. We previously reported a reliable method for the isolation of intestinal crypts from the small intestine and robust three-dimensional (3D) expansion of intestinal organoids (basal-out) in adult bovines. The present study aimed to establish next-generation intestinal organoids for practical applications in disease modeling-based host-pathogen interactions and feed efficiency measurements. In this study, we developed a rapid and convenient method for the efficient generation of intestinal organoids through the modulation of the Wnt signaling pathway and continuous apical-out intestinal organoids. Remarkably, the intestinal epithelium only takes 3-4 days to undergo CHIR (1 µM) treatment as a Wnt activator, which is much shorter than that required for spontaneous differentiation (7 days). Subsequently, we successfully established an apical-out bovine intestinal organoid culture system through suspension culture without Matrigel matrix, indicating an apical-out membrane on the surface. Collectively, these results demonstrate the efficient generation and next-generation of bovine intestinal organoids and will facilitate their potential use for various purposes, such as disease modeling, in the field of animal biotechnology.

Three-dimensional (3D) organoids act as model systems because they mimic in vivo tissue morphology. Recent advancements in the field have demonstrated that organoids derived from various organs have assisted in understanding the underlying mechanisms of disease modeling and expanded our knowledge of organ development in vitro. Furthermore, these organoids have become a promising biomaterial in regenerative medicine for therapeutic purposes as well as in nutritional research for feed efficiency measurement in livestock. Intestinal organoids of livestock, including pigs, cattle, chickens, and horses, have been developed. These could be used to examine host-pathogen interactions, such as interaction between enteric viruses and epithelial cells, and are potential alternatives to in vivo systems. However, there are very limited studies regarding species-specific medium to cultivate and establish intestinal organoids of livestock. Species-specific medium is applied differently between species for the cultivation of intestinal organoids, and its modification is important for the maintenance of specific cell types or genes from the cellular diversity of the intestinal epithelium. In this study, we introduce the histological development of a 3D culture system and a species-specific medium for the cultivation of intestinal organoids in livestock. Finally, we discuss the importance and future perspectives of intestinal organoids in the fields of agriculture and biotechnology for various purposes.