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.

INTRODUCTION
MATERIALS AND METHODS
    Intestinal organoid growth, passage, andcryopreservation
    Experimental design and animals
    Intestinal crypt isolation and 3D cultivation
    RNA isolation
    Library preparation and sequencing
    Data processing and analysis
    Differential gene expression analysis
    Immunocytochemistry
    Epithelial barrier permeability assay using FITCdextran
    Statistical analysis
RESULTS
    Growth performance and long-term maintenance ofporcine jejunum-derived intestinal organoids
    Gene expression profiling of porcine jejunum-derivedintestinal organoids
    Characterisation and paracellular permeability ofporcine jejunum-derived intestinal organoids
DISCUSSION
CONCLUSION
REFERENCES

• Bo Ram Lee(Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea) Corresponding author
• Sun A Ock(Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea)
• Mi Ryung Park(Animal Genetic Resources Research Center, National Institute of Animal Science, Rural Development Administration, Hamyang 50000, Korea)
• Min Gook Lee(Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju 55365, Korea)
• Sung June Byun(Poultry Research Institute, National Institute of Animal Science, Rural Development Administration, Pyeongchang 25342, Korea)