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.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Maintenance and cultivation of bovine intestinalorganoids
    Morphological classification of bovine intestinalorganoids and CHIR treatment
    RNA isolation
    Quantitative RT-PCR
    Generation of apical-out intestinal organoids
    Epithelial barrier permeability assay usingFITC-dextran
    Statistical analysis
RESULTS AND DISCUSSION
    Morphological classification of bovine intestinalorganoids
    Effect on Wnt activator treatment in bovine intestinalorganoids
    Gene expression profiling and generation of apical-outintestinal organoids
CONCLUSION
REFERENCES

• Kang Won Park(Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea)
• Hyeon Yang(Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea)
• Hayeon Wi(Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea)
• Sun A Ock(Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea)
• Poongyeon Lee(Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea)
• In-Sul Hwang(Columbia Center for Translational Immunology, Columbia University Irving Medical Center, Columbia University, New York 10032, USA)
• Bo Ram Lee(Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea) Corresponding Author