Three-dimensional (3D) organoid cultures from pancreatic tissue are emerging tools for studying pancreatic epithelial development, differentiation, and disease. While models derived from human fetal tissue or adult murine pancreas have provided key insights, their clinical translatability is limited by ethical and species-related differences. The domestic dog offers a valuable intermediate model for translational research, yet pancreatic organoid systems in this species remain underdeveloped. We successfully established long-term organoid cultures from adult canine pancreas and demonstrated their stable ductal phenotype. These organoids exhibited limited but detectable expression of acinar and endocrine markers, indicating partial trilineage potential. We further developed two-dimensional (2D) monolayers from organoids and assessed epithelial barrier integrity using transepithelial electrical resistance (TEER). Monolayers maintained tight junctions and showed media-dependent changes in morphology and KRT19 expression. Peak TEER values exceeded 4000 Ω·cm², confirming robust epithelial barrier function. This study presents a reproducible protocol for deriving organoids and monolayers from adult canine pancreas, offering a physiologically relevant and ethically accessible model for investigating pancreatic epithelial biology. The combination of 3D and 2D systems enables both lineage characterization and functional assessment, providing a foundation for future applications in regenerative medicine, disease modeling, and comparative translational research.
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