Abstract: Background Three-dimensional (3D) pancreatic organoids recapitulate native tissue architecture, but their closed structure limits access to apical and basolateral surfaces, making functional assays challenging. Two-dimensional (2D) monolayers derived from organoids provide direct epithelial access for assessing barrier function, ion transport, and other physiological processes. Canine pancreatic 2D monolayer systems, however, remain underdeveloped. Hypothesis/Objectives To establish a canine pancreatic monolayer and validate its utility as an in vitro model reflecting the molecular complexity of native pancreas. Animals Residual pancreatic tissue from two dogs euthanized due to non-pancreatic disorders. Methods Pancreatic monolayers were cultured in four media: an expansion medium and three differentiation media. Epithelial integrity was assessed by transepithelial electrical resistance (TEER). KRT-19 expression, a ductal cell marker, was quantified measuring immunofluorescence intensity. Results 2D monolayers were successfully derived from canine pancreatic organoids, maintaining tight junctions with peak TEER values exceeding 4000 Ω·cm², indicating robust barrier function. Morphology and KRT-19 expression were media-dependent; pancreas-specific differentiation produced a maximum 2.83-fold decrease in KRT-19 immunofluorescence (p < 0.001). Conclusions and Clinical Importance This study establishes a reproducible protocol for deriving 2D monolayers from adult canine pancreas. The system enables both lineage characterization and functional assessment, offering a physiologically relevant and ethically accessible model for investigating pancreatic epithelial biology. These monolayers provide a foundation for applications in regenerative medicine, disease modeling, and comparative translational research.
