Abstract: Background Disorders of the exocrine pancreas, such as pancreatitis, exocrine pancreatic insufficiency, and pancreatic neoplasia, lack adequate models to study their molecular pathophysiology. Canine pancreatic organoids provide a promising platform for investigating disease mechanisms and evaluating therapeutic interventions, with significant translational potential. Hypothesis/Objectives The aims of the study were to i) establish a reproducible protocol for generating canine pancreatic organoids, ii) assess their viability following 1–3 days of shipment on ice, and iii) validate their utility as an in vitro model that mimics the molecular complexity of native pancreatic tissue. Animals Residual pancreatic tissue from 4 dogs that were recently euthanized due to non-pancreatic disorders. Methods Pancreatic tissue was shipped on ice and used for organoid generation. Cells were embedded in 3D matrices and cultured in optimized media. The expression of endocrine (glucagon, somatostatin), exocrine (pancreatic α-amylase), and ductal cell marker (cytokeratin 19: KRT19) was evaluated in organoids using RT-qPCR and compared with pancreatic tissue. Results Organoids were successfully generated from all samples and exhibited cystic or bleb-like morphologies, requiring passaging every 7–8 days. Apical-in polarity, lumenal F-actin staining, and E-cadherin expression confirmed structural integrity and epithelial polarization. Organoids showed a 7-fold increase in ductal marker KRT19 expression but reduced endocrine and exocrine marker expression compared to donor tissue. Conclusions and Clinical Importance Canine pancreatic organoids can be reproducibly generated from post-euthanasia tissue and are primarily composed of ductal cells in Wnt-rich media. These organoids provide a foundation for creating advanced models to study pancreatic biology and disease mechanisms.
