Generation of canine gallbladder cholangiocyte organoids from healthy and gallbladder mucocele patient dogs for functional analysis of anion channels

Gallbladder mucocele (GBM) is one of the most common gallbladder disorders in dogs. Recent studies have suggested a potential association between GBM development and reduced expression of anion channel-related molecules in gallbladder epithelial cells. However, further investigation has been limited due to the lack of an effective in vitro culture system to validate the functions of the molecules. This study aimed to establish gallbladder cholangiocyte organoids (GCOs) from canine gallbladders and evaluate anion channel functions. GCOs were generated from gallbladders of three clinically healthy dogs and three GBM-affected dogs, followed by morphological evaluation, comparisons of anion channel-related gene expressions, and functional assessment of anion channels, specifically cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCCs), using organoid swelling assays. Most GCOs derived from normal gallbladder (N-GCOs) exhibited a round, cystic morphology, whereas GCOs from GBM-affected gallbladder (GBM-GCOs) predominantly displayed a budding morphology with narrowed lumen. Although no significant differences in anion channel-related gene expression were observed between the two groups, the functions of both CFTR and CaCCs were significantly impaired in GBM-GCOs. The morphological differences between the groups might be attributed to the reduced ion secretions and subsequent shortages of water transport into the organoid lumen in GBM-GCOs, leading to insufficient lumen expansion. These findings highlighted a potential link between anion channel dysfunction and GBM pathogenesis. Further studies utilizing canine GCOs are warranted to deepen our understanding of the role of anion channel dysfunction in GBM development.