PS6-21. Characterization of Canine Colon Organoids.

Abstract

Organoids are self-organizing three-dimensional cell culture models that provide a unique opportunity to study organ physiology. Unlike conventional cell lines, this culture system allows them to grow as structures that closely resemble the complex cellular composition and heterogeneity of the in vivo tissue. The objective of this study was to establish canine colon organoid cultures from tissue-resident stem cells and characterize their gene expression profile. Colon Organoids (CO; n = 3) generated from frozen necropsy tissue of end-of-life (EOL) dogs at Hill's Pet Nutrition Center (PNC) were compared to frozen EOL colon tissues (CT; n = 6).Culture growth and morphology were monitored, and mucin-2 (MUC2) secretion was assessed on day 12 for functional viability using ELISA. For gene expression analysis, total RNA was extracted, on day 12 from CO, and analyzed using a custom-made TaqMan PCR array canine metabolic panel (43 target genes). Statistical analysis for fold change (ΔΔCT) was performed following normalization to GAPDH, the house keeping gene. Cultured organoids successfully formed three-dimensional structures, with growth density increasing rapidly after Day 3. Organoids secreted MUC2 in media by day 12, levels ranging from 2720 to 3669 pg/ml (n = 3). Gene expression analysis revealed distinct patterns when comparing colon organoids (COs) to their tissue of origin. There was a substantial increase in stem cell and growth markers genes in CO including LGR5, a stem-cell marker (20.6-fold increase, p = 0.06), SOX9, a transcription factor involved in development (9.5-fold increase, p = 0.05), and FASN, an enzyme that synthesizes fatty acids (5.3-fold increase, p = 0.09) when compared to colon tissue. Core metabolic genes were conserved in CO, comparable to that of the primary tissue. Including GPT, an enzyme involved in amino acid and energy metabolism (0.87-fold change); ELOVL6, a gene involved in long chain fatty acid elongation (1.26-fold change); and PPARD, a transcription factor for fatty acid oxidation (1.11-fold change). In contrast, several major metabolic regulators were significantly downregulated in organoids (all p < 0.05). Including the bile acid sensor FXR (11.4-fold decrease), suggesting impaired bile acid sensing, as well as HK2 (9.6-fold decrease), DGAT1 (7.6-fold decrease), PGC-1a (7.2-fold decrease), and PPARA (5.1-fold decrease, ns). Decrease in PGC-1a and PPARA indicates a shift from mitochondrial fatty acid oxidation likely due to minor differences in energy modulation in CO compared to CT. Our results indicate that the colon organoids have both morphological integrity and functional viability and are suitable for targeted research involving metabolic function and energy balance. Characterization of colon organoids contributes to the advancement of studies investigating cellular and molecular mechanisms of action of nutritional ingredients in vitro.