Functional characteristics of a pig 2D intestinal organoid model as an in vitro platform for nutritional studies

Intestinal epithelial cell lines have been widely used in the field of biomedical and livestock research, and recently, the use of organoid systems has been attempted. However, they have several limitations as an <i>in vitro</i> platform in particularly in nutrition-related studies. Thus, this study aimed to compare the existing <i>in vitro</i> platform (IPEC-J2 cell line) with a three-dimension (3D) organoid model, and to understand the nutritional phenomena occurring in the intestinal lumen through the establishment and characterization of a two-dimension (2D) organoid model. By comparing the IPEC-J2 cell line and 3D intestinal organoids, we found differences in intestinal epithelial cell types, including nutrient-related enteroendocrine cells and enterocytes. 3D organoids have most of gut epithelial cell types, but IPEC-J2 did not. We further established a 2D organoid model with an exposed apical membrane and compared it with a 3D organoid model. The established 2D organoids had higher expression of enteroendocrine cells and enterocyte marker genes, and most genes were related to nutritional properties (nutrient transporters, hormones, and digestive enzymes). Fatty acids, one of the nutrients, were added to the two organoid models for comparison. Fluorescence image analysis confirmed that more fatty acids were absorbed by 2D organoids. Treatment with a long-chain fatty acid mixture increased the expression of fatty acid receptor (<i>FFAR1</i> and <i>FFAR4</i>) and hormone (<i>GCG</i>, <i>CCK</i>, and <i>PYY</i>) genes in 2D organoids but not in 3D organoids, leading to the activation of metabolic responses. The more facilitated metabolic process was observed in 2D organoids by increased mitochondria activity and ATP production. Our findings emphasize that pig intestinal organoid systems, particularly 2D organoid model, is better <i>in vitro</i> platform, particularly in nutrition-related studies. Compared with other <i>in vitro</i> platforms, 2D organoids can be used for studying intestinal epithelial cell-nutrient interactions structurally and characteristically. Our study provides a basis for utilizing a pig 2D intestinal organoid model as a potentially advanced <i>in vitro</i> system for intestinal epithelial cell-based nutritional research in domestic animals.