Plenge, M.; Schnepel, N.; Muesken, M.; Rohde, J.; Goethe, R.; Breves, G.; Mazzuoli-Weber, G.; Benz, P.

The porcine colon epithelium plays a crucial role in nutrient absorption, ion transport, and barrier function. However ethical concerns necessitate the development of alternatives to animal models for its study. The objective of this study was to develop and characterise a two?dimensional (2D) in vitro model of porcine colonic organoids that closely mimics native colon tissue, thereby supporting in vitro research in gastrointestinal physiology, pathology, and pharmacology. Porcine colonic crypts were isolated and cultured in three-dimensional (3D) organoid systems, which were subsequently disaggregated to form 2D monolayers on transwell inserts. The integrity of the monolayers was evaluated through the measurement of transepithelial electrical resistance (TEER) and electron microscopy. The functional prerequisites of the model were evaluated through the measurement of the mRNA expression of key ion channels and transporters, using quantitative RT-PCR. Ussing chamber experiments were performed to verify physiological activity. The 2D monolayer displayed robust TEER values and retained structural characteristics, including microvilli and mucus-secreting goblet cells, comparable to those observed in native colon tissue. Gene expression analysis revealed no significant differences between the 2D organoid model and native tissue with regard to critical transporters. Ussing chamber experiments demonstrated physiological responses that were consistent with those observed in native colonic tissue. In conclusion, 2D porcine colonic organoid model can be recommended as an accurate representation of the physiological and functional attributes of the native colon epithelium. This model offers a valuable tool for investigating intestinal barrier properties, ion transport, and the pathophysiology of gastrointestinal diseases, while adhering to the 3R principles.