Integration of Stromal Cells and Hydrogel Below Epithelium Results in Optimal Barrier Properties of Small Intestine Organoid Models

<b>Background/Objectives</b>: The barrier properties of the human small intestine play a crucial role in regulating digestion, nutrient absorption and drug metabolism. Current in vitro organotypic models consist only of an epithelium, which does not take into account the possible role of stromal cells such as fibroblasts or the extracellular matrix (ECM) which could contribute to epithelial barrier properties. Therefore, the aim of this study was to determine whether these stromal cells or ECM were beneficial or detrimental to barrier function when incorporated into an organotypic human small intestine model. <b>Methods</b>: Intestinal epithelial cell lines or primary cell organoids derived from the epithelial stem cells of the small intestine were cultivated either on a porous Transwell membrane (epithelial model) or on a primary small intestinal stromal cell-populated collagen-fibrin hydrogel (full thickness model). <b>Results</b>: Both models expressed villin (enterocytes), lysozyme (Paneth cells), Ki67 (proliferative cells) and zonula occludens-1 (tight junctions). The polarized epithelial barriers of the full thickness models demonstrated a significant decrease in transepithelial electrical resistance (TEER) with values comparable to that found in the native small intestine in contrast to the higher TEER values observed in the epithelial models. This correlated to an increase in secreted zonulin, a regulator of intestine permeability, in the full thickness models. The decreased TEER values were due to both the stromal cells and the choice of the hydrogel versus the Transwell membrane. Moreover, erythropoietin and epithelial growth factor secretion, which have roles in regulating barrier integrity, directly correlated with the changes in TEER and permeability. <b>Conclusions</b>: This study emphasizes the importance of different cell types being incorporated into small intestine models and, also, the influence of the scaffold or matrix used.