Culture media and format alter cellular composition and barrier integrity of porcine colonoid-derived monolayers

In vitro models of the intestine are used to study the complex in vivo intestinal processes in a simplified context. As such, these models need to be representative of their tissue of origin. Here, we demonstrate that porcine colonoids and colonoid-derived monolayers that have comparable stem cells and differentiated cell types to those of the native tissue can be developed but are influenced by cell seeding density, culture format, and medium formulation. Intestinal organoid technology has revolutionized our approach to in vitro cell culture due in part to their three-dimensional structures being more like the native tissue from which they were derived with respect to cellular composition and architecture. For this reason, organoids are becoming the new gold standard for undertaking intestinal epithelial cell research. Unfortunately, their otherwise advantageous three-dimensional geometry prevents easy access to the apical epithelium, which is a major limitation when studying interactions between dietary or microbial components and host tissues. To overcome this problem, we developed porcine colonoid-derived monolayers cultured on both permeable Transwell inserts and tissue culture treated polystyrene plates. We found that seeding density and culture format altered the expression of genes encoding markers of specific cell types (stem cells, colonocytes, goblets, and enteroendocrine cells), and barrier maturation (tight junctions). Additionally, we found that changes to the formulation of the culture medium altered the cellular composition of colonoids and of monolayers derived from them, resulting in cultures with an increasingly differentiated phenotype that was similar to that of their tissue of origin.