The intestinal epithelium plays a crucial role in regulating the immune response and maintaining homeostasis. It serves as a barrier that restricts the passage of water and macromolecules, and this barrier function is integral to its role. The barrier function is provided by a single layer of diverse epithelial cells, connected by cell junctions, supported by the extracellular matrix, and covered by a mucus layer. The study of epithelium has been limited for a long time to standard cell lines and synthetic substrates under conditions that do not mimic the physiological state. Thankfully, the next generation of intestinal models was developed with the advancement of organoids, small intestinal submucosa substrates, and microfluidic systems. In our study, we utilized these newly developed approaches in cell culture to investigate the barrier function in a better in vivo-mimicking state. Firstly, given the key role of mucus in the barrier function, we aimed to gain insights into mucin expression in human intestines, both in health and inflammatory bowel disease, by leveraging large-scale omic datasets generated from RNA-sequenced samples from ileum and colon tissues. In this thesis, we analyzed the mucin expression profiles within the dataset and provided an atlas of mucin expression for Crohn’s disease, ulcerative colitis, and the healthy state. We also identified a subgroup of Crohn's patients who exhibited de novo expression of secreted mucins and explored this further. Additionally, we implemented a new method for cell culture, in which we seeded intestinal stem cell-derived monolayers generated from human organoids onto hydrogels developed from porcine small intestinal submucosa enriched with native extracellular matrix proteins. We compared the cellular behavior of cells cultured on hydrogels to that of cells cultured on tissue culture plastic coated with Matrigel at early and late time points, and we employed bulk RNA sequencing to obtain a comprehensive overview of gene expression under these conditions. We found that the hydrogels supported the long-term survival of culture and resulted in the consistent upregulation of key survival, proliferation, and differentiation pathways. In contrast, these pathways were downregulated when the cells were cultured on tissue culture plastic, resulting in a short-lived culture. Furthermore, we utilized a custom-designed dynamic chamber within a millifluidic system to expose cells cultured on the hydrogels to physiological shear stress. Similarly, we applied bulk RNA sequencing at early and late time points. Our findings demonstrated that shear stress induced the differentiation of intestinal stem cells without the need for exogenous differentiation factors and increased the metabolic activity of these cells. Shear stress also induced the upregulation of pathways involved in the innate immune response, as well as an early downregulation of mechanotransduction pathways that had been previously upregulated on hydrogels. Interestingly, we observed an upregulation of stem cell markers at early exposure to shear stress, suggesting a potential role for short-term shear stress in stimulating cell stemness. In summary, our study represents a significant step forward in developing advanced in vitro models that yield more transferable results, thereby enhancing our understanding of intestinal barrier function and facilitating their application in disease research and drug discovery.
