Project Summary The mucus layer is the first line of defense against infiltration of microorganisms, digestive enzymes and acids, digested food particles, microbial by-products, and food-associated toxins. This layer coats the interior surface of the GI tract, lubricates luminal contents and acts as a physical barrier to bacteria and other antigenic substances present in the lumen. The moist, nutrient-rich mucus layer adjacent to the epithelial barrier of the GI tract is also essential in the maintenance of intestinal homeostasis and contains a thriving biofilm including beneficial and pathogenic microbial populations. However, mucus has proven difficult to study, owing to it compositional and functional complexity and a substantial lack of either high-quality purified samples or cell- based culture systems containing mucus. This project will establish a gastrointestinal (GI) model for both harvesting physiologically relevant mucus and an epithelial culture system that possess a native mucus layer. Building on Altis’ capabilities to generate models with greater physiological relevance using donor-derived cells, this program will establish key protocols and workflows to ensure a reliable and reproducible mucus layer to additionally replicate in vivo GI morphology and function. In Aim 1, we will finalize in vitro protocols for mucus production in epithelia from both the small and large intestine (jejunum and transverse colon), which are well-described to production functionally distinct mucus. These data will establish key quality metrics enabling Altis to explore R&D studies with our industry and academic clients in diverse aspects of early stage of drug development and the development of inflammatory disease and microbiome research. Based on prior discussions with our pharmaceutical development customers, we view the commercial potential for this model as strong, especially given the unique capabilities of Altis’ current platform.