ENTEROID CORE – Project Summary The Enteroid Core (EC) is a multi-service resource that provides Program Project and Core Investigators and their laboratories the tools and advice needed to establish and utilize human intestinal stem cell-derived enteroids/colonoids for understanding the pathogenesis of enteric bacterial infections. The Enteroid Core provides established enteroid and colonoid cultures from JHU biobank, expertise and training in the use of human small intestinal and colonic enteroid monolayers, growth factor-conditioned media required for propagation, maintenance, and differentiation of enteroid cultures, including M cells, as well as assistance in troubleshooting growth, differentiation, and use of enteroids for study of host-pathogen interactions in each project. The Enteroid Core works in close collaboration with the Immunology Core to develop novel co-culture models of human enteroids/colonoids with primary human innate immune cells. Our collaborative work during the current award period resulted in the first human immune-enteroid co-culture model that included monocyte-derived macrophages. In addition, the Enteroid Core developed techniques and protocols to assist Project laboratories achieve their proposed Aims including lentiviral transduction of human enteroids/colonoids for stable knockdown (via shRNA) of target genes (e.g. MUC2 for Project 1 (EAEC); MRP5 for Project 3 (ETEC)) and differentiation of specialized intestinal epithelial cell types (e.g. M cells for Project 2 (Shigella)). This renewal application seeks to continue these collaborative efforts to provide Projects 2 (Shigella) and 3 (ETEC) and the Immunology Core with media necessary for propagating human enteroids/colonoids and growing them as monolayers as well as media to normally differentiate enteroids/colonoids as well as to express M cells (Project 2 (Shigella)). Project 1 (EAEC) investigators have been trained to make conditioned media independently and will receive quality control support from the EC. The EC will continue to help with troubleshooting problems with culturing enteroids as well as use of enteroids for each project. In collaboration with the Immunology Core, the EC will continue developing innate immune cell-enteroid co-culture models to include human monocyte-derived dendritic cells and intraepithelial lymphocytes. In addition to new co-culture models, the EC and Immunology Core will increase the complexity of co-cultures through increased direct physical interaction of enteroid monolayers with immune cells normally present in the lamina propria, using commercially available 3D cell culture scaffolds, perfusion systems, and recently developed anaerobic chamber used for human enteroid monolayers.