Enteroid Core

企业核心--项目摘要 肠道核心(EC)是一个多服务资源，为计划项目和核心调查人员提供 他们的实验室需要建立和利用人类肠道干细胞所需的工具和建议 肠样/结肠样体有助于了解肠道细菌感染的发病机制。肠状核 提供来自JHU生物库的已建立的肠样和结肠样培养，人类使用方面的专业知识和培训 小肠和结肠肠样单层，繁殖所需的生长因子调节的培养基， 包括M细胞在内的肠样培养物的维持和分化，以及协助故障排除 在每个项目中用于研究宿主-病原体相互作用的肠样生长、分化和使用。肠样体 CORE与免疫学CORE密切合作，开发新的人类共培养模型 肠样/集落样体与原代人类先天免疫细胞。我们在本届大奖期间的协作工作 Period导致了第一个包括单核细胞来源的人类免疫-肠样共培养模型 巨噬细胞。此外，肠道核心制定了协助项目实验室的技术和方案。 实现他们提出的目标，包括慢病毒转导人类肠样/结肠样物质以稳定击倒 (通过shRNA)目标基因(例如，项目1的MUC2(EAEC)；项目3的MRP5(ETEC))和分化 专门化肠上皮细胞类型(例如，项目2的M细胞(志贺氏菌))。此续期申请旨在 继续这些合作努力，为项目2(志贺氏菌)和项目3(ETEC)和免疫学核心提供 繁殖人类肠样/结肠样细胞并将其作为单层生长所必需的介质和介质 正常分化肠样/结肠样细胞以及表达M细胞(项目2(志贺氏菌))。项目1(EAEC) 调查人员已经接受了独立制作条件媒体的培训，并将得到质量控制支持 欧盟委员会的。欧共体将继续帮助解决与培养肠样和使用有关的问题 每个项目的肠样体的数量。在与免疫学核心的合作下，欧共体将继续开发先天 免疫细胞-肠样共培养模型包括人单核细胞来源的树突状细胞和上皮内细胞 淋巴细胞。除了新的共培养模式，EC和免疫学核心将增加 通过增加肠样单层与免疫细胞正常的直接物理相互作用进行共培养 存在于固有层，使用商业上可获得的3D细胞培养支架、灌流系统和 新近开发的用于人体肠道单层的厌氧室。

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.