Enteroid Core

ENTEROID CORE – 项目摘要 Enteroid Core (EC) 是一种多服务资源，为项目和核心研究人员提供 他们的实验室提供建立和利用人类肠道干细胞所需的工具和建议 肠类/结肠类用于了解肠道细菌感染的发病机制。肠类核心 提供来自 JHU 生物库的已建立的肠样和结肠样培养物、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条件培养基， 肠样培养物（包括 M 细胞）的维护和分化，以及故障排除方面的帮助 每个项目中肠类的生长、分化和使用用于研究宿主-病原体相互作用。肠样 Core 与免疫学核心密切合作，开发新型人类共培养模型 具有初级人类先天免疫细胞的肠样/类结肠。我们在当前获奖期间的合作工作 时期产生了第一个人类免疫肠样共培养模型，其中包括单核细胞来源的 巨噬细胞。此外，Enteroid Core 还开发了技术和协议来协助项目实验室 实现他们提出的目标，包括慢病毒转导人肠类/类结肠以实现稳定敲低 （通过 shRNA）靶基因（例如项目 1 (EAEC) 的 MUC2；项目 3 (ETEC) 的 MRP5）和分化 特殊的肠上皮细胞类型（例如项目 2 的 M 细胞（志贺氏菌））。此续签申请旨在 继续这些合作努力，为项目 2（志贺氏菌）和 3（ETEC）以及免疫学核心提供 传播人类肠类/类结肠并将其培养为单层所需的培养基以及培养基 正常区分肠类/类结肠以及表达 M 细胞（项目 2（志贺氏菌））。项目1（东亚经济共同体） 研究人员接受过独立制作条件培养基的培训，并将获得质量控制支持 来自欧盟委员会。欧盟委员会将继续帮助解决肠类培养和使用问题 每个项目的 Enteroid 数。 EC 将与免疫学核心合作，继续开发先天性 免疫细胞-肠样共培养模型，包括人单核细胞衍生的树突状细胞和上皮内细胞 淋巴细胞。除了新的共培养模型之外，EC 和免疫学核心还将增加 通常通过增加肠样单层与免疫细胞的直接物理相互作用进行共培养 存在于固有层中，使用市售的 3D 细胞培养支架、灌注系统和 最近开发的用于人肠样单层的厌氧室。

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.