Enteroid Core

项目简介-项目简介 肠状核心（EC）是一种多服务资源，提供计划项目和核心研究者， 他们的实验室提供了建立和利用人类肠道干细胞所需的工具和建议， 肠样/结肠样用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物， 小肠和结肠类肠单层，繁殖所需的生长因子条件培养基， 包括M细胞在内的肠样培养物的维持和分化，以及故障排除方面的协助 在每个项目中，肠状体的生长、分化和用于宿主-病原体相互作用的研究。肠状体 Core与Immunology Core密切合作，开发新的人类共培养模型。 肠样/结肠样与原代人先天免疫细胞。我们在本届颁奖典礼上的合作 期间产生了第一个人免疫-肠共培养模型，包括单核细胞来源的 巨噬细胞此外，肠状核心开发了技术和协议，以协助项目实验室 实现了他们提出的目的，包括慢病毒转导人类肠/类结肠以稳定敲除 (via靶基因（例如项目1的MUC 2（EAEC）;项目3的MRP 5（ETEC））的shRNA）和靶基因的分化 专门的肠上皮细胞类型（例如项目2的M细胞（志贺氏菌））。本续期申请旨在 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 繁殖人肠样/结肠样细胞并使其生长为单层所必需的培养基以及培养基 正常分化肠样/结肠样以及表达M细胞（项目2（志贺氏菌））。项目1（EAEC） 研究人员已接受独立制作条件培养基的培训，并将获得质量控制支持 从EC。欧盟委员会将继续帮助解决培养肠上皮细胞以及使用 每一个项目的肠细胞。与免疫学核心合作，EC将继续开发先天性 免疫细胞-类肠共培养模型，包括人单核细胞衍生的树突细胞和上皮内 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的直接物理相互作用的共培养通常 存在于固有层中，使用可商购的3D细胞培养支架、灌注系统和 最近开发的用于人肠单层的厌氧室。

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.