Enteroid Core

肠样核心

• 批准号: 10427390
• 负责人: Nicholas Constantine Zachos
• 金额: $ 46.01万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10427390
• 关键词: Adopted; Adult; Anaerobic Bacteria; Apical; Award; Bacterial Infections; Biopsy; Cell Line; Cells; Childhood; Coculture Techniques; Collaborations; Colon; Confocal Microscopy; Culture Media; Dendritic Cells; Emergency Situation; Enteral; Enterobacteriaceae; Environment; Epithelial; Epithelial Cells; Escherichia coli; Excision; Exposure to; Fibrinogen; Freezing; Genes; Growth; Growth Factor; Human; Immune; Immune response; Immunology; Innate Immune Response; Intestines; Laboratories; Lamina Propria; Lymphocyte; M cell; Maintenance; Measurement; Methods; Modeling; Mucin-2 Staining Method; Operative Surgical Procedures; Oxygen; Pathogenesis; Pathologic; Patients; Perfusion; Protocols documentation; Publishing; Quality Control; Research Personnel; Research Project Grants; Research Support; Resources; Services; Shigella; Shigella Infections; Side; Small Intestines; Study models; Surface; System; Techniques; Time; Tissues; Training; Work; biobank; cell type; cytokine; enteroaggregative Escherichia coli; enterotoxigenic Escherichia coli; human model; intestinal epithelium; intraepithelial; knock-down; macrophage; monocyte; monolayer; novel; pathogen; pediatric patients; programs; response; scaffold; small hairpin RNA; stem cells; three dimensional cell culture; tool

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.

Enteroid Core - 项目摘要 Enteroid Core（EC）是一种多服务资源，可提供程序项目和核心调查人员以及 他们的实验室建立和利用人类肠干细胞衍生所需的工具和建议 用于了解肠道细菌感染的发病机理的肠动物/结肠。 Enteroid Core 提供JHU生物库的既定肠和肠结肠培养物，专业知识和人类使用的培训 小肠道和结肠肠单层，生长因子条件的传播所需的培养基， 维护和分化的肠道培养物，包括M细胞，以及在故障排除方面的帮助 在每个项目中，用于研究宿主病原体相互作用的肠道疾病的生长，分化和使用。肠道 核心与免疫学核心密切合作，以开发人类的新型共培养模型 带有原发性人类免疫力的肠托动物/结肠素。我们在当前奖励期间的合作工作 周期导致了第一个人类免疫 - 肠道共培养模型，其中包括单核细胞来源 巨噬细胞。此外，Enteroid Core开发了协助项目实验室的技术和协议 实现他们提出的目标，包括人类肠to虫/菌肠的慢病毒转导稳定敲低 （通过shRNA）的靶基因（例如项目1的MUC2（EAEC）；项目3（ETEC）的MRP5）和分化的分化 专门的肠上皮细胞类型（例如，项目2（Shigella）的M细胞）。此续签申请寻求 继续提供这些合作努力，以提供项目2（Shigella）和3（ETEC）以及免疫学核心 传播人类肠动物/结肠素的媒体并将它们作为单层和媒体种植所必需的媒体 通常会区分肠动物/结石和表达M细胞（Project 2（Shigella））。项目1（EAEC） 调查人员已接受培训以独立制作条件媒体，并将获得质量控制支持 来自EC。 EC将继续有助于解决文化摄影的问题以及使用 每个项目的肠toid。与免疫学核心合作，EC将继续发展先天 免疫细胞 - 肠道共培养模型包括人类单核细胞衍生的树突状细胞和上皮细胞 淋巴细胞。除了新的共培养模型外，EC和免疫学核心还将增加 通过增加肠托体单层与免疫细胞的直接物理相互作用的共同培养通常 使用市售的3D细胞培养支架，灌注系统和 最近开发的厌氧腔用于人肠托动物单层。