Core C: Advanced Co-Culture Engineering and Single Cell Statistics of Gut Immunology

项目摘要-核心C ACCESS-GI(肠道免疫学高级共培养工程和单细胞统计)核心 C将为斯坦福NAMSED调查人员提供一系列尖端方法(项目1、2和 飞行员)分析肠/胃上皮细胞及其与免疫细胞的相互作用 没有感染。我们将与有机B芯联合开展开发工作，以期 整合粘膜免疫细胞的类器官的产生。具体地说，在目标1中，我们将高度利用 多路传输、单细胞分析(CyTOF质量细胞术)和成像(多路传输离子束成像-MIBI) 由肖恩·本德尔在斯坦福大学合作开发的模式，为项目提供细胞分析分析 穿过NAMSED中心(项目1、2)。这些分析将是独一无二的好处，允许 人体肠道类器官结构、免疫细胞整合及其影响的同时表征 病原体对细胞行为的影响。此外，核心C将促进访问其他分析模式 可通过斯坦福大学的人类免疫监测核心(HIMC)获得，包括63-plex Luminex 人类细胞因子和趋化因子的定量测量，非Luminex靶标的标准ELISA，以及 用于单细胞定量聚合酶链式反应分析的荧光生物标记。核心将利用肖恩·本德尔及其团队的专业知识， 以及HIMC，用于分析由这些平台生成的数据，并在适当的情况下用于多平台数据 整合。在《目标2》中，萨拉·海尔肖恩和她的团队将为NAMSED调查人员提供新的分析方法 (项目1、2)，使用微流体设备，允许研究细胞/细胞和细胞/病原体之间的相互作用 高度受控的微环境。在《目标3》中，伊丽莎白·梅林和她的团队与 类有机物核心，将为项目1提供M细胞样类有机物，并开发表达类有机物的II类 产生具有生理水平的M细胞的有机体。她的团队还将提供同种免疫细胞 对NAMSED研究人员进行实验，以解决感染背景下的免疫反应 肠道类器官(项目1)以及选定的抗原提呈细胞与 未受感染和受感染的有机物。后者将成为一项关键工作的基础，与 有机体核心，以开发更紧密地复制生理的人类肠道有机体 肠道感染时病原体所处的微环境。

PROJECT SUMMARY – CORE C The ACCESS-GI (Advanced Co-culture Engineering and Single cell Statistics of Gut Immunology) Core C will provide a range of cutting edge approaches to Stanford NAMSED investigators (Projects 1, 2 and pilots) to analyze intestinal/gastric epithelial cells and their interactions with immune cells both with and without infection. In association with the Organoid Core B, we will carry out development work towards the generation of organoids that integrate mucosal immune cells. Specifically, in Aim 1, we will leverage highly multiplexed, single cells analysis (CyTOF mass cytometry) and imaging (multiplexed ion beam imaging – MIBI) modalities collaboratively developed at Stanford by Sean Bendall to provide cell analysis assays for projects across the NAMSED center (Projects 1, 2). These assays will be uniquely beneficial, allowing the simultaneous characterization of human gut organoid structure, the integration of immune cells, and the effects of pathogens on cellular behavior. Further to this, Core C will facilitate access to other analysis modalities available through Stanford's human immune monitoring core (HIMC), including 63-plex Luminex for quantitative measurement of human cytokines and chemokines, standard ELISA for non-Luminex targets, and Fluidigm Biomark for single cell qPCR analysis. The core will leverage the expertise of Sean Bendall, his team, and the HIMC for analysis of data generated by these platforms and, where appropriate, for multi-platform data integration. In Aim 2, Sarah Heilshorn and her team will provide novel assays to NAMSED investigators (Projects 1, 2), using microfluidic devices that allow investigation of cell/cell and cell/pathogen interactions in highly controlled micro-environments. In Aim 3, Elizabeth Mellins and her team, in collaboration with the Organoid Core, will provide M-cell-like organoids to Project 1, and class II expressing organoids and develop production of organoids with physiologic levels of M cells. Her team will also provide homogenous immune cell populations to NAMSED investigators for experiments addressing immune reactivity in the context of infected intestinal organoids (Project 1) as well as study interactions of selected antigen presenting cells with uninfected and infected organoids. The latter will form the foundation of a key effort, in collaboration with the Organoid Core, to develop human intestinal organoids that more closely replicate the physiologic microenvironment encountered by pathogens during enteric infection.