A 3D Tissue Map of the Human Lymphatic System

淋巴系统有五个主要作用：1）清除多余的体液； 2）吸收和运输 脂肪酸/乳糜进入循环系统； 3）血液过滤； 4）建立主要防御措施 通过免疫细胞的产生和激活引起的感染和癌症； 5) 的生成和激活 调节性免疫细胞可预防自身免疫/自身炎症性疾病。鉴于这些关键 生理功能，我们认为为三个主要功能开发三维（3D）组织图很重要 人体免疫系统的淋巴器官：脾、胸腺和淋巴结（器官特定项目 1- 分别为 3）。作为我们的主要优势之一，我们在采购方面拥有超过 10 年的经验 用于研究的移植质量器官，包括淋巴系统的器官。作为这项 24/7/365 全天候工作的一部分， 我们与美国器官采购组织 (OPO) 建立了良好的关系，有推荐 从过去十年的 56/58 来看。我们的标准操作程序 (SOP)，包括已发布的和基于网络的， 包括正常性评估、质量控制测定和系统解剖解剖/存储。我们 还参与国家 QA/QC 计划以评估组织处理/存储程序。我们的方法 开发检测管道以实现 3D 组织图谱的共同目标将首先涉及获取 利用我们在磁共振方面的优势，获得完整组织的宏观图像并解决组织形态问题 成像（MRI）。拟议的光学显微镜管道将使用福尔马林解决显微解剖特征 固定石蜡包埋 (FFPE) 和最佳切割温度 (OCT) 复合包埋切片 作为组织光学清除和扩张。这些样本将使用纳米到毫米的分辨率进行研究 随机光学重建显微镜 (STORM)、共焦、多光子和光片荧光 显微镜 (LSFM)，所有管道共享通用文件格式，以简化 3D 重建。基于 关于淋巴器官在免疫细胞的产生和运输中的独特作用，荧光激活 对血液和每个淋巴器官中的细胞进行细胞分选（FACS）将提供患者的比较- 特异性免疫细胞库并用于随后的单细胞 RNA-seq 分析。共同注册 生物分子与其同源细胞，我们将采用成像质量细胞术（IMC），多重单分子 荧光原位杂交 (smFISH) 和多重抗误差 FISH (MERFISH) 来绘制细胞图谱 最终在每个 3D 组织图谱上进行蛋白质和 mRNA 表达。我们在器官获取方面的经验 国家强磁场实验室 (NHML) 先进磁共振成像的可用性 UF 的光谱学 (AMRIS) 设施、多光子/共焦显微镜、LSFM、最先进的 FACS、10X GENOMICS Chromium 控制器和大学用于 RNA-Seq 的高通量 cDNA 文库测序 佛罗里达州以及苏黎世大学世界领先的 IMC 和 3D 数据分析设施，理想情况下 使我们能够提供最高质量的淋巴器官 3D 人类生物分子图谱 (HuBMAP)。

The lymphatic system serves five primary roles: 1) removal of excess body fluids; 2) absorption and transport of fatty acids/chyle to the circulatory system; 3) blood filtration; 4) mounting the primary defense against infections and cancer through immune cell production and activation; and 5) generation and activation of regulatory immune cells that protect against autoimmune/autoinflammatory disease. Given these key physiological functions, we deem it important to develop a three-dimensional (3D) tissue map for three major lymphoid organs of the human immune system: spleen, thymus and lymph nodes (Organ Specific Projects 1- 3, respectively). As one of our primary strengths, we have over 10 years of experience in the procurement of transplant-quality organs for research, including those of the lymphatic system. As part of this 24/7/365 effort, we have well-established relationships with the U.S. Organ Procurement Organizations (OPO), having referrals from 56/58 over the last decade. Our standard operating procedures (SOPs), both published and web-based, include assessment of normality, quality control assays, and systematic anatomical dissection/storage. We also participate in National QA/QC programs to evaluate tissue processing/banking procedures. Our approach to developing assay pipelines towards the common goal of a 3D tissue map will initially involve acquiring a macro image of the intact tissue and addressing tissue morphology, using our strengths in magnetic resonance imaging (MRI). The proposed optical microscopy pipeline will address microanatomical features using formalin fixed paraffin embedded (FFPE) and optimal cutting temperature (OCT) compound embedded sections as well as tissue optical clearing and expansion. These specimens will be studied from nm to mm resolution using stochastic optical reconstruction microscopy (STORM), confocal, multiphoton and light sheet fluorescence microscopies (LSFM), with all pipelines sharing a common file format for simplified 3D reconstruction. Based on the unique role for lymphatic organs in production and trafficking of immune cells, fluorescence activated cell sorting (FACS) of cells from blood and each lymphatic organ will provide a comparison of the patient- specific immune cell repertoire and serve in subsequent single cell RNA-seq analyses. To co-register biomolecules to their cognate cells, we will employ imaging mass cytometry (IMC), multiplex single molecule fluorescence in situ hybridization (smFISH) and multiplexed error-robust FISH (MERFISH) to map cellular protein and mRNA expression, ultimately on each 3D tissue atlas. Our experience in organ procurement, the availability of the National High Magnetic Field Laboratory (NHML) Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) Facility at UF, multiphoton/confocal microscopy, LSFM, state of the art FACS, 10X GENOMICS Chromium controller, and high throughput cDNA library sequencing for RNA-Seq at the University of Florida, as well as world leading IMC and 3D data analysis facility at the University of Zurich, ideally positions us to provide the highest quality 3D Human BioMolecular Atlas (HuBMAP) of the lymphatic organs.