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/乳酪对循环系统的影响；3)血液过滤；4)加强对 通过免疫细胞的产生和激活而导致感染和癌症；以及5)产生和激活 调节免疫细胞，防止自身免疫/自体炎症疾病。给出这些关键字 生理功能，我们认为重要的是开发一个三维(3D)组织图 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- 3)。作为我们的主要优势之一，我们拥有10多年的采购经验 移植质量的研究器官，包括淋巴系统的器官。作为这项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。我们的标准操作程序(SOP)，包括发布的和基于网络的， 包括正常性评估、质量控制分析和系统解剖解剖/储存。我们 还参与国家QA/QC计划，以评估组织处理/银行程序。我们的方法 为了朝着3D组织图的共同目标开发化验管道，最初将涉及获得 完整组织的宏观图像和处理组织形态，利用我们在磁共振中的优势 成像(MRI)。拟议的光学显微镜管道将使用福尔马林处理显微解剖特征 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。这些样品将使用从纳米到毫米的分辨率进行研究 随机光学重建显微镜(STORM)、共聚焦、多光子和薄片荧光 显微镜(LSFM)，所有管道共享一种通用文件格式，以简化3D重建。基座 论淋巴器官在免疫细胞产生和运输中的独特作用 对血液和每个淋巴器官的细胞进行细胞分类(FAC)将提供患者- 特异性免疫细胞库，并在随后的单细胞RNA-SEQ分析中服务。共同注册 生物分子到它们的同源细胞，我们将采用成像质谱仪(IMC)，多重单分子 荧光原位杂交(SmFISH)和多重误差稳健FISH(MerFish)定位细胞 蛋白质和信使核糖核酸的表达，最终在每个3D组织图谱上。我们在器官采购方面的经验， 国家强磁场实验室(NHML)先进磁共振成像的可用性 和光谱(AMRIS)设备在UF，多光子/共聚焦显微镜，LSFM，最先进的FACS，10X 基因组学、铬控制器和高通量RNA-Seq文库测序 以及苏黎世大学世界领先的IMC和3D数据分析设施，最好是 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.