Nanowell-based single-cell technology for characterizing clinical samples ex vivo

基于纳米孔的单细胞技术，用于离体表征临床样品

• 批准号: 8517895
• 负责人: John Christopher Love
• 金额: $ 40.63万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-09 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8517895
• 关键词: Address; Adoption; Affect; Antigens; Area; Autoimmune Diseases; Autoimmunity; Automation; Biological Assay; Biology; Biopsy; Blood; Body Fluids; Cell Count; Cell physiology; Cells; Childhood; Clinical; Clinical Immunology; Cloning; Collaborations; Communicable Diseases; Communities; Complex; Computational Biology; Computer software; Core Facility; Cytometry; Data; Data Analyses; Deposition; Development; Diabetes Mellitus; Diagnostic; Disease; Engineering; Feedback; Flow Cytometry; Gene Expression; Genes; HIV; HIV vaccine; Heterogeneity; Human; Human Biology; Ice; Image; Imagery; Immune; Immunologist; Immunophenotyping; Individual; Infection; Institutes; Intervention; Knowledge; Link; Love; Malignant Neoplasms; Measures; Mediating; Methods; Modification; Monitor; Multiple Sclerosis; Nature; Pathology; Pathway interactions; Patient Care; Patients; Performance; Pharmaceutical Preparations; Phenotype; Physicians; Preparation; Process; Proteins; Protocols documentation; Proxy; Recovery; Relative (related person); Research; Research Personnel; Resolution; Sampling; Science; Scientist; Software Tools; Solutions; Sorting - Cell Movement; Source; Speed; Sum; System; T-Lymphocyte; Technology; Testing; Time; Tissues; Training; Translating; Translations; United States National Institutes of Health; Vaccines; Validation; Walkers; base; cell type; computerized tools; cytokine; drug development; experience; flexibility; human disease; improved; innovation; interest; mucosal site; nanolitre scale; new technology; operation; response; single cell analysis; software development; success; tool

Many immune-mediated diseases-infectious diseases like HIV and autoimmune diseases like multiple sclerosis or diabetes-mediate pathology in specific tissues, yet most of our knowledge about them has resulted from studying cells circulating in blood. These cells have been a convenient proxy because blood is the most accessible compartment and the number of cells recovered can be large. Increasing evidence suggests, however, that the biology of diseases in affected tissues can vary substantially from that in the blood, and understanding these differences may be critical to develop new drugs, vaccines, and diagnostics to improve patient care. The significant heterogeneities among cells resident in tissues necessitates characterizing such samples with single-cell resolution, but existing technologies routinely employed by clinical immunologists (flow cytometry, ELISpot) typically require an excess of cells to use for analysis. Their inefficiencies have hindered the ability to pursue science understanding the human biology of diseases and treatments in tissues because biopsies yield very few cells. This research will optimize, validate, and deploy a unique nanowell-based platform to address this unmet need for characterizing single cells from clinical biopsies with minimal manipulations. The project is a collaboration amongst: the Love and Lauffenburger Labs (MIT) with expertise in applying microfabricated technologies to resolve single-cell heterogeneities and in developing computational tools for analyzing such data; the Kwon and Walker Labs (Ragon Institute) with expertise on the clinical immunology of HIV and vaccines; the Mesirov and Wong Labs (Broad Institute) with expertise in developing software tools for data analysis and means of visualizing complex data; and the Roederer Lab (NIH VRC) with expertise in single-cell technologies for characterizing immunophenotypes and gene expression. Together, this interdisciplinary team spanning engineering, computational biology, clinical immunology, and data visualization will 1) improve the experience of end-users using nanowells to study cells from biopsies by increasing the number of samples each user can process through engineering and automation, by streamlining the process for extracting, integrating, analyzing and viewing data, and by enhancing the ability to recover rare cells; 2) validate modular nanowell-based operations for determining the types of cells present (cytometry) and their secreted proteins (microengraving) and the efficiencies of recovering cells and genes expressed relative to current standards; and 3) deploy the platform as a core facility at the Ragon Institute, making the technology broadly available for the first time to the community of end-users (scientists and physicians studying phenotypic diversity in clinical samples). The success of the project will yield a quantitative increase in the number of samples analyzed in nanowells per user, define protocols for executing assays comparable to conventional technologies, and establish a publicly-accessible platform for end-users, opening up new biology in all areas of human cellular disease and treatments.

许多免疫介导的疾病感染疾病，如艾滋病毒和自身免疫性疾病，如多种 在特定组织中的硬化症或糖尿病 - 中等病理学，但我们对它们的大多数了解 是由于研究细胞在血液中循环。这些细胞一直是方便的代理 最容易接近的隔室和回收的单元数量可能很大。越来越多的证据 但是，表明受影响组织中疾病的生物学可能与血液中的生物学有很大不同， 理解这些差异对于开发新药，疫苗和诊断可能至关重要 改善患者护理。居住在组织中的细胞之间的显着异质性需要 通过单细胞分辨率来表征此类样品，但是现有的技术通常通过临床使用 免疫学家（流式细胞仪，ELISPOT）通常需要过量的细胞进行分析。他们的 低效率阻碍了追求科学理解人类疾病生物学和的能力 由于活检产生的细胞很少，因此在组织中的处理。这项研究将优化，验证和部署 唯一基于纳米韦尔的平台，以解决这种未满足的对临床表征的单个单元的需求 活检最少的操作。该项目是一个合作：Love and Lauffenburger 实验室（麻省理工学院）在应用微生物技术方面具有专业知识来解决单细胞异质性和 开发用于分析此类数据的计算工具；权和沃克实验室（Ragon Institute）与 艾滋病毒和疫苗临床免疫学专业知识； Mesirov和Wong Labs（Broad Institute）与 开发用于数据分析和可视化复杂数据的手段的软件工具方面的专业知识；和 Roederer Lab（NIH VRC）具有单细胞技术专业知识，用于表征免疫表型和 基因表达。这支跨学科团队跨越工程，计算生物学，临床 免疫学和数据可视化将1）改善使用纳米维尔研究细胞的最终用户的经验 从活检中增加每个用户可以通过工程和 自动化，通过简化提取，集成，分析和查看数据的过程以及 增强恢复稀有细胞的能力； 2）验证基于模块化的纳米维尔操作以确定 存在的细胞类型（细胞仪）及其分泌的蛋白质（微果）及其效率 相对于当前标准表达的细胞和基因； 3）将平台部署为核心设施 在拉贡研究所 （研究临床样本中表型多样性的科学家和医生）。该项目的成功将 每位用户中分析的纳米线的样品数量的定量增加，定义协议 执行与传统技术相当的测定法，并为公开访问的平台建立 最终用户，在人类细胞疾病和治疗的所有领域开放新的生物学。