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-感染性疾病，如艾滋病毒和自身免疫性疾病，如多重 硬化症或糖尿病介导的病理在特定的组织，但我们大多数的知识， 这是研究血液中循环细胞的结果。这些细胞一直是一个方便的代理，因为血液是 最易接近的隔室和回收的细胞数量可能很大。越来越多的证据 然而，这表明，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， 了解这些差异对于开发新药、疫苗和诊断方法至关重要， 改善患者护理。组织中存在的细胞之间的显著异质性需要 用单细胞分辨率表征这些样品，但临床常规使用的现有技术 免疫学家（流式细胞术，ELISpot）通常需要过量的细胞来进行分析。他们的 效率低下阻碍了追求科学的能力，理解人类疾病的生物学， 因为活组织检查产生的细胞很少。本研究将优化、验证和部署 独特的基于细胞的平台，以解决这一未满足的需求，表征单细胞从临床 只需最少的操作即可进行活检。该项目是一个合作之间：爱和劳芬伯格 实验室（麻省理工学院）在应用微加工技术解决单细胞异质性和 开发用于分析此类数据的计算工具; Kwon和步行者实验室（Ragon研究所） 在艾滋病毒和疫苗的临床免疫学的专业知识; Mesirov和黄实验室（布罗德研究所）， 开发数据分析软件工具和复杂数据可视化方法的专业知识;以及 Roederer实验室（NIH VRC）拥有单细胞技术的专业知识，用于表征免疫表型， 基因表达。这个跨学科的团队跨越了工程学、计算生物学、临床 免疫学和数据可视化将1）改善最终用户使用细胞研究细胞的体验 通过增加每个用户可以通过工程处理的样本数量， 自动化，通过简化提取、集成、分析和查看数据的流程，以及通过 增强回收稀有细胞的能力; 2）验证基于模块化纳米孔的操作以确定 存在的细胞类型（细胞术）和它们分泌的蛋白质（微刻）以及 回收相对于当前标准表达的细胞和基因;以及3）将平台部署为核心设施 在Ragon研究所，首次将该技术广泛提供给最终用户社区 （科学家和医生研究临床样本中的表型多样性）。该项目的成功将 产生每个用户在细胞中分析的样品数量的定量增加，定义用于 执行与传统技术相当的测定，并建立一个公共访问平台， 最终用户，在人类细胞疾病和治疗的所有领域开辟新的生物学。