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

许多免疫介导的疾病--像艾滋病毒这样的传染病和像多发性硬化症这样的自身免疫性疾病 硬化症或糖尿病在特定组织中介导病理，然而我们对它们的大多数知识已经 是通过研究血液中循环的细胞而产生的。这些细胞一直是一个方便的替代品，因为血液是 最容易接近的隔间和回收的细胞数量可能很大。越来越多的证据 然而，这表明，受影响组织中的疾病生物学可能与血液中的疾病有很大不同， 了解这些差异对于开发新的药物、疫苗和诊断方法可能至关重要 改善患者护理。驻留在组织中的细胞之间的显著异质性使得 用单细胞分辨率表征这样的样本，但临床上常规使用的现有技术 免疫学家(流式细胞仪，ELISpot)通常需要过多的细胞来进行分析。他们的 效率低下阻碍了从事科学研究了解疾病的人类生物学和 组织中的治疗，因为活组织检查产生的细胞非常少。这项研究将优化、验证和部署 独特的基于纳米细胞的平台，以满足这一尚未满足的需求，以表征临床上的单个细胞 用最少的操作做活组织检查。该项目的合作伙伴：The Love and Lauffenburger 实验室(麻省理工学院)在应用微制造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；权和沃克实验室(Ragon Institute)与 在艾滋病毒和疫苗的临床免疫学方面的专业知识；梅西罗夫和王实验室(博德研究所) 在开发数据分析软件工具和将复杂数据可视化的方法方面的专业知识；以及 罗德勒实验室(NIH VRC)，在单细胞技术方面拥有专业知识，用于表征免疫表型和 基因表达。总而言之，这个跨学科的团队横跨工程学、计算生物学、临床 免疫学和数据可视化将1)改善终端用户使用纳米细胞研究细胞的体验 通过工程和技术增加每个用户可以处理的样本数量 自动化，通过简化提取、集成、分析和查看数据的流程，以及 增强回收稀有细胞的能力；2)验证基于模块化纳米细胞的操作，以确定 存在的细胞类型(细胞术)及其分泌的蛋白质(微雕)和效率 恢复相对于当前标准表达的细胞和基因；以及3)将该平台部署为核心设施 在拉贡研究所，将这项技术首次广泛提供给最终用户社区 (科学家和内科医生研究临床样本的表型多样性)。该项目的成功将 使每个用户分析的样本数量定量增加，定义以下协议 执行可与传统技术媲美的分析，并建立一个公共可访问的平台 最终用户，在人类细胞疾病和治疗的所有领域开辟了新的生物学。

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.