Droplet-based Analysis of Secretion and Sorting (DASS) for characterization and selection of individual immune cells

描述(由申请人提供)：分析单细胞分泌产物的方法将对免疫系统的研究具有特别的价值。成功的抗体反应需要激活编码有用抗体的单个细胞。同样，成功的细胞毒性T淋巴细胞(CTL)反应需要激活编码适当T细胞受体的单个细胞。研究单细胞的分泌一直很困难，因为细胞及其分泌的产物必须保存在同一隔室中。多孔板不适合，因为单个电池的输出太稀薄，不能在相对较大的井体积(&gt；2 ul)中进行测量。我们已经开发出油包水液滴作为研究单细胞分泌分子的强大工具：在这些小体积(40pl)液滴中的单个细胞可以在一小时内分泌出可测量的分子浓度。这些液滴可以以每秒约1000个液滴的速度形成、分析和选择，从而实现高速分析。我们将开发一个检测单细胞分泌肿瘤坏死因子α和颗粒酶B的平台，这两个分子是免疫系统的关键功能和调节分子。与基于良好基础的系统相比，我们的油包水滴平台的一个主要优势是可以快速识别和收集感兴趣的活细胞。然后这些细胞可以用于进一步的研究，或者它们可以扩大到更高的细胞数量，用于过继细胞治疗研究。这些检测通常对研究免疫系统的激活和调节是有用的。此外，这些检测将使分离出具有杀死感染或癌细胞能力的单个T细胞成为可能。这将为确定有用的T细胞受体的蛋白质序列提供强有力的手段。这些序列可用于嵌合T细胞疗法，或用于产生抗体样分子，以识别其癌细胞靶标，其中一些可能被证明是有用的癌细胞标记物。在第二个应用中，液滴选择的CTL将被释放和扩大，由此产生的扩大的群体将极大地丰富具有自体抗癌细胞活性的细胞。这一过程或其特点，对于开发更有效的主动采用细胞治疗方案可能是重要的。在第二阶段的研究中，我们将通过将原型微流控检测和仪器商业化，以及通过销售质量受控分析试剂来提供这些工具。

DESCRIPTION (provided by applicant): Methods to analyze the products secreted from single cells will be of particular value for studies of the immune system. A successful antibody response requires activation of the individual cells that encode useful antibodies. Similarly, a successful Cytotoxic-T-Lymphocyte (CTL) response requires activation of the individual cells that encode appropriate T-cell receptors. It has been difficult to study secretion from single cell because the cell and its secreted products must be maintained in the same compartment. Multi-well plates are not suitable because the output of a single cell is too dilute for measurement in the relatively large well volumes (>2 uL). We have developed water-in-oil droplets as a powerful tool to study molecules secreted from single cells: A single cell in one of these small volume (40 pL) droplets can secrete molecules to measurable concentrations in < one hour. These droplets can be formed, analyzed and selected at rates ~1000 droplets per second, allowing for high-speed analysis. We will develop a platform to measure single-cell secretion of Tumor Necrosis Factor Alpha (TNF-� Interferon Gamma (IFN-?), and Granzyme B, key functional and regulatory molecules of the immune system. A major advantage of our water-in-oil droplet platform, relative to well-based systems, is that living cells of interest can be rapidly identifie and collected. These cells can then be used for further study, or they can be expanded to high cell numbers for use in adoptive cell therapy research. These assays will be generally useful to study immune system activation and regulation. In addition, the assays will enable the isolation of individual T-cells with the ability to kill infected or cancerous cells. This will provide a powrful means to determine the protein sequence of useful T- cell receptors. These sequences can be used for Chimeric T-Cell therapy, or used to create antibody-like molecules to enable identification of their cancer-cell targets, some of which might prove to be useful cancer-cell markers. In a second application, droplet-selected CTLs will be released and expanded, and the resulting expanded population will be greatly enriched for cells with autologous anti-cancer-cell activity. This process, or features of it, might be important in developing more effective active adoptive cell therapy protocols. In Phase II research, we will make these tools available through commercializing prototype microfluidic detection and instruments, and by selling quality-controlled assay reagents.