Sample Sparing Chambers for Imaging of T cell Response and Function

 描述（由申请人提供）：基于显微镜的细胞功能测定提供了对人类免疫学和疾病的重要见解，这是通过其他形式的分析无法实现的。重要的是，当代显微镜平台非常适合从少量细胞中提供详细的空间和功能信息，当感兴趣的细胞频率或可用性有限时，这是一种战略属性。不幸的是，在样品制备和处理期间，特别是当工程化表面用于测试细胞功能时，样品中的细胞损失到死体积空间，严重限制了这种潜力的实现。为了解决这种低效率的细胞分析，我们寻求开发两个互补的显微镜载玻片室，允许使用复杂的模型系统进行细胞刺激，同时提高细胞加载的效率。第一个系统采用了改进的井设计，将细胞接种集中在一个小的基底区域上，在该基底区域上进行细胞成像和分析。第二个系统使用磁性捕获定位细胞接种到一个类似的指定区域，非常适合系统中，积极选择是用来准备细胞的目标是从灵活的，但低通量的方法用于制造初始原型过渡到更高的吞吐量系统。具体来说，我们将从基于光刻机的软光刻过渡到传统实验室器皿中常用的塑料注塑成型。作为这些小室的一个具体应用，我们将研究调节性T细胞对TCR/CD 28/LFA-1刺激的微观组织的反应能力。 该项目的成功完成将为免疫功能的细胞和分子研究提供一个新的检测平台。这些系统在整个研究界的战略部署将大大加快基础研究转化为对免疫疾病的更清晰理解。

 DESCRIPTION (provided by applicant): Microscopy-based assays of cell function provide important insights into human immunology and disease that are not attainable through other forms of analysis. Importantly, contemporary microscopy platforms are well suited for providing detailed spatial and functional information from small numbers of cells, a strategic property when the cells of interest are of limited frequency or availability. Unfortunately, loss of cells i a sample to dead volume spaces during sample preparation and handling, particularly when engineered surfaces are used to test cell function, severely limits the realization of this potentil. To address this low efficiency of cell analysis, we seek to develop two complementary microscopy slide chambers that allow the use of complex model systems for cell stimulation while increasing the efficiency of cell loading. The first system incorporates a modified well design that focuses cell seeding onto a small substrate area on which cell imaging and analysis takes place. The second system uses magnetic capture to localize cell seeding onto a similarly specified area, and is well suited for systems in which positive selection is used to prepare cells The goal of this project is to transition from the flexible but low-throughput methods used to fabricate the initial prototypes to higher-throughput systems. Specifically, we will transition fro elastomer-based soft lithography to injection molding of plastics commonly used in conventional labware. As a specific application of these chambers, we will examine the ability of regulatory T cells to respond to the microscale organization of TCR/CD28/LFA-1 stimulation. Successful completion of the proposed project will provide a new assay platform that is well suited for cellular and molecular studies of immune function. Strategic deployment of these systems throughout the research community will greatly accelerate the translation of basic research into clearer understanding of immune-based diseases.