Cell-based sensors for measuring impact of microsystems on cell physiology

描述（由申请人提供）：用于操作、测量、培养和分离细胞的微系统的发展稳步增长。对于这些系统的设计和采用来说，重要的是了解系统本身如何改变细胞状态。这类测量可能难以执行，原因要么是用户缺乏复杂性，要么是难以在小型设备中使用有限数量的细胞进行常规测定。因此，测量微系统对细胞生理影响的分析通常局限于一般特征，如活力、形态和生长。这一建议的重点是开发一套细胞系，这将为压力对细胞生理学的影响提供更细致的观点。在我们实验室创建热休克细胞传感器的先前工作的基础上，我们建议创建一组细胞系，报告与基于细胞的微流体装置相关的应激反应途径的转录激活。具体来说，我们相信报道DNA损伤、剪切应力和热休克的细胞系将为微系统的设计者和用户提供一套有价值的试剂。我们建议创造这些细胞系，使它们在光谱上不同，从而可以立即混合和分析。我们将制定一套使用这些试剂和解释结果的标准。因此，我们建议不仅要生成细胞系，还要生成必要的协议，成像和数据解释算法，以及供其他人遵循的案例研究。我们的具体目标是：(1)在热休克、DNA损伤和剪切应力诱导启动子的控制下，用可诱导荧光蛋白创建细胞系。每个细胞系都有三种细胞类型，代表了微系统中可能使用的细胞的多样性；(2)确定细胞系对输入应力（如剪切、热和光）的响应；(3)对这些试剂的使用进行个案研究，并向社区分发。

DESCRIPTION (provided by applicant): There is a steady increase in the development of microsystems for manipulating, measuring, culturing, and separating cells. Important to the design and adoption of these systems is knowledge of how the systems themselves alter cell state. Such measurements can be difficult to perform, due to either lack of sophistication on the part of the user, or difficulty performing conventional assays with limited numbers of cells in small devices. As a result, assays to measure the impact of microsystems on cell physiology are typically limited to general characteristics such as viability, morphology, and growth. This proposal focuses on developing a suite of cell lines that will provide a more nuanced view of the effects of stress on cell physiology. Building on prior work in our lab creating a heat shock cell sensor, we propose to create a set of cell lines that report on transcriptional activation of stress response pathways relevant to cell-based microfluidic devices. Specifically, we believe that lines reporting on DNA damage, shear stress, and heat shock will provide a valuable set of reagents for designers & users of microsystems. We propose to create these cell lines so that they are spectrally distinct and thus can be mixed and assayed at once. We will formulate a set of standards for applying these reagents and interpreting results from them. As such, we propose to not only generate the cell lines, but the requisite protocols, imaging and data interpretation algorithms, as well as case studies for others to follow. Our specific aims are to (1) create cell lines with inducible fluorescent proteins under the control heat shock-, DNA damage-, and shear stress-inducible promoters. Each cell line will be created in three cell types, representing the diversity of cells one may use in a microsystem; (2) Determine the responses of the cell lines to input stresses, such as shear, heat, and light; (3) Undertake a case study of the use of these reagents and distribute them to the community. PUBLIC HEALTH RELEVANCE: Microsystems that can analyze small numbers of cells could have wide use for point-of- care diagnostics and biotechnology. Currently, there are no standardized metrics by which such microsystems can be designed or used to ensure that they are not harmful to the cells they are trying to analyze. We are proposing to develop cell "sensors" that would glow colors if they are subjected to stresses from microsystems.