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开发稳步增长。对于这些系统的设计和采用来说，重要的是系统本身如何改变细胞状态的知识。这样的测量可能难以执行，这是由于用户方面缺乏复杂性，或者难以在小装置中用有限数量的细胞执行常规测定。因此，测量微系统对细胞生理学影响的测定通常限于一般特征，如活力、形态和生长。这项提案的重点是开发一套细胞系，这将提供一个更微妙的观点，压力对细胞生理的影响。在我们实验室先前创建热休克细胞传感器的工作的基础上，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，线报告DNA损伤，剪切应力和热休克将提供一套有价值的试剂的设计者和用户的微系统。我们建议创建这些细胞系，使它们在光谱上是不同的，从而可以混合和测定一次。我们将制定一套标准来应用这些试剂并解释其结果。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，以及其他人可以遵循的案例研究。我们的具体目标是（1）创建具有在热休克、DNA损伤和剪切应力诱导型启动子控制下的诱导型荧光蛋白的细胞系。每个细胞系将在三种细胞类型中创建，代表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的响应，例如剪切，热和光;（3）进行这些试剂使用的案例研究并将其分发给社区。 公共卫生相关性：可以分析少量细胞的微系统可以广泛用于即时诊断和生物技术。目前，还没有标准化的指标，通过这些指标可以设计或使用这样的微系统来确保它们对它们试图分析的细胞无害。我们建议开发细胞“传感器”，如果它们受到来自微系统的压力，就会发出颜色。

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.