Ultrahigh throughput cellular manipulation via massively parallel microinjection

描述(由申请人提供)：通过引入外源材料对细胞的操纵是广泛应用的关键推动因素，包括药物发现、转基因和基于细胞的治疗。然而，在许多应用中，进展仍然受到当前操纵技术的限制。这项提议的长期目标是通过开发用于基于显微注射的操作的先进仪器来解决这一问题。尽管微注射是细胞操作的“黄金标准”，但由于依赖熟练的操作员和系列化的注射方法，它的劳动密集型和低吞吐量(~3个细胞/分钟)在很大程度上已被降级为利基应用。这项建议的目标是开发超高通量(UHT)微注射仪器，通过自动化、大规模并行化和单片集成来解决这些限制。该仪器将基于微电子机械系统(MEMS)设备核心，由大量平行的细胞捕获点阵列和单片集成注射器组成，这将使每分钟同时捕获和注射数千个细胞，而最不需要人工或机器人参与。在初步研究证明这一仪器的第一个功能要素，即超高温细胞捕获的可行性的指导下，拟议的努力寻求通过追求逐步引入额外功能的阶段性研究计划来推进下一步。具体目标是：1)开发用于UHT细胞捕获和渗透的原型；2)开发用于UHT显微注射的原型。将为MEMS器件核心开发新的微制造工艺，并将开发添加高速单元处理功能的计算机控制的外部子系统。然后将使用活细胞测试(即捕获、注射、释放和转染率以及活性)来验证仪器的功能。这项拟议的研究具有创新性，因为它不仅通过自动化，而且通过将所有功能大规模并行化和单片集成到单个MEMS设备中，首次尝试解决微注射的局限性。这项研究具有重要意义，因为它可以：a)充分简化微注射，使更多的研究人员能够使用它；b)大大增强当前的应用，在这些应用中，吞吐量通常是一个限制因素，例如转基因；以及c)对于进展受到与当前操作技术相关的安全性或有效性问题限制的应用，例如基于基因修改的体外细胞疗法，可以起到根本的促进作用。 公共卫生相关性(由申请者提供)：拟议的努力寻求开发用于细胞操纵的创新工具，具有在许多学科中开辟比以前更大规模的生物学调查的新途径的潜力。它还可以实现治疗遗传病的新技术和设计其他疾病的细胞疗法。因此，拟议的努力与NIH促进对生物系统的了解、改善疾病控制和增强健康的使命的相关性是显而易见的。

DESCRIPTION (provided by applicant): The manipulation of cells via introduction of exogenous materials serves as a critical enabler for a broad spectrum of applications, including drug discovery, transgenics, and cell-based therapeutics. However, in many applications progress is nevertheless constrained by the limitations of current manipulation techniques. The long-term goal of this proposal is to address this issue through development of advanced instrumentation for microinjection-based manipulation. Although microinjection represents the "gold standard" for cellular manipulation, it has been largely relegated to niche applications by its labor-intensiveness and low throughput (~3 cells/min), which result from reliance upon skilled operators and serialized injection methodologies. The objective of this proposal is to develop ultrahigh throughput (UHT) microinjection instrumentation that addresses these limitations through automation, massive parallelization, and monolithic integration. This instrumentation will be based upon a microelectromechanical systems (MEMS) device core, composed of a massively parallel array of cell Capture Sites with monolithically integrated Injectors, which will enable simultaneous capture and injection of many thousands of cells/min with minimal need for human or robotic involvement. Guided by preliminary studies demonstrating feasibility of the first functional element of this instrumentation, namely UHT cell capture, the proposed effort seeks to take the next steps forward through pursuit of a staged research plan that gradually introduces additional functionalities. The Specific Aims are: 1) Develop prototype for UHT cell capture and permeabilization; and 2) Develop prototype for UHT microinjection. Novel microfabrication processes will be developed for the MEMS device cores and computer-controlled external subsystems will be developed that add high-speed cell handling functionality. Instrument functionality will then be validated using live cell testing (i.e. capture, injection, release, and transfection efficiency, as well as viability). The proposed research is innovative because it represents the first attempt to address microinjection's limitations through not only automation, but also massive parallelization and monolithic integration of all functionality into a single MEMS device. This research is significant because it may: a) simplify microinjection sufficiently to make it accessible to a broader range of researchers; b) considerably enhance current applications where throughput is often a limiting factor, e.g. transgenics; and c) serve as a fundamental enabler for applications where progress is constrained by safety or efficacy concerns associated with current manipulation techniques, e.g. ex vivo cell therapies based on genetic modification. PUBLIC HEALTH RELEVANCE (provided by applicant): The proposed effort seeks to develop innovative instrumentation for cellular manipulation that holds potential for opening new avenues of biological investigation at larger scales than previously possible across many disciplines. It may also enable realization of novel techniques for curing genetic diseases and engineering cellular therapies for other diseases. As such, the relevance of the proposed effort to NIH's mission to advance understanding of biological systems, improve control of disease, and enhance health is apparent.