Ultrahigh throughput cellular manipulation via massively parallel microinjection

描述（由申请人提供）：通过引入外源性物质对细胞进行操作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。该提案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个问题。尽管显微注射代表了细胞操作的“黄金标准”，但由于其劳动密集型和低通量（~3个细胞/分钟），其在很大程度上被降级为利基应用，这是由于依赖于熟练的操作员和系列化的注射方法。本提案的目的是开发微通量（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。该仪器将基于微机电系统（MEMS）设备核心，由具有单片集成注射器的细胞捕获位点的大规模平行阵列组成，这将使数千个细胞/分钟的同时捕获和注射成为可能，而对人类或机器人的参与需求最小。在初步研究的指导下，证明了这种仪器的第一个功能元素，即UHT细胞捕获的可行性，拟议的努力旨在通过追求逐步引入其他功能的分阶段研究计划，采取下一步。具体目标是：1）开发UHT细胞捕获和透化的原型; 2）开发UHT显微注射的原型。将为MEMS器件核心开发新的微加工工艺，并开发计算机控制的外部子系统，以增加高速细胞处理功能。然后将使用活细胞测试（即捕获、注射、释放和转染效率以及活力）验证仪器功能。拟议的研究是创新的，因为它代表了第一次尝试，以解决微注射的限制，不仅通过自动化，而且还大规模并行化和单片集成的所有功能到一个单一的MEMS设备。这项研究是重要的，因为它可以：a）充分简化显微注射，使其可用于更广泛的研究人员; B）显著提高目前的应用，其中通量通常是一个限制因素，例如转基因;以及c）作为进展受到与当前操纵技术相关的安全性或有效性问题限制的应用的基本使能器，例如基于遗传修饰的离体细胞疗法。 公共卫生相关性（由申请人提供）：拟议的努力旨在开发用于细胞操作的创新仪器，这些仪器有可能在比以前可能跨许多学科的更大规模上开辟新的生物学研究途径。它还可以实现治疗遗传疾病和工程细胞疗法治疗其他疾病的新技术。因此，NIH的使命，以促进对生物系统的理解，改善疾病控制，并提高健康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.