High throughput microfluidic intracellular delivery platform

描述(申请人提供)：诱导多能干细胞(IPSCs)及其在组织工程和疾病建模中的应用具有改变当前医疗实践的巨大潜力。目前的研究主要集中在设计高效的无病毒协议来生产大量的ipscs。蛋白质的直接输送避免了突变插入的风险，并能够更准确地控制高度敏感的重新编程过程。然而，细胞穿透肽方法目前提供的重编程效率太低，无法临床使用。所提出的微流控技术已经证明了它能够高效地将蛋白质输送到人成纤维细胞，并且它不需要化学修饰或使用外源化合物。此外，初步结果表明，该技术可以发展成为一种通用的递送方法，能够将一系列大分子递送到不同类型的细胞，而目前的技术无法满足这一要求。目前的原型能够提供10,000-20,000个细胞/S的高吞吐量，并且每次运行可以产生高达100,000个细胞。这种单细胞水平控制和宏观吞吐量的组合使该设备相对于现有的输送方法处于独特的地位。目的1：研究蛋白质递送和细胞回收的机制，以更好地了解该系统并指导其优化。初步结果表明，大分子的释放是通过孔形成机制进行的。为了验证这一假设，将在旨在控制内吞作用和直接成像膜孔的实验中使用荧光大分子和蛋白质模型。结果将用于开发输送系统的预测模型，并进行优化研究，以提高输送效率、一致性和细胞活力。未来几代设备的设计 将以获得的机械理解为指导，并将致力于纳入诸如与电穿孔耦合的特征。还将开发该系统的简化版本，以供合作实验室使用。目的2：细胞内给药方法将被优化为基于蛋白质的成纤维细胞向ipSCs的重编程。该设备强大的输送能力将使人们能够研究重新编程过程本身的生物学方面，例如产生最大重新编程效率的转录因子的最佳组合，以及确定单个因素在整个过程中的作用。此外，该设备将被用于通过将其他大分子，如microRNA和信使核糖核酸，与基于蛋白质的重新编程相结合，来研究潜在的改进。除了对应用程序进行重新编程外，这种高通量微流控设备平台能够以最小的细胞死亡提供一系列大分子，可以实现对细胞功能的前所未有的控制。因此，在未来，它可以应用于疾病机制的研究、大分子治疗候选者的鉴定、干细胞分化以及报告细胞系的诊断应用。

DESCRIPTION (provided by applicant): Induced pluripotent stem cells (iPSCs) and their application to tissue engineering and disease modeling have great potential to change current medical practices. Current research is largely focused on devising efficient virus-free protocols to produce large numbers of iPSCs. Direct delivery of proteins obviates the risk of mutagenic insertion and enables more accurate control of the highly sensitive reprogramming process. However, cell-penetrating peptide methods currently provide reprogramming efficiencies that are too low for clinical use. The microfluidic delivery technology proposed has demonstrated its ability to deliver proteins at high efficiencies to human fibroblasts and it eliminates the need fo chemical modification or the use of exogenous compounds. Moreover, preliminary results indicate that the technique can be developed into a universal delivery method capable of delivering a range of macromolecules to different cell types underserved by current technologies. The current prototype is capable of delivering high throughput rates of 10,000-20,000 cells/s and can yield up to 1 million delivered cells per run. This combination of single-cell level control and macro-scale throughput places this device in a unique position relative to existing delivery methods. Aim 1: The mechanism of protein delivery and cell recovery will be investigated to better understand the system and direct its optimization. Preliminary results indicate macromolecular delivery occurs through a pore formation mechanism. To validate this hypothesis, model fluorescent macromolecules and proteins will be used in experiments designed to control against endocytosis and image membrane pores directly. Results will be used to develop a predictive model of the delivery system and conduct optimization studies to improve delivery efficiency, uniformity and cell viability. The design of future device generations will be guided by the gained mechanistic understanding and will aim to incorporate features such as coupling with electroporation. A streamlined version of the system will also be developed for use in collaborating laboratories. Aim 2: The intracellular delivery method will be optimized for protein-based reprogramming of fibroblasts to iPSCs. The robust delivery capabilities of the device will allow studies on the biological aspects of the reprogramming process itself, such as the optimal combination of transcription factors to produce maximum reprogramming efficiency and identification of the role of individual factor in the overall process Moreover, the device will be used to investigate potential improvements by combining other macromolecules, such as microRNA and mRNA, with protein-based reprogramming. In addition to reprogramming applications, such a high throughput microfluidic device platform capable of delivering a range of macromolecules with minimal cell death could enable unprecedented control over cellular function. Hence, in the future, it can be implemented in studies of disease mechanisms, identification of macromolecular therapeutic candidates, stem cell differentiation, and diagnostic applications with reporter cell lines.