High throughput microfluidic intracellular delivery platform

描述（由申请人提供）：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有效的无病毒方案来生产大量的多能干细胞。蛋白质的直接递送消除了诱变插入的风险，并且能够更准确地控制高度敏感的重编程过程。然而，细胞穿透肽方法目前提供的重编程效率太低，无法用于临床。提出的微流体输送技术已经证明了它能够高效地将蛋白质输送到人类成纤维细胞，并且它消除了化学修饰或使用外源化合物的需要。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当前技术无法满足的不同细胞类型。目前的原型机能够提供10,000-20,000个单元/秒的高吞吐率，每次运行可产生高达100万个单元。这种单细胞水平控制和宏观规模吞吐量的结合使该设备相对于现有的输送方法处于独特的位置。目的1：研究蛋白质传递和细胞恢复的机制，以更好地了解该系统并指导其优化。初步结果表明，大分子的传递是通过孔隙形成机制进行的。为了验证这一假设，模型荧光大分子和蛋白质将用于控制内吞作用和直接成像膜孔的实验中。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、均匀性和细胞活力。未来设备世代的设计

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.