High throughput microfluidic intracellular delivery platform

高通量微流控细胞内递送平台

基本信息

批准号：
8839787
负责人：
DANIEL G ANDERSON
金额：
$ 51.09万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8839787
关键词：
Alzheimer&apos s Disease Behavior Biological Cell Death Cell Line Cell Survival Cell physiology Cells Chemicals Chromosomes Clinical Combined Modality Therapy Confocal Microscopy Coupling Cytoplasm Cytoplasmic Protein DNA DNA Integration Dependence Development Device Designs Devices Diabetes Mellitus Diagnostic Disease Disease model Electroporation Endocytosis Family Fibroblasts Future Generations Genome Goals Health Human Image In Vitro Individual Label Laboratories Medical Membrane Messenger RNA Methods MicroRNAs Microfluidic Microchips Microfluidics Modeling Modification Mutagens Nature Neurons Parkinson Disease Patients Peptides Plasmid Cloning Vector Plasmids Plastics Positioning Attribute Process Production Proteins Protocols documentation RNA Recovery Relative (related person)Reporter Research Research Project Grants Risk Role Running Scanning Electron Microscopy Staging System Techniques Technology Testing Therapeutic Tissue Engineering Transfection Transmission Electron Microscopy Transplantation Viral Viral Vector Virus Work base cell type design disease mechanisms study dopaminergic neuron effective therapy human disease improved in vivo induced pluripotent stem cell macromolecule meetings novel predictive modeling prototype research study stem cell differentiation success sugar trafficking transcription factor two-photon

项目摘要

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.

描述（由申请人提供）：诱导多能干细胞（iPSC）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 iPSC。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对于临床使用而言太低。所提出的微流体递送技术已证明其能够高效地将蛋白质递送至人类成纤维细胞，并且无需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当前技术服务不足的不同细胞类型。目前的原型能够提供 10,000-20,000 个细胞/秒的高吞吐率，每次运行最多可产生 100 万个细胞。单细胞水平控制和宏观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。目标 1：研究蛋白质递送和细胞恢复的机制，以更好地了解系统并指导其优化。初步结果表明大分子递送是通过孔形成机制发生的。为了验证这一假设，模型荧光大分子和蛋白质将用于旨在直接控制内吞作用和图像膜孔的实验中。结果将用于开发递送系统的预测模型并进行优化研究，以提高递送效率、均匀性和细胞活力。未来几代设备的设计将以所获得的机械理解为指导，旨在整合诸如与电穿孔耦合等特征。还将开发该系统的简化版本以供合作实验室使用。目标 2：细胞内递送方法将针对基于蛋白质的成纤维细胞重编程为 iPSC 进行优化。该设备强大的递送能力将允许对重编程过程本身的生物学方面进行研究，例如转录因子的最佳组合以产生最大的重编程效率以及识别单个因子在整个过程中的作用此外，该设备将可用于通过将其他大分子（例如 microRNA 和 mRNA）与基于蛋白质的重编程相结合来研究潜在的改进。除了重编程应用之外，这种能够以最小的细胞死亡提供一系列大分子的高通量微流体装置平台可以实现对细胞功能的前所未有的控制。因此，未来它可以应用于疾病机制的研究、大分子治疗候选物的鉴定、干细胞分化以及报告细胞系的诊断应用。