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：
9061704
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 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9061704
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 Positioning Attribute Process Production Proteins Protocols documentation RNA Recovery 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。当前的研究主要集中在设计有效的无病毒方案上生产大量IPSC。蛋白质的直接递送消除了诱变插入的风险，并可以更准确地控制高度敏感的重编程过程。但是，当前细胞穿透肽方法提供的重编程效率太低，无法用于临床使用。提出的微流体递送技术证明了其以高效率为人类成纤维细胞递送蛋白质的能力，并消除了化学修饰或使用外源化合物的需求。此外，初步结果表明，该技术可以发展为一种通用输送方法，能够将一系列大分子传递到当前技术不足的不同细胞类型。当前的原型能够输送10,000-20,000个单元/s的高吞吐量率，并且每次运行最多可产生100万个单元。单细胞级控制和宏观尺度吞吐量的这种组合将该设备相对于现有交付方法的独特位置。目标1：将研究蛋白质递送和细胞回收的机制，以更好地了解系统并指导其优化。初步结果表明大分子通过孔形成机制发生。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。结果将用于开发传递系统的预测模型，并进行优化研究以提高递送效率，均匀性和细胞活力。未来设备的设计将以获得的机械理解为指导，并将旨在结合诸如耦合与电穿孔之类的特征。该系统的简化版本还将用于协作实验室。 AIM 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。该设备的强大输送能力将允许研究重编程过程本身的生物学方面，例如转录因子的最佳组合，以产生最大的重编程效率，并鉴定单个因素在整体过程中的作用，此外，该设备将用于研究潜在的改进，通过将MacroMoRNA和其他基于Protein Reprogrampommpommprampommpommprammprammpommpommprammpommpommpommpommpommpommpommpommpommprammogmongommpommmpommpommpommpommpommpommongommpommongommpommongommengommengommengomements进行研究。除了重编程应用外，如此高的吞吐量微流体设备平台能够传递一系列具有最小细胞死亡的大分子，可以实现对细胞功能的前所未有的控制。因此，将来它可以在疾病机制的研究，大分子治疗候选物，干细胞分化以及记者细胞系的诊断应用中实施。