High throughput microfluidic intracellular delivery platform

高通量微流控细胞内递送平台

• 批准号: 8706186
• 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
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8706186
• 关键词: Alzheimer' 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; 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; public health relevance; research study; stem cell differentiation; success; sugar; trafficking; transcription factor; two-photon

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的iPSC。蛋白质的直接递送避免了诱变插入的风险，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来说太低。所提出的微流体递送技术已经证明了其以高效率将蛋白质递送至人成纤维细胞的能力，并且其消除了对化学修饰或使用外源性化合物的需要。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目前技术所缺乏的不同细胞类型。目前的原型能够提供10，000 - 20，000个细胞/s的高通量速率，每次运行可产生高达100万个递送细胞。这种单细胞水平控制和大规模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位置。目的1：研究蛋白质递送和细胞回收的机制，以更好地理解该系统并指导其优化。初步结果表明，大分子传递发生通过孔形成机制。为了验证这一假设，模型荧光大分子和蛋白质将被用于设计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。未来几代器械的设计 将由所获得的机械理解指导，并将致力于结合功能，如电穿孔耦合。还将开发该系统的精简版，供合作实验室使用。目的2：将优化细胞内递送方法以用于将成纤维细胞基于蛋白质重编程为iPSC。该装置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，并鉴定单个因子在整个过程中的作用。此外，该装置将用于研究通过组合其他大分子，例如microRNA和mRNA，基于蛋白质的重编程。除了重编程应用之外，这种能够以最小的细胞死亡递送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，在未来，它可以在疾病机制的研究，大分子治疗候选人的鉴定，干细胞分化和报告细胞系的诊断应用中实施。