Electrosonic Actuation Microarray: High-Throughput Tool for Transfection of Diffi

电声驱动微阵列：用于 Diffi 转染的高通量工具

• 批准号: 8058167
• 负责人: John Mark Meacham
• 金额: $ 48.63万
• 依托单位: OPENCELL TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-15 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8058167
• 关键词: Address; Area; Basic Science; Biological; Biological Products; Biological Sciences; Cell Line; Cell Size; Cell Survival; Cell membrane; Cell physiology; Cells; Cellular biology; Collaborations; Development; Devices; Drops; Drug Delivery Systems; Drug Design; Electrodes; Electronics; Electroporation; Gene Transfer; Gene Transfer Techniques; Genes; Genetic; Glioblastoma; Goals; Guidelines; Image; Institutes; Investigation; Laboratories; Life; Lipofectamine; Malignant Neoplasms; Mechanics; Mediating; Methods; Microfabrication; Microscopic; Modification; Nucleic Acids; Patients; Performance; Pharmaceutical Preparations; Phase; Plasmids; Play; Population; Production; Protocols documentation; RNA Interference; Reagent; Research; Research Personnel; Role; Sampling; Shapes; Small Business Innovation Research Grant; Solutions; Specific qualifier value; Staging; Stem Cell Research; Stem cells; System; Techniques; Technology; Transfection; Treatment Efficacy; Universities; Work; base; cancer stem cell; cell type; clinical application; cost effective; established cell line; experience; gene therapy; improved; lipofection; neoplastic cell; operation; prototype; scale up; stem cell therapy; tool; uptake

DESCRIPTION (provided by applicant): The ability to introduce drugs, genes, nucleic acids, and/or imaging agents into living cells is critical to drug design and delivery, as well as many cell biology and genetic modification protocols; however, currently available physical and reagent-based techniques are inadequate for applications requiring transfection of difficult-to-transfect cells (e.g., primary and stem cells). For this reason, transfection of nucleic acids into cells has become a significant challenge in the development of RNAi therapies and stem cell clinical applications. The technology that is the subject of this project proposal has demonstrated the potential to significantly impact these areas by enabling investigations of difficult-to-transfect cells, which are not currently feasible. The proposed work addresses this challenge through development of a microfabricated technology that enables treatment of arbitrarily sized cell populations on a cell-by-cell basis. STEAM (Single-sample Treatment via Electrosonic Actuation Microarray) ejects biological cells through microscopic nozzles with incorporated electroporation electrodes, thereby opening pores by concurrent mechanical and electrical disruption of the cell membrane. The parallel microarray format is scalable to accommodate discrete sample volumes from ~100 nl to tens of ml; however, in continuous-flow mode, the same device can rapidly process cells at 1 to 100 million cells per second. The critical advantage of STEAM is the uniformity of treatment experienced by each cell in a population, which is the key to achieving high transfection efficiency. During the SBIR Phase I project a prototype STEAM device demonstrated successful treatment of laboratory established cell lines. Device operating parameters were optimized using a small fluorescent molecule to evaluate uptake and cell viability. In addition, STEAM achieved trasfection efficiencies of 80% (mechanical poration) and >90% (mechanical + electroporation) for GFP-encoding plasmid into HEK293 cells with cell viability >70%, which is on par with lipofection and the best commercially available electroporation systems. The primary objectives of this SBIR Phase II project are further device refinement and optimization towards development of a production prototype and direct comparison with available physical and reagent-based techniques for transfection of difficult cells. To achieve these objectives, (1) a stand-alone STEAM system with disposable cartridge-based sample handling and on-board electronic control of both mechanical and electroporation parameters will be developed, and (2) a direct comparison of STEAM, commercial electroporation systems, lipofectamine-mediated transfection, and lentiviral gene transfer in difficult cells (including primary cancer stem cells from glioblastoma multiforme) will be performed. PUBLIC HEALTH RELEVANCE: Development of the STEAM (Single-sample Treatment via Electrosonic Actuation Microarray) platform will address the current need for alternative gene transfer solutions for use with difficult-to-transfect cells (e.g., primary and stem cells). The lack of successful commercial gene transfer solutions limits research in the life sciences and biomedical fields. STEAM addresses the need for effective, high-throughput, and scalable techniques to achieve transfection of difficult cells.

描述（由申请人提供）：将药物，基因，核酸和/或成像剂引入活细胞的能力对于药物设计和递送以及许多细胞生物学和遗传修饰方案至关重要；但是，目前可用的基于物理和试剂的技术不足以用于转染难以转染细胞的应用（例如，初级细胞和干细胞）。因此，将核酸转染到细胞中已成为RNAi疗法和干细胞临床应用的开发中的重大挑战。该项目提案的主题的技术证明了通过对难以转化的细胞进行调查，这可能会对这些领域产生重大影响，而这些细胞目前不可行。拟议的工作通过开发微观生育技术来应对这一挑战，该技术可以通过细胞细胞的基础来治疗任意大小的细胞群体。 Steam（通过电体驱动微阵列进行的单样本处理）通过与掺入的电穿孔电极的微观喷嘴驱逐生物细胞，从而通过同时的机械和电气破坏来打开孔。平行的微阵列格式可扩展以适应从〜100 nl到数十毫升的离散样品体积；但是，在连续流模式下，同一设备可以以每秒1至1亿个单元的形式迅速处理细胞。蒸汽的关键优势是每个细胞在人群中经历的治疗均匀性，这是实现高转染效率的关键。在SBIR阶段投影期间，一个原型蒸汽装置显示了实验室已建立的细胞系的成功处理。使用小的荧光分子优化了设备工作参数，以评估摄取和细胞活力。此外，蒸汽达到了80％（机械孔隙）和> 90％（机械 +电穿孔）的TrasFection效率，用于将GFP编码质粒质化质粒到HEK293细胞中，其细胞活力> 70％，这与唇彩相吻合，与唇彩相吻合，并且是最佳的市售电力系统。该SBIR II期项目的主要目标是进一步的改进和优化生产原型的开发，并与可用的物理和基于试剂的可用技术直接比较，用于转染困难细胞。为了实现这些目标，（1）将开发具有基于盒式墨盒的样品处理和机械和电穿孔参数的板载电子控制的独立蒸汽系统，并且（2）直接比较蒸汽，商业电穿孔系统，Lipofofectamine介导的转型转染，以及在困难的细胞中（包括原始的癌症）群（包括原始癌症）的元素（包括元素）的元素转移。 公共卫生相关性：蒸汽的开发（通过电驱动微阵列通过单样本处理）平台将解决当前对与难以传递细胞（例如，初级和干细胞）一起使用的替代基因转移溶液的需求。缺乏成功的商业基因转移解决方案限制了生命科学和生物医学领域的研究。 Steam解决了有效，高通量和可扩展技术以实现困难细胞转染的需求。