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

描述（由申请人提供）：将药物、基因、核酸和/或显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基因修饰方案至关重要；然而，目前可用的物理和试剂技术对于需要转染难以转染的细胞（例如，原代细胞和干细胞）的应用是不够的。因此，将核酸转染到细胞中已成为RNAi疗法发展和干细胞临床应用的重大挑战。该项目提案的主题技术已经证明了通过对难以转染的细胞进行研究来显著影响这些领域的潜力，这在目前是不可行的。提出的工作通过开发一种微加工技术来解决这一挑战，该技术可以在细胞的基础上处理任意大小的细胞群。蒸汽（Single-sample Treatment via Electrosonic Actuation Microarray）通过带有电穿孔电极的微型喷嘴喷射生物细胞，从而通过同时对细胞膜进行机械和电破坏来打开毛孔。并行微阵列格式是可扩展的，以适应从~100毫升到几十毫升的离散样品体积；然而，在连续流模式下，同样的设备可以以每秒1到1亿个细胞的速度快速处理细胞。STEAM的关键优势在于群体中每个细胞处理的均匀性，这是实现高转染效率的关键。在SBIR一期项目中，一个原型STEAM设备成功地处理了实验室建立的细胞系。使用小荧光分子对设备操作参数进行优化，以评估摄取和细胞活力。此外，STEAM将gfp编码质粒转染到HEK293细胞中，转染效率为80%（机械穿孔）和>90%（机械+电穿孔），细胞存活率为>70%，与脂肪转染和最好的市购电穿孔系统相当。SBIR二期项目的主要目标是进一步改进和优化设备，以开发生产原型，并与现有的物理和试剂技术进行直接比较，用于转染困难的细胞。为了实现这些目标，(1)将开发一个独立的蒸汽系统，该系统具有一次性样品处理和机械和电穿孔参数的机载电子控制，(2)将直接比较蒸汽、商业电穿孔系统、脂质体介导的转染和在困难细胞（包括多形性胶质母细胞瘤的原发性癌症干细胞）中的慢病毒基因转移。

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.