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

描述(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药物设计和输送以及许多细胞生物学和遗传修饰方案至关重要；然而，目前可用的基于物理和试剂的技术不足以应用于需要对难以转染的细胞(例如，原代细胞和干细胞)进行转染的应用。因此，将核酸导入细胞已成为发展RNAi疗法和干细胞临床应用的重大挑战。作为本项目提案主题的技术已经证明，通过对目前尚不可行的难以转染性细胞的研究，有可能对这些领域产生重大影响。拟议的工作通过开发一种微制造技术来解决这一挑战，该技术能够逐个细胞地处理任意大小的细胞群体。STEAM(通过电声驱动微阵列的单一样品处理)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气破坏来打开毛孔。并行微阵列格式可扩展，以适应从~100 nL到数十毫升的离散样品体积；然而，在连续流动模式下，同一设备可以以每秒1到1亿个细胞的速度快速处理细胞。STEAM的关键优势是群体中每个细胞所经历的处理都是一致的，这是实现高转染率的关键。在SBIR第一阶段项目期间，一个原型蒸汽装置展示了对实验室建立的细胞系的成功处理。使用一种小的荧光分子来评估摄取和细胞活力，以优化设备的操作参数。此外，STEAM还获得了80%(机械穿孔)和90%(机械+电穿孔)的GFP编码质粒对HEK293细胞的转染率，细胞存活率为70%，与脂质体和商业上最好的电穿孔系统相当。这个SBIR第二阶段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，并直接与现有的基于物理和试剂的技术进行比较，以导入困难的细胞。为了实现这些目标，(1)将开发一个独立的STEAM系统，该系统具有基于一次性墨盒的样品处理和机载电子控制的机械和电穿孔参数，以及(2)将STEAM、商用电穿孔系统、脂质体介导的转染法和在困难细胞(包括来自多形性胶质母细胞瘤的原代癌症干细胞)中的慢病毒基因转移进行直接比较。 与公共卫生相关：STEAM(通过电声驱动微阵列进行单一样本治疗)平台的开发将满足当前对用于难以转化的细胞(例如，原代细胞和干细胞)的替代基因转移解决方案的需求。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满足了对高效、高通量和可扩展技术的需求，以实现困难细胞的转基因。

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.