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遗传修饰方案是至关重要的;然而，目前可用的基于物理和试剂的技术不足以用于需要转染难以转染的细胞（例如，原代细胞和干细胞）。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开发中的重大挑战。作为本项目提案主题的技术已证明有可能对这些领域产生重大影响，因为它可以对目前尚不可行的难以检测的细胞进行研究。拟议的工作通过开发一种微制造技术来解决这一挑战，该技术能够在逐个细胞的基础上处理任意大小的细胞群。STEAM（单样品处理通过超声致动微阵列）通过具有结合的电穿孔电极的显微喷嘴来激发生物细胞，从而通过细胞膜的同时机械和电破坏来打开孔。平行微阵列格式是可扩展的，以适应离散的样品体积从约100 nl到几十ml;然而，在连续流模式下，相同的设备可以快速处理细胞在1至100百万细胞每秒。STEAM的关键优势是群体中每个细胞所经历的处理的均匀性，这是实现高转染效率的关键。在SBIR第一阶段项目期间，原型STEAM设备证明了对实验室建立的细胞系的成功治疗。使用小荧光分子优化装置操作参数以评估摄取和细胞活力。此外，STEAM实现了GFP编码质粒到HEK 293细胞中的80%（机械穿孔）和>90%（机械+电穿孔）的转染效率，细胞活力> 70%，这与脂质转染和最好的市售电穿孔系统相当。该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，并与现有的物理和基于试剂的技术进行直接比较，用于转染困难的细胞。为了实现这些目标，（1）将开发一种独立的STEAM系统，该系统具有基于一次性支架的样品处理和机械和电穿孔参数的机载电子控制，以及（2）将在困难细胞（包括来自多形性胶质母细胞瘤的原代癌症干细胞）中进行STEAM、商业电穿孔系统、脂质体介导的转染和慢病毒基因转移的直接比较。 公共卫生相关性：STEAM（通过超声致动微阵列进行单样本处理）平台的开发将解决目前对用于难以转染细胞（例如，原代细胞和干细胞）。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解决了对有效、高通量和可扩展技术的需求，以实现困难细胞的转染。

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.