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

基本信息

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
项目类别：
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

项目摘要

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，目前可用的物理和基于试剂的技术不足以满足需要转染难以转染的细胞（例如原代细胞和干细胞）的应用。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。作为该项目提案主题的技术已证明有可能通过对难以转染的细胞进行研究而对这些领域产生重大影响，而这在目前尚不可行。拟议的工作通过开发微加工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。 STEAM（通过电声驱动微阵列进行单样品处理）通过带有电穿孔电极的微型喷嘴喷射生物细胞，从而通过同时机械和电破坏细胞膜来打开孔。并行微阵列格式可扩展，以容纳从约 100 nl 到数十 ml 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 至 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的一致性，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 设备展示了对实验室建立的细胞系的成功处理。使用小荧光分子优化设备操作参数以评估摄取和细胞活力。此外，STEAM 将 GFP 编码质粒转染至 HEK293 细胞中的效率为 80%（机械穿孔）和 >90%（机械 + 电穿孔），细胞活力 >70%，与脂转染和最佳商用电穿孔相当系统。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，并与用于转染困难细胞的现有物理和基于试剂的技术进行直接比较。为了实现这些目标，(1) 将开发一种独立的 STEAM 系统，该系统具有一次性基于盒的样品处理和机械和电穿孔参数的机载电子控制，以及 (2) 直接比较 STEAM、商业电穿孔系统、脂质体介导的转染以及困难细胞（包括来自多形性胶质母细胞瘤的原代癌症干细胞）的慢病毒基因转移。公共健康相关性：STEAM（通过电声驱动微阵列进行单样本处理）平台的开发将解决当前对用于难以转染细胞（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 满足了对有效、高通量和可扩展技术的需求，以实现困难细胞的转染。