Microfluidic Device for Efficient Gene-Transfection of Adult Stem Cells

描述（由申请人提供）：TheraCell公司提议开发一种用于快速分离和电穿孔单个间充质成体干细胞（MSCs）的新设备，作为其CellLabTM干细胞产品套件的一部分，用于脊柱融合和其他基于干细胞的骨骼再生治疗。该干细胞处理装置将由三个关键模块组成，其中电穿孔模块是其中之一，这也是本提案的动机。（另外两个关键模块，同样采用单细胞操作技术，正在由TheraCell在不同的项目下开发。）成体间充质干细胞是一种多能细胞，可以沿成骨、成脂、成肌和其他途径分化，是细胞和基于细胞的组织工程的一种很有前途的工具。我们建议开发一种技术来选择单个MSCs，然后在很短的时间内对多达107个MSCs进行单细胞基因转染（通过电穿孔）。与目前使用的整体电穿孔方法相比，新的单细胞电穿孔方法将产生一种效率更高、效果更好的基因转染方法。在第一阶段，我们将设计和制造一个基于新型MEMS（微机电系统）技术的微流控微电穿孔芯片原型。该芯片将包含几个测试结构，包括设计的单通道版本，以及多通道版本。设计将以这样的方式开发，使其易于扩展到最终产品所需的数百个渠道。原型微电穿孔芯片将使用荧光标记的微球来评估基本的流体流动特性，然后将干细胞与荧光蛋白电穿孔，使用荧光显微镜进行分析。一旦确定了最佳的电穿孔参数，该芯片将用经BMP-2蛋白电穿孔的干细胞进一步评估，并分析这些细胞的成骨潜能。在第二阶段，我们将在第一阶段的基础上进一步优化微电穿孔芯片的设计。通道的数量将从几个通道扩展到数十或数百个通道，以容纳目标应用程序所需的大量单元。我们将建立一个完整的预生产原型单细胞微电穿孔装置。在第二阶段结束时，我们计划展示一个完整的原型，可以作为一个独立的研究应用产品快速商业化，也可以集成到一个完整的干细胞处理设备中，其中包括其他细胞操作模块，用于未来的临床应用。公共卫生相关性：我们建议开发一种新的设备，用于电穿孔和高效的个体间充质成体干细胞（MSCs）基因转染，用于脊柱融合和其他骨骼再生治疗。成人间充质干细胞是一种多能细胞，是细胞和基于细胞的组织工程的一种很有前途的工具。与目前使用的整体电穿孔方法相比，新的单细胞电穿孔方法将产生一种效率更高、效果更好的基因转染方法，从而改善干细胞治疗的效果。

DESCRIPTION (provided by applicant): TheraCell Inc. proposes to develop a new device for the rapid isolation and electroporation of individual mesenchymal adult stem cells (MSCs), as part of its CellLabTM stem cell product suite, for application in spine fusion and other stem cell-based skeletal regeneration therapies. This stem cell processing device will consist of three critical modules, of which the electroporation module is one, and which is the motivation for this proposal. (The other two critical modules, also employing single cell manipulation techniques, are being developed by TheraCell under separate projects). Adult MSCs are pluripotent cells that can differentiate along osteogenic, adipogenic, myogenic, and other pathways, and represent a promising tool for cell and cell-based tissue engineering. We propose to develop the technology to select the individual MSCs, and then to perform single-cell gene-transfection (via electroporation) for up to ~107 individual MSCs in a very short period of time. The new single-cell electroporation method will result in a significantly more efficient and effective gene-transfection method compared with the bulk electroporation methods currently in use. In Phase I, we will design and fabricate a prototype microfluidic micro-electroporation chip, based on new MEMS (micro-electro-mechanical systems) technology. The chip will contain several test structures, including single-channel versions of the design, as well as a multi-channel version. The design will be developed in such as way as to lend itself easily for scale up to hundreds of channels required for the final product. The prototype micro-electroporation chips will be evaluated for basic fluid flow characteristics using fluorescently labeled microspheres, and then with stem cells that will be electroporated with a fluorescence protein for analysis using a fluorescence microscope. Once the optimum electroporation parameters are determined, the chip will be further evaluated with stem cells that have been electroporated with the BMP-2 protein, and those cells will be analyzed for osteogenic potential. In Phase II we will further optimize the design of the micro-electroporation chip, based on the Phase I results. The number of channels will be scaled up from a few to tens or hundreds of channels, to accommodate the large numbers of cells required for the targeted application. We will build a complete pre-production prototype single-cell micro-electroporation device. At the end of Phase II, we plan to demonstrate a completed prototype that can be rapidly commercialized as a stand-alone product for research applications, and also integrated into a complete stem cell processing device, which includes the other cell manipulation modules, for future clinical applications. PUBLIC HEALTH RELEVANCE: We propose to develop a new device for the electroporation and efficient gene-transfection of individual mesenchymal adult stem cells (MSCs) for application in spine fusion and other skeletal regeneration therapies. Adult MSCs are pluripotent cells that represent a promising tool for cell and cell-based tissue engineering. The new single-cell electroporation method will result in a significantly more efficient and effective gene-transfection method compared with the bulk electroporation methods currently in use, for improved stem cell therapy outcomes.