Microfluidic Device for Efficient Gene-Transfection of Adult Stem Cells

用于成体干细胞高效基因转染的微流体装置

• 批准号: 7743966
• 负责人: Carolyn Rossington Tull
• 金额: $ 18.22万
• 依托单位: THERACELL, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-25 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7743966
• 关键词: Adipose tissue; Adult; Area; Biological Preservation; Bone Marrow; Bone Regeneration; Cell Count; Cell Survival; Cell physiology; Cells; Characteristics; Collection; Development; Devices; Electroporation; Fluorescence; Future; Genes; Glass; Individual; Injection of therapeutic agent; Label; Lead; Liquid substance; Marketing; Measures; Mechanics; Medical center; Mesenchymal; Methods; Microfluidic Microchips; Microfluidics; Microspheres; Monitor; Motivation; Organ; Pathway interactions; Phase; Polymers; Population; Power Sources; Preparation; Principal Investigator; Process; Product R; Production; Property; Protein Analysis; Proteins; Pump; RNA; Research; Sampling; Series; Site; Source; Spinal Fusion; Stem cells; Structure; Surgeon; Suspension substance; Suspensions; Syringes; System; Techniques; Technology; Testing; Time; Tissue Engineering; Tissue Sample; Tissues; Transfection; Vertebral column; Work; adult stem cell; base; bone morphogenetic protein 2; cell preparation; clinical application; design; fluid flow; fluorescence microscope; improved; osteogenic; programs; prototype; public health relevance; research and development; scaffold; scale up; skeletal regeneration; stem cell therapy; therapy outcome; tissue processing; tissue regeneration; tool; transgene expression; voltage

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.

描述（由申请人提供）：TheraCell Inc.建议开发一种新的设备，用于快速分离和电穿孔单个间充质成体干细胞（MSC），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疗法。该干细胞处理装置将由三个关键模块组成，电穿孔模块是其中之一，并且这是该提议的动机。(The另外两个关键模块也采用单细胞操作技术，由TheraCell在单独的项目下开发）。成体间充质干细胞是一种多能性细胞，可以沿着成骨、成脂、成肌和其他途径分化，并代表了细胞和基于细胞的组织工程的有前途的工具。我们建议开发技术来选择个体MSC，然后在很短的时间内对多达107个个体MSC进行单细胞基因转染（通过电穿孔）。新的单细胞电穿孔方法将导致一个显着更有效和更有效的基因转染方法相比，目前使用的批量电穿孔方法。在第一阶段，我们将设计和制造一个原型的微流控微电穿孔芯片，基于新的MEMS（微机电系统）技术。该芯片将包含几个测试结构，包括单通道版本的设计，以及多通道版本。该设计将以这样的方式开发，以便轻松扩展到最终产品所需的数百个通道。将使用荧光标记的微球评估原型微电穿孔芯片的基本流体流动特性，然后使用荧光显微镜用荧光蛋白电穿孔的干细胞进行分析。一旦确定了最佳电穿孔参数，将用已经用BMP-2蛋白电穿孔的干细胞进一步评估芯片，并分析这些细胞的成骨潜力。在第二阶段，我们将在第一阶段结果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增加到几十个或几百个通道，以适应目标应用所需的大量细胞。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，该原型可以作为研究应用的独立产品快速商业化，并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装置，用于电穿孔和有效的基因转染个体间充质成体干细胞（MSC），用于脊柱融合和其他骨骼再生治疗。成体间充质干细胞是多能细胞，代表了细胞和基于细胞的组织工程的有前途的工具。与目前使用的批量电穿孔方法相比，新的单细胞电穿孔方法将导致显著更高效和有效的基因转染方法，以改善干细胞治疗结果。