Development of a novel low-cost capillary-based cell and tissue acquisition syste

描述（由申请方提供）：组织异质性是对该疾病进行合理的细胞特异性分子研究（包括基因组或蛋白质组学分析）的严重限制因素。组织显微切割和细胞分选技术在过去的十年中已经从简单的手动组织切割发展到复杂的激光捕获显微切割（LCM）仪器和高速荧光辅助细胞分选系统（FACS）。与基因组学和蛋白质组学技术相结合，现在可以生成细胞特异性转录组/蛋白质组数据，推进疾病生物标志物和新型治疗靶点的鉴定。目前，LCM和FACS是用于分离特定组织和细胞类型的两种主要技术。然而，由于其高成本和复杂的接口，这些技术不足以完全支持对细胞特异性分子数据日益增长的需求。因此，非常需要一种低成本且易于使用的显微切割装置，其将提供类似于LCM和FACS的能力。SBIR项目的总体目标是开发一种新的低成本显微切割仪器，具有细胞分辨率。在该项目的第一阶段，我们提出了建立一个原型，并测试一种新的毛细管为基础的真空辅助细胞和组织采集系统（CTAS）的可行性，设想作为一个附件倒置显微镜。拟议的CTAS将能够以细胞分辨率解剖组织，并收集材料（RNA或蛋白质）用于下游应用（例如表达微阵列）。该项目的第一阶段非常成功。我们开发了一个功能齐全的原型，并展示了其用于从小鼠中枢神经系统（脊髓和大脑）收集特定细胞类型的用途。对CTAS的结构和主要部件，包括毛细管保持器、收集器、真空源、CTAS保持器和光源进行了开发、测试和优化。第二阶段的具体目标包括：1）进一步开发CTAS的关键组件; 2）开发控制单元和可调参数; 3）在组织切片和细胞培养物上进一步测试CTAS。此外，原型将在不同的实验室环境中进行测试，包括组织解剖和从异质细胞培养来源中进行细胞特异性收集。NeuroInDx将完成这项工作，这对于成功评价申报的CTAS是必要的，并将在本项目的第三阶段将该仪器商业化。 公共卫生相关性：细胞特异性分选/捕获技术是精确表征特定细胞类型以了解其功能和代谢调节以及临床前转化研究的先决条件。目前，用于获取特定细胞的两种主要方法是可用的：荧光辅助细胞分选（FACS）和激光捕获显微切割（LCM）。这些技术都很复杂，仪器不仅非常昂贵，而且维护成本也很高。在该项目的第一阶段，我们开发了一种低成本的真空辅助毛细管为基础的细胞和组织采集系统（CTAS），并证明了其可行性和适用性的组织显微切割和下游应用。它是一种简单的非侵入性技术（与LCM不同，它不需要组织固定和干燥），可以轻松自动化，并提供广泛的细胞和组织特异性分离参数。我们估计CTAS将比LCM或FACS仪器便宜至少5-10倍。在SBIR应用的第二阶段，我们建议进一步开发仪器，优化和测试的应用范围，包括组织显微切割和细胞特异性收集异质细胞培养物。作为第二阶段的一部分，CTAS的beta测试将在包括学术实验室和工业在内的几个地点进行。这项工作将使其在下一个第三阶段充分商业化。这种低成本的显微切割仪器将是负担得起的，几乎任何研究实验室，因此，需求可能会非常高，鉴于细胞特异性分析的需求不断增长。

DESCRIPTION (provided by applicant): Tissue heterogeneity is a serious limiting factor for sound cell-specific molecular studies of the disease including genomic or proteomic analysis. Tissue microdissection and cell sorting technologies have advanced tremendously over the last decade from simple manual tissue dissection to sophisticated laser capture microdissecting (LCM) instruments and high speed fluorescence assisted cell sorting systems (FACS). In combination with genomics and proteomics technologies it is now possible to generate cell specific transcriptome/proteome data, advancing the identification of disease biomarkers and novel therapeutic targets. Currently, LCM and FACS are the two main technologies for the isolation of specific tissues and cell types. However, due to their high costs and often sophisticated interface, these technologies are not sufficient to fully support the growing need for cell specific molecular data. Therefore, there is a tremendous need for a low-cost and simple-to-use microdissection device that would offer capabilities similar to LCM and FACS. The overall goal of this SBIR project is to develop a new low-cost microdissection instrument with cellular resolution. In phase I of this project we proposed to build a prototype and test the feasibility of a novel capillary- based vacuum-assisted cell and tissue acquisition system (CTAS) that was envisioned as an attachment to inverted microscopes. The proposed CTAS would be able to dissect tissues at cellular resolution and collect material (RNA or protein) for downstream applications (e.g. expression microarrays). Phase I of this project was highly successful. We developed a fully functional prototype and demonstrated its use for collection of specific cell types from mouse central nervous system (spinal cord and brain). The architecture and major components of CTAS, including the capillary holder, collector, vacuum source, CTAS holder and light source, were developed, tested and optimized. Phase II specific aims include 1) further development of the critical components of CTAS; 2) development of control unit and adjustable parameters; 3) further testing of CTAS on tissue sections; and cell cultures. In addition, the prototype will be tested in different laboratory settings including tissue dissection and cell specific collection from heterogeneous cell culture sources. NeuroInDx will complete this work, which will be necessary to successfully evaluate proposed CTAS, and will commercialize the instrument in phase III of this project. PUBLIC HEALTH RELEVANCE: Cell specific sorting/capture technology is a prerequisite for precise characterization of the specific cell types for understanding their function and regulation of the metabolism, as well as for preclinical translational research. Currently two major approaches for the acquisition of specific cells are available: fluorescence assisted cell sorting (FACS) and laser-capture microdissection (LCM). These technologies are sophisticated and the instruments are not only very expensive but have high maintenance costs. In phase I of this project, we developed a low-cost vacuum-assisted capillary-based cell and tissue acquisition system (CTAS) and demonstrated its feasibility and applicability for tissue microdissection and downstream applications. It is a simple, non-invasive (unlike LCM it does not require tissue fixing and drying) technology that can be easily automated and offers a wide range of cell- and tissue-specific separation parameters. We estimate that CTAS will be at least 5-10 times cheaper than LCM or FACS instruments. In phase II of this SBIR application, we propose further development of the instrument, its optimization and testing for the range of applications including tissue microdissection and cell specific collection from heterogeneous cell cultures. As part of Phase II, beta testing of CTAS will be carried out in several sites including academic laboratories and industry. This work will result in its full commercialization in the following phase III. This low-cost microdissection instrument will be affordable for virtually any research laboratory, and therefore, the demand will likely be very high given the growing need for cell specific analysis.