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

描述（由申请人提供）：组织异质性是疾病细胞特异性分子研究的严重限制因素，包括基因组或蛋白质组学分析。组织显微解剖和细胞分选技术在过去十年中取得了巨大的进步，从简单的手工组织解剖到复杂的激光捕获显微解剖（LCM）仪器和高速荧光辅助细胞分选系统（FACS）。结合基因组学和蛋白质组学技术，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治疗靶点的鉴定。目前，LCM和FACS是分离特定组织和细胞类型的两种主要技术。然而，由于它们的高成本和复杂的界面，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，需要一种低成本和简单易用的显微解剖设备，提供类似于LCM和FACS的功能。SBIR项目的总体目标是开发一种具有细胞分辨率的新型低成本显微解剖仪器。在这个项目的第一阶段，我们建议建立一个原型并测试一种新型的基于毛细管的真空辅助细胞和组织采集系统（CTAS）的可行性，该系统被设想为倒置显微镜的附件。拟议的CTAS将能够以细胞分辨率解剖组织并收集下游应用（例如表达微阵列）的材料（RNA或蛋白质）。该项目的第一期非常成功。我们开发了一个功能齐全的原型，并演示了其用于收集小鼠中枢神经系统（脊髓和脑）的特定细胞类型。对毛细管支架、集热器、真空源、CTAS支架和光源的结构和主要部件进行了开发、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)控制单元和可调参数的开发；3) CTAS在组织切片上的进一步测试；细胞培养。此外，原型将在不同的实验室环境中进行测试，包括组织解剖和来自异质细胞培养源的细胞特异性收集。NeuroInDx将完成这项工作，这将是成功评估拟议CTAS所必需的，并将在该项目的第三阶段将该仪器商业化。

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.