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

描述（由申请人提供）：中枢神经系统（CNS）的组织异质性是疾病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。当必须建立特定细胞和区域的原代神经祖细胞培养时，这尤其具有挑战性。虽然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），但没有可靠的集成方法或仪器可用于分离和后续培养细胞。LCM通常在固定染色或未染色的组织上进行。随着神经干细胞技术的进步，人们迫切需要一种低成本、简单易用的设备，用于非固定脑组织的显微解剖和体外操作。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，该仪器将允许采购和跟踪特定活细胞的培养。在这里，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新鲜组织，并将这些细胞用于下游应用（例如原代细胞培养）。我们开发了CTAS的“原理验证”功能原型，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。第一阶段的具体目标包括：1)开发CTAS的关键组件；2) CTAS运行参数的制定；3) CTAS在组织切片和细胞培养上的检测。这项工作完成后，CTAS将在该项目的第二期商业化。

DESCRIPTION (provided by applicant): Tissue heterogeneity of central nervous system (CNS) is a serious limiting factor for sound cell-specific molecular studies of the disease including genomic or proteomic analysis. This is especially challenging when cell and region specific primary neural progenitor cultures have to be established. Although 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), no reliable integrated methods or instruments are available that would allow isolation and subsequent culturing of cells. LCM is typically performed on fixed stained or unstained tissues. With the advancement of neural stem cell technologies there is a tremendous need for a low- cost and simple-to-use device that would offer microdissection of unfixed brain tissues and manipulation in vitro. The overall goal of this SBIR project is to develop a new low-cost microdissection instrument with cellular resolution that would allow procurement and follow up cultivation of specific live cells. Here we propose to build a prototype and test the feasibility of a novel capillary-based vacuum-assisted cell and tissue acquisition system (CTAS) that is envisioned as an attachment to inverted microscopes. The proposed CTAS would be able to dissect fresh tissues at cellular resolution and use these cells for downstream applications (e.g. primary cell cultures). We developed a "proof of principle" functional prototype of CTAS and demonstrated its use for collection of specific cell types from mouse central nervous system (spinal cord and brain). Phase I specific aims include 1) development of the critical components of CTAS; 2) development of CTAS operational parameters; 3) testing of CTAS on tissue sections and cell cultures. After completion of this work, CTAS will be commercialized in phase II of this project. PUBLIC HEALTH RELEVANCE: Cell specific sorting/capture technology is a prerequisite for precise characterization of the specific cell classes and types for understanding their function and regulation of the metabolism, as well as for preclinical translational research. However, isolation of live brain cells for the purpose of their culturing and in vitro manipulation is still challenging. This is especially demanding when region specific neural progenitors are targeted. In phase I of this project, we will develop a low-cost vacuum-assisted capillary-based cell and tissue acquisition system (CTAS) and demonstrate its feasibility and applicability for collection of live cells from various brain regions. Collected live cells will be used to establish primary cell cultures including neural progenitor cultures (NPCs). 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. In phase I of this SBIR application, we propose the development of the instrument's critical components, optimization and testing for the range of applications including region specific NPCs and cell specific collection from heterogeneous cell cultures and subsequent molecular characterization of the cells. 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 rapid cell specific culturing methods in neural stem cell biology. It is also a versatile instrument that can be applied to fixed tissue sections and used to collect larger tissue areas in lieu to LCM. Unlike fluorescence-activated cell sorting (FACS), which requires dissociation of tissue, CTAS preserves tissue integrity and microenvironment of the cells to be isolated.