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)的组织异质性是包括基因组或蛋白质组分析在内的健全的针对该疾病的细胞特异性分子研究的严重限制因素。当必须建立细胞和区域特定的原代神经前体细胞培养时，这尤其具有挑战性。尽管组织显微解剖和细胞分选技术在过去十年中取得了巨大进步，从简单的手动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)，但目前还没有可靠的集成方法或仪器来分离和随后培养细胞。LCM通常在固定的、染色的或未染色的组织上进行。随着神经干细胞技术的进步，对一种低成本、易用的设备的巨大需求，这种设备可以对未固定的脑组织进行显微解剖和体外操作。这个SBIR项目的总体目标是开发一种新的低成本、具有细胞分辨率的显微解剖仪器，使采购和后续培养特定的活细胞成为可能。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“原则性证明”功能原型，并演示了它用于从小鼠中枢神经系统(脊髓和脑)收集特定类型的细胞。第一阶段的具体目标包括：1)开发CTAS的关键部件；2)开发CTAS的操作参数；3)在组织切片和细胞培养上测试CTAS。这项工作完成后，四氯化碳将在该项目的第二阶段实现商业化。 公共卫生相关性：细胞特异性分选/捕获技术是精确描述特定细胞类别和类型、了解其功能和新陈代谢调节以及临床前转化研究的先决条件。然而，为了培养和体外操作的目的分离活的脑细胞仍然是具有挑战性的。当针对特定区域的神经前体细胞时，这一要求尤其苛刻。在该项目的第一阶段，我们将开发一种低成本的基于真空辅助毛细血管的细胞和组织采集系统(CTAS)，并展示其用于收集不同脑区活细胞的可行性和适用性。收集的活细胞将用于建立原代细胞培养，包括神经前体细胞培养(NPC)。这是一种简单、非侵入性的技术(与LCM不同，它不需要组织固定和干燥)，可以很容易地实现自动化，并提供广泛的细胞和组织特定分离参数。在这项SBIR应用的第一阶段，我们建议开发仪器的关键部件，优化和测试一系列应用，包括区域特定的NPC和从异种细胞培养中收集特定细胞，以及随后的细胞分子表征。这种低成本的显微解剖仪器几乎对任何研究实验室都是负担得起的，因此，鉴于神经干细胞生物学中对快速细胞特异性培养方法的需求日益增长，需求可能会非常高。它也是一种多功能的仪器，可以应用于固定的组织切片，并用于收集更大的组织区域，以代替LCM。与需要分离组织的荧光激活细胞分选(FACS)不同，CTAS保留了组织的完整性和待分离细胞的微环境。

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.