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

开发一种新型低成本毛细管细胞和组织采集系统

• 批准号: 7999706
• 负责人: Stanislav Karsten
• 金额: $ 35万
• 依托单位: NEUROINDX INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-18 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7999706
• 关键词: Adult; Affect; Animals; Architecture; Area; Blood capillaries; Brain; Brain region; Budgets; Cell Culture Techniques; Cell Separation; Cell Survival; Cells; Cellular Morphology; Cellular Structures; Collection; Computer software; Cultured Cells; Development; Device or Instrument Development; Devices; Disease; Dissection; Dissociation; Electronics; Ensure; Fluorescence; Fluorescence-Activated Cell Sorting; Freezing; Fresh Tissue; Future; Gene Expression; Genomics; Genotype; Goals; Heterogeneity; Histocompatibility Testing; In Vitro; Industry; Label; Laboratories; Laboratory Research; Lasers; Life; Manuals; Metabolism; Methods; Microdissection; Microscope; Modeling; Molecular; Mus; Nature; Neuraxis; Nucleic Acids; Performance; Pharmacologic Substance; Phase; Price; Primary Cell Cultures; Process; Proteins; Proteomics; Rattus; Regulation; Research; Resolution; Sampling; Small Business Innovation Research Grant; Sorting - Cell Movement; Specimen; Speed; Spinal Cord; Staining method; Stains; System; Techniques; Technology; Testing; Time; Tissues; Translational Research; Vacuum; Work; base; brain cell; brain tissue; capillary; cell type; cost; design; flexibility; follow-up; gel electrophoresis; improved; instrument; interest; laser capture microdissection; meetings; nerve stem cell; novel; pre-clinical; prototype; sample collection; sound; stem cell biology; stem cell technology; tissue culture; tissue fixing; tissue preparation; tool; user-friendly

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.

描述（由申请方提供）：中枢神经系统（CNS）的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。当必须建立细胞和区域特异性原代神经祖细胞培养物时，这尤其具有挑战性。尽管组织显微切割和细胞分选技术在过去十年中从简单的手动组织切割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）已经取得了巨大的进步，但是没有可靠的集成方法或仪器可以允许分离和随后培养细胞。LCM通常在固定的染色或未染色组织上进行。随着神经干细胞技术的发展，迫切需要一种低成本且易于使用的装置，该装置将提供未固定脑组织的显微切割和体外操作。该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，可以采购和后续培养特定的活细胞。在这里，我们建议建立一个原型和测试的可行性，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），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“原理证明”功能原型，并证明了其用于从小鼠中枢神经系统（脊髓和脑）收集特定细胞类型。第一阶段的具体目标包括：1）开发CTAS的关键组件; 2）开发CTAS操作参数; 3）在组织切片和细胞培养物上测试CTAS。这项工作完成后，CTAS将在该项目的第二阶段商业化。 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别和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。然而，为了培养和体外操作目的而分离活脑细胞仍然具有挑战性。当靶向区域特异性神经祖细胞时，这是特别需要的。在该项目的第一阶段，我们将开发一种低成本的真空辅助毛细血管细胞和组织采集系统（CTAS），并证明其可行性和适用性，从不同的大脑区域收集活细胞。收集的活细胞将用于建立原代细胞培养物，包括神经祖细胞培养物（NPC）。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。在SBIR应用的第一阶段，我们提出了仪器关键组件的开发，优化和测试应用范围，包括区域特异性NPC和来自异质细胞培养物的细胞特异性收集以及随后的细胞分子表征。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，鉴于神经干细胞生物学中快速细胞特异性培养方法的需求不断增长。它也是一种多功能仪器，可应用于固定的组织切片，并用于代替LCM收集更大的组织区域。与需要组织解离的荧光激活细胞分选（FACS）不同，CTAS保留了待分离细胞的组织完整性和微环境。