High throughput cell migration assay amenable to high content imaging

高通量细胞迁移测定适合高内涵成像

• 批准号: 7800228
• 负责人: Keren Isaac Hulkower
• 金额: $ 10.01万
• 依托单位: PLATYPUS TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7800228
• 关键词: Adoption; Affect; Antineoplastic Agents; Area; Arteriosclerosis; Benchmarking; Biocompatible; Biological Assay; Caliber; Cell Adhesion; Cell Count; Cell Line; Cell Survival; Cell-Matrix Junction; Cells; Cellular Morphology; Chemicals; Communities; Consult; Data; Data Analyses; Deposition; Detection; Development; Ensure; Exclusion; Fluorescence; Funding; Generations; Goals; Image; In Vitro; Industry; Institutes; Knowledge; Laboratories; Lead; Letters; Libraries; Life; Liquid substance; Malignant Neoplasms; Measurable; Measures; Membrane; Methods; Microscope; Migration Assay; Molecular Bank; Neoplasm Metastasis; Optics; Ornithorhynchus anatinus; Pattern; Phase; Polymers; Positioning Attribute; Process; Proliferating; Reader; Reading; Reagent; Reporting; Research; Research Infrastructure; Research Personnel; Robotics; Screening procedure; Seeds; Silicones; Staining method; Stains; System; Techniques; Technology; Testing; Therapeutic; Time; Translations; United States National Institutes of Health; Universities; Wisconsin; Wound Healing; base; cancer cell; cell motility; combinatorial chemistry; cost; culture plates; design; drug candidate; drug development; drug discovery; experience; follow-up; high throughput screening; human disease; improved; inhibitor/antagonist; innovation; instrumentation; migration; miniaturize; public health relevance; skills; small molecule; small molecule libraries; success; therapeutic development; tissue culture; wound

DESCRIPTION (provided by applicant): The long term goal of this project is to develop a 384-well plate-based cell migration assay suitable for high throughput screening (HTS) of chemical libraries. The principal barrier to performing HTS to discover cancer drugs affecting cell migration is the lack of affordable assays that are robust, reproducible and cost-effective to perform. The further advancement of OrisTM technology, as described in this proposal, will form the basis of an affordable, easy to use cell-based assay capable of providing relevant data for quickly screening drug candidates for their ability to impact cell migration. The availability of a HTS 384 well cell-based assay that requires minimal numbers of cells and minute volumes of test compounds will facilitate primary screens and better drug development for cancer therapeutics. The proposed HTS assay format will be compatible with automated liquid handling systems and high content screening (HCS) platforms. It will be amenable to primary screens which can be read quickly by plate reader instrumentation. It will also permit subsequent secondary screens in the same assay wells that can be easily quantitated via imaging platforms. This HTS assay is based on the innovative use of a chemically derived cell exclusion zone that will be deposited in a defined central area at the bottom of a tissue culture well. Cells are then seeded and attach at the perimeter of the well, the exclusion zone dissolves and reveals a zone that is now permissible for cell migration. The first generation OrisTM Cell Migration Assay provides 96 wells for investigating the effects of cell movement modulators. It uses silicone stoppers to create exclusion zones. Using the Oris" assay, we demonstrated measurable migration of A-549 cells and reported z-factors of > 0.46 for migration to support claims of assay robustness. We have shown that the assay is suitable for testing modulators of cell motility. We further showed the ability to collect multiple pieces of information from a single test well (i.e., high content screening capable) and data analysis compatible with fluorescence microplate readers and imaging platforms. Finally, we provided recent data to support the creation of a reversible polymeric exclusion zone that eliminates the need for a silicone stopper and makes the assay highly amenable for use with automated liquid handlers employed by HTS laboratories. It appears that the polymer completely dissolves as evidenced by the full migration of cells into the previously restricted area and has no obvious deleterious effects on cell viability or test compounds. These data strongly support the feasibility of modifying the Oris" 96-well cell migration assay into a 384-well, high throughput cell migration assay. In this phase I assay, we propose to first develop a 96-well cell migration assay to allow for greater amounts of both primary and secondary data to be obtained from a single assay well by using multiplexed staining techniques with different fluorophor conjugates. The assay will enjoy a wide range of compatibility with a variety of HCS platforms as well as standard fluorescent plate readers and microscopes. Our intended product, a 384 well assay, will streamline the drug discovery process to facilitate quicker screening of molecular libraries for development of therapeutics that block cancer cell metastasis or promote wound healing. PUBLIC HEALTH RELEVANCE: The principal barrier to performing high throughput screening (HTS) to discover cancer drugs affecting cell migration is the lack of affordable assays that are robust, reproducible and cost-effective to perform. Our proposed HTS assay format will be compatible with automated liquid handling systems and high content screening platforms. This assay format will be amenable to primary screens which can be read quickly by plate reader instrumentation while permitting subsequent secondary screens in the same assay wells that can be easily quantitated via imaging platforms. Based on proven success in launching the Oris" product line, Platypus has the skills, knowledge, and infrastructure to develop, validate and manufacture products for cell-based assays. The goal of this Phase I proposal, is to 1) show feasibility for creating consistently sized and placed polymer deposits in multiwell plates to create chemically derived cell exclusion zones and 2) ability of cells to proliferate and migrate in the presence of motility modulating compounds on these plates.

描述(由申请人提供)：该项目的长期目标是开发一种适用于高通量筛选(HTS)的384孔板细胞迁移试验。进行HTS以发现影响细胞迁移的抗癌药物的主要障碍是缺乏负担得起的可靠、可重复性和成本效益高的分析方法。如本提案中所述，OrisTM技术的进一步发展将构成一种负担得起的、易于使用的基于细胞的分析的基础，该分析能够为快速筛选候选药物影响细胞迁移的能力提供相关数据。HTS 384良好细胞化验法的问世，需要最少的细胞数量和极少量的测试化合物，将促进癌症治疗的初步筛选和更好的药物开发。拟议的HTS检测格式将与自动化液体处理系统和高含量筛选(HCS)平台兼容。它将适用于主要屏幕，可以通过平板阅读器仪器快速读取。它还将允许在相同的化验井中进行后续的二级筛查，这些二级筛查可以通过成像平台轻松地进行定量。这种HTS分析是基于化学衍生细胞隔离区的创新使用，该隔离区将存放在组织培养井底部的指定中心区域。然后，细胞被播种并附着在井的周围，隔离区溶解，显露出现在允许细胞迁移的区域。第一代OrisTM细胞迁移分析为研究细胞运动调节剂的作用提供了96个孔。它使用硅胶塞子来创建隔离区。使用Oris“测试，我们展示了A-549细胞的可测量迁移，并报告了迁移的z因子为0.46，以支持测试稳健性的声明。我们已经证明，该方法适用于细胞运动调节剂的检测。我们进一步展示了从单个测试井收集多条信息的能力(即高含量筛选能力)以及与荧光微板阅读器和成像平台兼容的数据分析。最后，我们提供了最新的数据，以支持创建一个可逆的聚合物隔离区，消除了对硅胶塞子的需要，并使该测试非常适合用于HTS实验室使用的自动液体处理机。似乎聚合物完全溶解，细胞完全迁移到以前的限制区域，对细胞活性或测试化合物没有明显的有害影响。这些数据有力地支持了将Oris“96孔细胞迁移实验修改为384孔、高通量细胞迁移实验的可行性。在这个第一阶段的测试中，我们建议首先开发一个96孔细胞迁移测试，通过使用不同荧光素偶联物的多重染色技术，允许从一次测试中获得更多的一次和二次数据。该分析将享受与各种HCS平台以及标准荧光平板读取器和显微镜的广泛兼容性。我们的预期产品，384孔分析，将简化药物发现过程，以促进更快地筛选分子文库，以开发阻止癌细胞转移或促进伤口愈合的治疗药物。 公共卫生相关性：进行高通量筛查(HTS)以发现影响细胞迁移的癌症药物的主要障碍是缺乏负担得起的可靠、可重复性和成本效益高的分析方法。我们建议的HTS检测格式将与自动化液体处理系统和高含量筛选平台兼容。这种分析形式将适用于一次筛查，可以通过平板阅读器仪器快速读取，同时允许在相同的化验井中进行后续二次筛查，可以通过成像平台容易地进行定量。基于Oris“产品线的成功推出，Platypus拥有开发、验证和制造基于细胞的分析产品的技能、知识和基础设施。这项第一阶段计划的目标是：1)证明在多孔板中创建尺寸一致且放置一致的聚合物沉积物的可行性，以创建化学衍生的细胞隔离区；2)在这些板上存在运动调节化合物的情况下，细胞增殖和迁移的能力。