High throughput cell migration assay amenable to high content imaging

描述（由申请人提供）：本项目的长期目标是开发一种适用于化学文库高通量筛选（HTS）的基于384孔板的细胞迁移试验。进行HTS以发现影响细胞迁移的癌症药物的主要障碍是缺乏可负担得起的分析，这些分析是稳健的，可重复的和具有成本效益的。如本提案所述，OrisTM技术的进一步发展将成为一种经济实惠、易于使用的基于细胞的测定方法的基础，该方法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孔基于细胞的测定法的可用性需要最小数量的细胞和微小体积的测试化合物，这将促进癌症治疗的初步筛选和更好的药物开发。拟定的HTS测定格式将与自动液体处理系统和高含量筛选（HCS）平台兼容。它将适用于可以通过酶标仪仪器快速读取的初级筛选。它还将允许在相同的测定威尔斯孔中进行随后的二次筛选，其可以通过成像平台容易地定量。该HTS测定基于化学衍生的细胞排除区的创新使用，该细胞排除区将沉积在组织培养孔底部的限定中心区域中。然后接种细胞并附着在孔的周边，排除区溶解并显示出现在允许细胞迁移的区域。第一代OrisTM细胞迁移试验提供了96个威尔斯孔，用于研究细胞运动调节剂的作用。它使用硅胶塞子来创建禁区。使用Oris分析，我们证明了A-549细胞的可测量迁移，并报告了迁移的z因子> 0.46，以支持分析稳健性的声明。我们已经表明，该测定法适用于测试细胞运动性的调节剂。我们进一步展示了从单个测试井收集多条信息的能力（即，能够进行高含量筛选）和与荧光酶标仪和成像平台兼容的数据分析。最后，我们提供了最近的数据，以支持创建一个可逆的聚合物排除区，消除了对硅胶塞的需要，并使测定非常适合与HTS实验室采用的自动液体处理器一起使用。通过细胞完全迁移到先前限制的区域中证明，聚合物似乎完全溶解，并且对细胞活力或供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的96孔细胞迁移分析仪改进为384孔高通量细胞迁移分析仪的可行性。在该I期试验中，我们建议首先开发96孔细胞迁移试验，以通过使用具有不同荧光团缀合物的多重染色技术从单个试验孔获得更大量的主要和次要数据。该检测试剂盒与各种HCS平台以及标准荧光酶标仪和显微镜具有广泛的兼容性。我们的预期产品，384孔测定，将简化药物发现过程，以促进更快地筛选分子库，用于开发阻断癌细胞转移或促进伤口愈合的治疗剂。 公共卫生相关性：进行高通量筛选（HTS）以发现影响细胞迁移的癌症药物的主要障碍是缺乏可负担得起的、稳健的、可重复的和具有成本效益的测定。我们提出的HTS分析格式将与自动化液体处理系统和高含量筛选平台兼容。该测定形式将适合于初级筛选，其可以通过读板器仪器快速读取，同时允许在相同的测定威尔斯孔中进行随后的次级筛选，其可以通过成像平台容易地定量。基于Oris产品线的成功，Platypus拥有开发、验证和制造细胞分析产品的技能、知识和基础设施。该I期提案的目标是：1）显示在多孔板中产生一致大小和放置的聚合物沉积物以产生化学衍生的细胞排除区的可行性，以及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。

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.