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: 8056324
• 负责人: Bharat R Acharya
• 金额: $ 39.55万
• 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 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8056324
• 关键词: 3-Dimensional; Affect; Antineoplastic Agents; Automation; Basement membrane; Biocompatible; Biological Assay; Caliber; Cell Count; Cell Line; Cell Survival; Cell-Matrix Junction; Cells; Collagen; Computer software; Culture Media; Data; Deposition; Detection; Development; Disease; Dose; Drug Formulations; Economics; Encapsulated; Environment; Equipment; Excision; Exclusion; Extracellular Matrix; Funding; Gel; Generations; Goals; Health; Human Resources; Image; Image Analysis; Industry; Institutes; Invaded; Investigation; Kinetics; Knowledge; Laboratory Personnel; Letters; Libraries; Liquid substance; Malignant Neoplasms; Manuals; Measurement; Measures; Membrane; Migration Assay; Miniaturization; Modeling; NCI Center for Cancer Research; Names; Neoplasm Metastasis; Ornithorhynchus anatinus; Phase; Positioning Attribute; Process; Provider; Reading; Reagent; Research; Research Infrastructure; Research Personnel; Resources; Robotics; Screening procedure; Silicones; Staining method; Stains; System; Technology; Testing; Therapeutic; Time; Titrations; Universities; base; cell motility; cost effective; cytotoxicity; drug candidate; drug development; drug discovery; experience; extracellular; high throughput screening; in vitro Assay; in vivo; inhibitor/antagonist; innovation; instrument; instrumentation; migration; miniaturize; novel; phase 1 study; preclinical evaluation; prevent; response; skills; small molecule libraries; success; surface coating; technology development; therapeutic evaluation; tissue culture

DESCRIPTION (provided by applicant): The long term goal of this project is to develop a 384-well cell invasion assay suitable for high throughput screening (HTS) of chemical libraries. The principal barrier to evaluating cancer drugs for cell invasion is the lack of affordable 3-dimensional assays that are robust, reproducible, physiologically relevant, suitable for automation 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 rapid and quantitative results that facilitates selection and evaluation of therapeutic candidates for cell invasion. The availability of a 384-well cell invasion assay that requires minimal numbers of cells and minute volumes of test compounds will accelerate drug development for cancer therapeutics. The proposed assay format will be compatible with automated liquid handling systems and high content analysis (HCA) instruments. The proposed assay will both be useful as a primary screen which can be read quickly by HTS instruments while also efficiently permitting subsequent secondary screens in the same assay wells that can be easily quantitated via HCA platforms. This is an extraordinary economic benefit that increases the knowledge-generating power of the research dollar; it conserves reagents (compounds, cells, etc) and resources (manpower) that would otherwise be consumed in repetitive testing required by other formats such as trans-membrane assays. The proposed 3D invasion assay is based on the innovative use of a biocompatible gel (BCG) centrally deposited in each well to exclude cells from adhering in the centers of the wells. As demonstrated in phase 1 activities, the BCG is an effective replacement for the Oris" silicone cell seeding stoppers used in our first generation cell motility assays. After cells are seeded, the BCG dissolves to reveal reproducible Detection Zones in the center of each well. A coating of extracellular matrix is then overlaid in the well and invasion in the x, y and z axes can begin. By eliminating the cell seeding stoppers, which both prevent access by automated liquid handling equipment and require a manual removal step to begin the assay, the BCG-based assay will offer the ability for robotic delivery of cells, media and test compounds thereby decreasing hands-on time required by laboratory personnel. In Phase 1 of this proposal, we developed a 96-well based Cell Migration Assay utilizing BCG to create a dissolving barrier that successfully formed Detection Zones in the center of assay wells. We effectively screened over 100 formulations of BCG and selected a suitable formulation that a) was compatible with both tissue culture treated and collagen I coated surfaces, b) had the ability to block cell attachment while completely dissolving in tissue culture media, c) permitted cell migration upon dissolution and d) did not interfere with cell viability or induce cytotoxicity. We developed and optimized robotic dispensing capabilities to achieve uniform and reproducible BCG deposition in a 96- well format and confirmed that BCG does not interfere with the efficacy of 4 different classes of inhibitors in this novel migration assay. The data presented from our Phase 1 studies clearly demonstrate the feasibility of a HTS-compatible cell motility assay based on accurate and precise deposition of a dissolvable, biocompatible gel that creates a temporary cell exclusion zone in 96-well assay plates. These results justify continued development of this technology for a 384-well, 3D high throughput cell invasion assay. Based on proven success in launching the Oris" cell-based assay product line, Platypus has the skills, knowledge, and infrastructure to develop, validate and manufacture products for cell-based assays. The major goals of this Phase 2 proposal are to 1) miniaturize the 96-well migration assay to a 384-well invasion assay format, 2) optimize conditions for an ECM overlay; and 3) validate the assays in 3-day variability studies and dose-response titrations with well characterized inhibitors. Successful completion of these goals will provide researchers with a cost-effective 384-well 3D invasion assay that will reduce labor and materials needed for assay set-up and offer the ability to efficiently capture additional information per well by using multiplexed staining, thereby maximizing research funds and human resources.

描述(由申请人提供)：该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384孔细胞侵袭试验。评估抗癌药物对细胞侵袭的主要障碍是缺乏负担得起的三维分析，这些分析是健壮的、可重复的、与生理相关的、适合自动化和执行成本效益的。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，该分析能够获得快速和定量的结果，便于选择和评估细胞侵袭的治疗候选者。一种需要最少细胞数量和极少量测试化合物的384孔细胞侵袭试验的问世，将加速癌症治疗药物的开发。拟议的分析格式将与自动化液体处理系统和高含量分析(HCA)仪器兼容。建议的化验既可用作一次筛查，可被HTS仪器快速读取，又可有效地在同一化验井中进行后续二次筛查，可通过HCA平台轻松定量。这是一个非同寻常的经济效益，它增加了研究资金的知识产生能力；它节省了试剂(化合物、细胞等)和资源(人力)，否则这些资源将被消耗在其他形式的重复测试中，如跨膜分析。拟议的3D侵袭分析基于创新地使用生物相容凝胶(BCG)集中沉积在每口井中，以排除细胞粘连在井的中心。正如在第一阶段的活动中所证明的那样，卡介苗是我们第一代细胞运动分析中使用的Oris“硅胶细胞播种器”的有效替代品。在细胞被播种后，卡介苗溶解，在每口井的中心显示可重复的检测区。然后在井中覆盖一层细胞外基质，并开始沿x、y和z轴侵入。通过消除阻止自动液体处理设备进入并需要手动移除步骤才能开始分析的细胞播种器，基于卡介苗的检测将提供自动输送细胞、培养液和测试化合物的能力，从而减少实验室人员所需的动手时间。在这项建议的第一阶段，我们开发了一种基于96孔的细胞迁移试验，利用卡介苗创建了一种溶解屏障，成功地在分析孔的中心形成了检测区。我们有效地筛选了100多个卡介苗配方，并选择了合适的配方：a)与组织培养处理的表面和I型胶原涂层表面都兼容，b)在组织培养介质中完全溶解时能够阻止细胞附着，c)溶解时允许细胞迁移，d)不干扰细胞活力或产生细胞毒性。我们开发和优化了机器人配药能力，以实现96孔格式的均匀和可重复性的卡介苗沉积，并证实在这一新的迁移试验中，卡介苗不会干扰4种不同类别的抑制剂的效果。我们第一阶段研究提供的数据清楚地证明了基于准确和精确地沉积可溶的、生物相容的凝胶的HTS兼容细胞运动性分析的可行性，该凝胶在96孔板上创建了临时的细胞隔离区。这些结果证明了继续开发这项技术以进行384孔、3D高通量细胞侵袭试验。在成功推出Oris“基于细胞的检测产品线”的基础上，Platypus拥有开发、验证和制造基于细胞的检测产品的技能、知识和基础设施。这一第二阶段提案的主要目标是：1)将96孔迁移分析小型化为384孔侵袭分析格式；2)优化ECM覆盖的条件；3)在3天的变异性研究和剂量反应滴定中验证具有良好特征的抑制剂的分析。成功完成这些目标将为研究人员提供具有成本效益的384孔3D侵袭分析，这将减少分析设置所需的人力和材料，并提供通过使用多重染色有效地捕获每孔额外信息的能力，从而最大限度地增加研究资金和人力资源。