High throughput cell migration assay amenable to high content imaging

描述（由申请人提供）：该项目的长期目标是开发一种384孔细胞侵袭试验，适用于化学文库的高通量筛选（HTS）。评估癌症药物对细胞侵袭的主要障碍是缺乏负担得起的三维分析，这些分析是可靠的，可重复的，生理相关的，适合自动化的，具有成本效益的。如本提案所述，OrisTM技术的进一步发展将形成一种经济实惠、易于使用的基于细胞的检测方法的基础，能够快速定量地获得结果，从而促进细胞入侵治疗候选药物的选择和评估。384孔细胞侵袭试验的可用性，需要最少数量的细胞和微量的测试化合物，将加速癌症治疗药物的开发。建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。所提出的分析方法既可以作为HTS仪器快速读取的主筛选，也可以有效地允许在相同的分析井中进行后续的二次筛选，可以通过HCA平台轻松定量。这是一个非凡的经济效益，增加了研究经费产生知识的能力；它节省了试剂（化合物，细胞等）和资源（人力），否则将被其他格式（如跨膜测定）所需的重复测试所消耗。提出的3D侵入试验是基于创新性地使用生物相容性凝胶（BCG）沉积在每个孔的中心，以防止细胞粘附在孔的中心。正如一期试验所证明的那样，BCG是第一代细胞运动试验中使用的Oris硅胶细胞播种塞的有效替代品。细胞播种后，卡介苗溶解，在每个孔的中心显示可重复的检测区。一层细胞外基质覆盖在孔中，开始向x、y和z轴侵入。通过消除细胞播种塞（既阻止自动液体处理设备进入，又需要手动移除步骤才能开始分析），基于bcg的分析将提供机器人递送细胞、培养基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。在该方案的第一阶段，我们开发了一种基于96孔的细胞迁移实验，利用BCG创建了一个溶解屏障，成功地在实验孔的中心形成了检测区。我们有效筛选了100多种卡介苗配方，并选择了一种合适的配方，a)与组织培养处理和胶原I包被表面兼容，b)在组织培养基中完全溶解时能够阻止细胞附着，c)允许细胞在溶解时迁移，d)不干扰细胞活力或诱导细胞毒性。我们开发并优化了机器人分配能力，以实现96孔格式的均匀和可重复的卡介苗沉积，并确认卡介苗不会干扰4种不同类型抑制剂在这种新型迁移试验中的功效。我们的1期研究数据清楚地证明了hts兼容细胞运动测定的可行性，该方法基于精确和精确地沉积可溶解的生物相容性凝胶，该凝胶在96孔测定板上形成临时细胞隔离区。这些结果证明了该技术在384口井、3D高通量细胞侵袭试验中的持续发展。基于成功推出Oris细胞检测产品线，Platypus拥有开发、验证和制造细胞检测产品的技能、知识和基础设施。第二阶段的主要目标是：1)将96口井的迁移分析缩小到384口井的入侵分析；2)优化ECM覆盖层的条件；3)在3天的变异性研究和具有良好特征的抑制剂的剂量反应滴定中验证分析。这些目标的成功完成将为研究人员提供具有成本效益的384口井3D入侵分析，这将减少分析设置所需的劳动力和材料，并能够通过使用多路染色有效地捕获每口井的额外信息，从而最大限度地利用研究资金和人力资源。

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.