Screening Device for Differentiated Primary Cell Models of Airway Epithelia

描述(由申请人提供)：药物筛选的方法通常涉及戏剧性的妥协，以实现高产量和降低成本。例如，使用永生化的异源细胞系和基因工程来过度表达目标蛋白和/或加入荧光替代报告程序来显示结果。至少在一定程度上，因为这样的系统经常产生与实际人类疾病状态无关的反应，从历史上看，高通量筛查并没有带来强劲的投资回报。更具代表性的原代细胞模型通常适用于组织和疾病，但由于缺乏技术解决方案和高昂的成本，这些模型在筛查中没有得到充分利用。出于类似的原因，更相关的检测技术，如内源性免疫细胞化学和电生理学，在HTS中并不常见。该项目的目标是将用于呼吸道上皮细胞的金标准、器官型细胞模型引入真正的高通量筛选，并使更多信息更丰富的高含量分析方法的使用成为可能，包括内源性CFTR转运和呼吸道表面液体高度。除了囊性纤维化和慢性阻塞性肺病的应用，这种设备还将在皮肤模型和药物转运研究中具有价值。该装置由96个标准微板格式的微腔阵列组成。微腔设计足够小，可以与384孔板密度兼容，因此每块板上可以很容易地实现384个微腔。该平板包括与标准液体处理机器人和高分辨率显微镜的兼容性和易用性的专门功能。重要的是，该设备非常简单，生产成本与HTS实验室的成本限制一致，并显著节省了主电池和介质的消耗。成功地完成了小规模装置的可行性研究，表明高度微型化的方法与气液界面器官型上皮细胞培养是兼容的。虽然这不是这项提议的目的，但该设备被设计成集成到专门为该培养系统设计的电生理学仪器中。

DESCRIPTION (provided by applicant): Approaches to drug screening typically involve dramatic compromises in order to achieve high throughput and hold down costs. Examples include the use of immortalized, heterologous cell lines, and genetic engineering to over-express the target protein and/or incorporate a fluorescent surrogate reporter to display the result. At least partly because such systems often produce responses that are not relevant to the actual human disease state, high throughput screening has not historically delivered a strong return on investment. More representative primary cell models are often available for tissues and diseases, but these models are underutilized in screening because of the lack of technological solutions and high costs. For similar reasons, more relevant assay technologies such as endogenous immunocytochemistry and electrophysiology are not commonly employed in HTS. The goal of this project is to bring the gold standard, organotypic cell model for airway epithelia into true high throughput screening, and enable the use of more informative high content assays including endogenous CFTR trafficking and airway surface liquid height. In addition to applications for cystic fibrosis and COPD, the resultant device will also be valuable for skin models and drug transport studies. The device consists of an array of 96 microchambers in standard microplate format. The microchamber design is small enough to be compatible with 384-well plate densities, so 384 microchambers per plate is readily achievable. The plate includes specialized features for compatibility and ease-of-use with standard liquid handling robotics, and high resolution microscopy. Importantly, the device is simple enough to be produced at a cost consistent with the cost constraints of HTS labs, and confers dramatic savings in primary cell and media consumption. The feasibility study on a small-scale device was successfully completed, indicating that the highly miniaturized approach is compatible with organotypic epithelial airway culture at air-liquid interface. Although not an aim of this proposal this device is designed to be integrated into an electrophysiology instrument designed specifically for this culture system.