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，以实现高通量和压低成本。实例包括使用永生化的异源细胞系和基因工程来过表达靶蛋白和/或掺入荧光替代报告物以显示结果。至少部分地因为这样的系统通常产生与实际人类疾病状态不相关的响应，高通量筛选在历史上没有提供强有力的投资回报。对于组织和疾病，通常可以获得更具代表性的原代细胞模型，但由于缺乏技术解决方案和成本高，这些模型在筛选中未得到充分利用。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，通常不采用HTS。该项目的目标是将气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，并能够使用更多信息的高含量测定，包括内源性CFTR运输和气道表面液体高度。除了囊性纤维化和COPD的应用外，所得到的设备也将对皮肤模型和药物转运研究有价值。该装置由标准微孔板格式的96个微室阵列组成。微室设计足够小以与384孔板密度兼容，因此每个板384个微室是容易实现的。该板包括与标准液体处理机器人和高分辨率显微镜兼容和易于使用的专门功能。重要的是，该装置足够简单，可以以与HTS实验室的成本限制一致的成本生产，并在原代细胞和培养基消耗方面提供了显着的节省。成功完成了小型装置的可行性研究，表明高度小型化的方法与气液界面的器官型上皮气道培养相容。虽然不是本提案的目的，但该器械设计为集成到专门为此培养系统设计的电生理仪器中。

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.