A Novel High Throughput Tumor Spheroid Microtechnology

描述（由申请人提供）：癌细胞的二维（2D）培养物通常用于药物发现，用于筛选和初步表征潜在药物化合物库的功效。尽管其简单性和与高通量筛选仪器的兼容性，但2D细胞测定通常无法预测化合物在体内的功效，使得药物开发和发现成为极其昂贵的过程。2D培养与体内癌细胞复杂的3D环境之间的差异是单层培养系统的主要缺点。新型化疗药物的开发需要在类似于3D肿瘤环境的环境中针对恶性肿瘤细胞进行化合物筛选。癌细胞球状体（CCS）是恶性细胞的3D簇，其被认为是实体瘤的生理模型;它们具有与无血管肿瘤相似的代谢和增殖梯度，并表现出实体瘤的临床表达谱。尽管CCS具有预测药物临床疗效的固有能力，但CCS的形成和维持所需的复杂且昂贵的方法学要求严重阻碍了将CCS纳入主流药物开发过程。我们通过开发一种技术平台来克服现有技术的局限性，以使用水性两相系统（ATPS）在标准384微孔板中产生一致大小的球状体。将一滴含有癌细胞的较浓水相自动分配到含有第二浸没相的每个孔中。液滴限制细胞并保持与浸没相不混溶，以促进细胞聚集成尺寸明确的紧凑CCS。重要的是，培养基的覆盖提供了营养物质，并最大限度地减少了众所周知的蒸发问题和其他测定中培养基渗透压摩尔浓度的变化。生成384个球体的整个过程是自动完成的，并且在单个步骤中完成。CCS的形成和维护前所未有的容易，并且与高通量筛选实验室中的标准设备完全兼容，使得这种微技术随时可供学术界和工业界的研究人员使用。我们预计，这种微技术将使药物测试和筛选与三维肿瘤模型的常规实验室技术之前，昂贵和繁琐的体内分析。此外，它将大大提高测试吞吐量和成本效益（增加测试化合物的数量和减少试剂消耗）和效率（减少动手时间），以加快药物发现。我们将通过两个具体目标来实现我们的目标：（i）用水性双相系统生成癌细胞球状体;（ii）用于化合物测试的ATPS球状体的初步验证。

DESCRIPTION (provided by applicant): Two-dimensional (2D) cultures of cancer cells are routinely used in drug discovery for screening and initial characterization of the efficacy of librry of potential drug compounds. Despite their simplicity and compatibility with high throughput screening instruments, 2D cell assays often fail to predict the efficacy of compounds in vivo, making drug development and discovery an extremely costly process. Disparity between 2D cultures and the complex 3D environment of cancer cells in vivo is the major shortcoming of monolayer culture systems. Development of novel chemotherapeutics requires compound screening against malignant tumor cells in a setting that resembles the 3D tumor environment. Cancer cell spheroids (CCS) are 3D clusters of malignant cells that are regarded as physiologic models of solid tumors; they possess similar metabolic and proliferative gradients to avascular tumors and exhibit the clinical expression profiles of solid tumors. Despite the inherent power of CCS to predict clinical efficacy of drugs, incorporation of CCS into the mainstream drug development process is severely hindered by complex and expensive methodological requirements for the formation and maintenance of CCS. We overcome the limitations of existing techniques by developing a technological platform to generate spheroids of consistent size in standard 384-microwell plates using an aqueous two- phase system (ATPS). A drop of the denser aqueous phase containing cancer cells is robotically dispensed into each well containing the second, immersion phase. The drop confines cells and remains immiscible from the immersion phase to facilitate aggregation of cells into a compact CCS of well-defined size. Importantly the overlay of culture media provides nutrients and minimizes the well-known problem of evaporation and changes in osmolality of media as in other assays. The entire process of generating 384 spheroids is done robotically and in a single step. The unprecedented ease of formation and maintenance of CCS and full compatibility with standard equipment in high throughput screening laboratories makes this microtechnology readily available to the researchers in academia and industry. We anticipate that this microtechnology will make drug testing and screening with 3D tumor models a routine laboratory technique prior to expensive and tedious in vivo analyses. In addition, it will dramatically improve testing throughput and cost-effectiveness (increasing numbers of tested compounds and reduced reagent consumption) and efficiency (reducing hands-on time) to expedite drug discovery. We will accomplish our goals through two specific aims: (i) Generation of cancer cell spheroids with aqueous biphasic systems; (ii) Initial validation of ATPS spheroids for compound testing.