A Novel High Throughput Tumor Spheroid Microtechnology

描述(由申请人提供)：癌细胞的二维(2D)培养通常用于药物发现，以筛选和初步表征潜在药物化合物文库的有效性。尽管2D细胞分析方法简单，并与高通量筛选工具兼容，但往往无法预测化合物在体内的疗效，使药物开发和发现成为一个极其昂贵的过程。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3D肿瘤环境的环境中对恶性肿瘤细胞进行化合物筛选。癌细胞球体(CCS)是实体肿瘤的生理模型，具有与非血管性肿瘤相似的代谢和增殖梯度，具有实体肿瘤的临床表现。尽管CCS具有预测药物临床疗效的内在力量，但CCS的形成和维护存在复杂而昂贵的方法学要求，严重阻碍了CCS纳入主流药物开发过程。我们克服了现有技术的局限性，开发了一种技术平台，使用双水相系统(ATPS)在标准384微孔板中生成尺寸一致的球体。一滴含有癌细胞的致密水相被自动分配到包含第二个浸泡相的每个井中。液滴限制细胞，并保持与浸泡相不相容，以促进细胞聚集成定义良好大小的致密CCS。重要的是，培养液的覆盖提供了营养，并将众所周知的蒸发和渗透压变化的问题降到最低，就像在其他检测中一样。生成384个椭球体的整个过程是机器人完成的，只需一步。CCS的形成和维护前所未有地容易，并且与高通量筛选实验室中的标准设备完全兼容，这使得学术界和工业界的研究人员随时可以使用这项微技术。我们预计，这项微技术将使3D肿瘤模型的药物测试和筛选成为一项常规的实验室技术，而不是昂贵和乏味的体内分析。此外，它将极大地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)用双水相系统产生癌细胞球体；(Ii)对用于化合物测试的ATPS球体进行初步验证。

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.