High-Throughput Spheroid Screening Platform

项目摘要 该提案将开发高分辨率球体筛选平台（HTSSP），以创建一个完全 这是研究组织和肿瘤中细胞间相互作用和微环境压力的新工具。等 相互作用在组织内细胞的药物反应中是关键的，但目前的方法不允许 在组织样微环境中高通量筛选（HTS）细胞应答。HTSSP正在 开发，使强大的多细胞球体3D组织/肿瘤模型系统的使用， 在真正的高通量筛选（HTS）应用中，在3D药物筛选中有许多潜在的应用。到 创建HTSSP，我们正在解决通过流式细胞术进行大颗粒分析的关键限制（目标1）， 大颗粒如球状体的连续样品处理（目标2），改进分析方法， 分析中的球体的空间/结构信息（目标3），并展示 真实的世界测定中的HTSSP（目标4）。为了改善流式细胞术分析，我们将优化我们目前的高 通过修改流动池和光学器件， 直径在100和1000微米之间的颗粒。该流式细胞仪将与创新的 悬浮培养和采样流体系统，以创建HTSSP的硬件。这些射流将保持 在孵育或暴露于药物期间，将多个球状体样品置于搅拌的悬浮液室中，然后使用 相同的腔室用于采样和回收，以供我们的流式细胞仪分析。最后的协同 技术成就将是改进数据采集系统的开发， 从球体内的探头提取3D位置信息。当与硬件结合时，这将 协同完成HTSSP，将用于关键的高通量药物筛选 应用，包括：凋亡/坏死诱导，共培养物中每种细胞类型的分数，荧光药物 摄取。总体而言，HTSSP将代表制药行业的重大技术进步 筛选，因为它将提供第一个真正的高通量方法来研究药物相互作用， 一个3D组织模型

PROJECT SUMMARY This proposal will develop the High-Throughput Spheroid Screening Platform (HTSSP) to create an entirely new tool to study cell-cell interactions and microenvironmental stresses that occur in tissues and tumors. Such interactions are critical in the pharmaceutical response of cells within tissues, but current methods do not allow high throughput screening (HTS) of cellular responses in a tissue-like microenvironment. The HTSSP is being developed to enable the use of the powerful multicellular spheroid 3D tissue/tumor model system, which has many potential applications in 3D drug screening, in true high-throughput screening (HTS) applications. To create the HTSSP, we are addressing critical limitations in large particle analysis via flow cytometry (Aim 1), continuous sample handling of large particles such as spheroids (Aim 2), improving analytical methods to obtain spatial/structural information from spheroids in analysis (Aim 3), and demonstrating key applications of the HTSSP in real world assays (Aim 4). To improve flow cytometry analysis, we will optimize our current high throughput parallel flow cytometer (the 10xFC) by modifying the flow cells and the optics for ideal use with particles between 100 and 1000 microns in diameter. This flow cytometer will be integrated with an innovative suspension culture and sampling fluidic system to create the hardware of the HTSSP. These fluidics will maintain multiple samples of spheroids in stirred suspension chambers during incubation or exposure to drugs, then use the same chambers for sampling and resampling for analysis by our flow cytometer. The final synergistic technical achievement will be the development of improved data acquisition systems that enable the rapid extraction of 3D location information from probes within a spheroid. When combined with the hardware, this will synergistically complete the HTSSP, which will be used for key high-throughput pharmaceutical screening applications, including: apoptosis/necrosis induction, fraction of each cell type in a co-culture, fluorescent drug uptake. Taken as a whole, the HTSSP will represent a significant technological advance in pharmaceutical screening as it will provide the first truly high throughput approach to the study of pharmaceutical interactions in a 3D tissue model.