High-Throughput Spheroid Screening Platform

项目总结 该提案将开发高通量球体筛选平台(HTSSP)，以创建完全 研究组织和肿瘤中发生的细胞间相互作用和微环境压力的新工具。是这样的 相互作用在组织内细胞的药物反应中至关重要，但目前的方法不允许 组织状微环境中细胞反应的高通量筛选(HTS)。HTSSP正在被 开发用于使用强大的多细胞椭球3D组织/肿瘤模型系统，该系统具有 在3D药物筛选、真正的高通量筛选(HTS)应用中有许多潜在的应用。至 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)， 连续处理球体等大颗粒样品(目标2)，改进分析方法，以获得 来自球体的空间/结构信息在分析中(目标3)，并展示了 现实世界中的HTSSP检测(目标4)。为了改进流式细胞仪分析，我们将优化我们目前的最高 吞吐量并行流式细胞仪(10xFC)，通过修改流动池和光学元件实现理想的使用 直径在100微米到1000微米之间的颗粒。这款流式细胞仪将与创新的 悬浮培养和采样流控系统创建了HTSSP的硬件。这些流体将保持 在孵化或接触药物期间，在搅拌的悬浮室中放置多个球体样本，然后使用 相同的采样和重采样小室，用于我们的流式细胞仪分析。最终的协同效应 技术成就将是开发改进的数据采集系统，使快速 从椭球体内的探头提取3D位置信息。当与硬件结合时，这将 协同完成HTSSP，用于关键的高通量药物筛选 应用，包括：凋亡/坏死诱导，共培养中每种细胞类型的分数，荧光药物 领悟。作为一个整体，HTSSP将代表着制药领域的重大技术进步 筛选，因为它将提供第一个真正高通量的方法来研究药物相互作用 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.