A Frequency-multiplexed flow cytometer for high throughput screening and drug discovery

用于高通量筛选和药物发现的频率复用流式细胞仪

• 批准号: 8970595
• 负责人: Dino Di Carlo
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8970595
• 关键词: Accounting; Address; Agreement; Air; Apoptosis; Area; Biological Assay; Calibration; Camptothecin; Cell Communication; Cells; Cellular biology; Collection; Color; Complex; Computer software; Coupled; Cytometry; Data; Detection; Disease; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescence; Frequencies; Goals; Individual; Information Systems; Jurkat Cells; Laboratories; Lateral; Libraries; Light; Liquid substance; Literature; Marketing; Measurement; Measures; Microfluidics; Molecular Bank; Monitor; Nature; Noise; Optics; Patients; Performance; Pharmaceutical Preparations; Population; Positioning Attribute; Preclinical Drug Evaluation; Process; Propidium Diiodide; Protocols documentation; Pump; Reader; Running; Sampling; Scheme; Shapes; Side; Speed; Staging; Staurosporine; Stream; Syringes; System; Techniques; Technology; Testing; Time; Tube; United States National Institutes of Health; annexin A5; base; cost; cytotoxicity; design; detector; drug development; drug discovery; fluorescence imaging; high throughput screening; instrument; optical emission; particle; photomultiplier; photonics; public health relevance; radio frequency; radiofrequency; repository; response; screening; small molecule; success; time use

 DESCRIPTION: Drug discovery is an extremely lengthy and expensive process. On average, drugs today cost more than $5 billion to develop, and take more than a decade to reach the market. Most drugs today are discovered using high throughput screening, in which millions of trial compounds are assayed against cells to determine if they interact with a disease target. This is typically performed using well-level screening techniques, such as fluorescence, chemiluminescence, and optical absorbance. While these techniques are high throughput, they lack the ability to interrogate wells at the single-cell level, which provides a much more comprehensive picture of the drug-cell interaction than population-averaged measurements. Flow cytometry is a well established and widely used technique used in most areas of cell biology that provides multi-parameter, cell-level information by measuring optical scatter and fluorescence information from individual cells in flow at a high throughput (~10,000 cells/second). While this cellular throughput is high, the sample throughput of flow cytometers is relatively low, when compared to fluorescence plate readers, for example, due to the serial sample handling approach typically employed by modern flow cytometers. If the sample throughput could be increased to appropriate levels, it would enable multi-parameter drug screening assays using flow cytometry, which would obviate certain further downstream assays in the drug discovery workflow (e.g. cytotoxicity assays). This improvement would increase the efficiency of drug discovery by better elucidating the complex drug-cell interactions, reducing the overall cost and time-to-market of new drugs. We propose here to develop a parallel flow cytometer system using a combination of two technologies developed in the laboratories of the co-PI's. Fluorescence Imaging using Radiofrequency-tagged Emission (FIRE) is a high-speed optical technique that enables a single photomultiplier tube detector to measure fluorescence or scatter from multiple points on a sample using radiofrequency-domain multiplexing. We will use a modified FIRE optical system to probe fluorescence and scatter from cells flowing in multiple parallel intertially focused streams, created by an Inertial Microfluidic Parallel Stream (IMPS) microfluidic chip. IMPS chips create ordered streams of cells using inertial flow field shaping without the complexity of multiple sheath fluids to direct cells, allowing high numerical aperture optics to detect fluorescence from all streams simultaneously. Combining these techniques, we will develop an optical and fluidic system capable of measuring multi-color flow cytometry data from 10 samples of cells at the same time. This order of magnitude increase in the sample throughput will transform the utility of flow cytometry in drug discovery, ultimately enabling its widespread use in high throughput screening (>100,000 wells/day). We will characterize the system (precision, linearity, sensitivity) using standard protocols, and perform a basic two-color apoptosis time course assay and compare our results in this assay to those obtained using a conventional commercial flow cytometer.

 描述：药物发现是一个极其漫长且昂贵的过程。平均而言，当今药物的开发成本超过 50 亿美元，并且需要十多年才能进入市场。如今，大多数药物都是通过高通量筛选发现的，其中对数百万种试验化合物进行针对细胞的分析，以确定它们是否与疾病靶标相互作用。这通常使用荧光、化学发光和光学吸光度等良好水平的筛选技术来进行。虽然这些技术具有高通量，但它们缺乏在单细胞水平上询问孔的能力，这提供了比群体平均测量更全面的药物-细胞相互作用的图像。 流式细胞术是一种成熟且广泛使用的技术，用于细胞生物学的大多数领域，通过以高通量（约 10,000 个细胞/秒）测量流中单个细胞的光学散射和荧光信息来提供多参数、细胞水平信息。虽然细胞通量很高，但与荧光读板机相比，流式细胞仪的样品通量相对较低，例如，由于现代流式细胞仪通常采用的串行样品处理方法。如果样品通量可以增加到适当的水平，则可以使用流式细胞术进行多参数药物筛选测定，这将避免药物发现工作流程中的某些进一步的下游测定（例如细胞毒性测定）。这一改进将通过更好地阐明复杂的药物-细胞相互作用来提高药物发现的效率，减少 新药的总体成本和上市时间。 我们在此建议结合联合 PI 实验室开发的两种技术来开发并行流式细胞仪系统。使用射频标记发射的荧光成像 (FIRE) 是一种高速光学技术，使单个光电倍增管探测器能够使用射频域多路复用测量样品上多个点的荧光或散射。我们将使用改进的 FIRE 光学系统来探测在多个平行惯性聚焦流中流动的细胞的荧光和散射，这些流是由惯性微流控并行流 (IMPS) 微流控芯片创建的。 IMPS 芯片使用惯性流场整形创建有序的细胞流，无需复杂的多种鞘液来引导细胞，从而允许高数值孔径光学器件同时检测来自所有流的荧光。结合这些技术，我们将开发一种光学和流体系统，能够同时测量 10 个细胞样本的多色流式细胞术数据。样品通量的这一数量级增加将改变流式细胞术在药物发现中的效用，最终使其在高通量筛选（> 100,000 孔/天）中得到广泛应用。我们将使用标准方案表征系统（精度、线性、灵敏度），并进行基本的双色细胞凋亡时间过程测定，并将该测定中的结果与使用传统商业流式细胞仪获得的结果进行比较。