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首页> 外文期刊>FEBS Open Bio >Paired image- and FACS-based toxicity assays for high content screening of spheroid-type tumor cell cultures
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Paired image- and FACS-based toxicity assays for high content screening of spheroid-type tumor cell cultures

机译：配对的基于图像和FACS的毒性测定法可用于高含量筛选球形肿瘤细胞培养物

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Novel spheroid-type tumor cell cultures directly isolated from patients' tumors preserve tumor characteristics better than traditionally grown cell lines. However, such cultures are not generally used for high-throughput toxicity drug screens. In addition, the assays that are commonly used to assess drug-induced toxicity in such screens usually measure a proxy for cell viability such as mitochondrial activity or ATP-content per culture well, rather than actual cell death. This generates considerable assay-dependent differences in the measured toxicity values. To address this problem we developed a robust method that documents drug-induced toxicity on a per-cell, rather than on a per-well basis. The method involves automated drug dispensing followed by paired image- and FACS-based analysis of cell death and cell cycle changes. We show that the two methods generate toxicity data in 96-well format which are highly concordant. By contrast, the concordance of these methods with frequently used well-based assays was generally poor. The reported method can be implemented on standard automated microscopes and provides a low-cost approach for accurate and reproducible high-throughput toxicity screens in spheroid type cell cultures. Furthermore, the high versatility of both the imaging and FACS platforms allows straightforward adaptation of the high-throughput experimental setup to include fluorescence-based measurement of additional cell biological parameters.
机译：直接从患者肿瘤中分离出来的新型球状肿瘤细胞培养物比传统生长的细胞系保留了更好的肿瘤特征。但是，这种培养物通常不用于高通量毒性药物筛选。此外，在此类筛选中通常用于评估药物诱导的毒性的测定法通常会测量细胞活力（例如线粒体活性或每个培养孔中的ATP含量）的指标，而不是实际的细胞死亡。这会在测定的毒性值中产生相当大的测定依赖性差异。为了解决这个问题，我们开发了一种可靠的方法，该方法可记录每个细胞而非每个孔的药物诱导的毒性。该方法包括自动配药，然后是基于细胞死亡和细胞周期变化的基于图像和FACS的配对分析。我们显示这两种方法以96孔格式生成高度一致的毒性数据。相比之下，这些方法与经常使用的基于良好分析的方法之间的一致性通常很差。所报道的方法可以在标准的自动显微镜上实施，并为在球状细胞培养物中进行准确且可重现的高通量毒性筛选提供了一种低成本方法。此外，成像和FACS平台的高度多功能性使高通量实验装置的直接适应性得以增强，包括基于荧光的其他细胞生物学参数测量。

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