Tahara Haruna, Matsuda Shun, Yamamoto Yusuke, Yoshizawa Hiroe, Fujita Masaharu, Katsuoka Yasuhiro, Kasahara Toshihiko
Safety Evaluation Center, Fujifilm Corporation, Kanagawa, Japan.
Safety Evaluation Center, Fujifilm Corporation, Kanagawa, Japan.
J Pharmacol Toxicol Methods. 2017 Nov;88(Pt 1):92-99. doi: 10.1016/j.vascn.2017.08.003. Epub 2017 Aug 12.
Various cytotoxicity assays measuring indicators such as enzyme activity, dye uptake, or cellular ATP content are often performed using 96-well microplates. However, recent reports show that cytotoxicity assays such as the ATP assay and MTS assay underestimate cytotoxicity when compounds such as anti-cancer drugs or mutagens induce cell hypertrophy whilst increasing intracellular ATP content. Therefore, we attempted to evaluate the reliability of a high-content image analysis (HCIA) assay to count cell number in a 96-well microplate automatically without using a cell-number indicator.
We compared cytotoxicity results of 25 compounds obtained from ATP, WST-8, Alamar blue, and HCIA assays with those directly measured using an automatic cell counter, and repeating individual experiments thrice.
The number of compounds showing low correlation in cell viability measured using cytotoxicity assays compared to automatic cell counting (r<0.8, at least 2 of 3 experiments) were follows: ATP assay; 7; WST-8 assay, 2; Alamar blue assay, 3; HCIA cytotoxicity assay, 0. Compounds for which correlation was poor in 3 assays, except the HCIA assay, induced an increase in nuclear and cell size. However, correlation between cell viability measured by automatic cell counter and the HCIA assay was strong regardless of nuclear and cell size. Additionally, correlation coefficients between IC values obtained from automatic cell counter and from cytotoxicity assays were as follows: ATP assay, 0.80; WST-8 assay, 0.84; Alamar blue assay, 0.84; and HCIA assay, 0.98.
From the above, we showed that the HCIA cytotoxicity assay produces similar data to the automatic cell counter and is highly accurate in measuring cytotoxicity.
各种细胞毒性测定方法，如测量酶活性、染料摄取或细胞ATP含量等指标，通常使用96孔微孔板进行。然而，最近的报告显示，当抗癌药物或诱变剂等化合物诱导细胞肥大同时增加细胞内ATP含量时，诸如ATP测定和MTS测定等细胞毒性测定会低估细胞毒性。因此，我们试图评估一种高内涵图像分析（HCIA）测定法在不使用细胞计数指标的情况下自动计数96孔微孔板中细胞数量的可靠性。
我们将从ATP、WST-8、alamar蓝和HCIA测定中获得的25种化合物的细胞毒性结果与使用自动细胞计数器直接测量的结果进行比较，并将每个实验重复三次。
与自动细胞计数相比，使用细胞毒性测定法测得的细胞活力相关性较低的化合物数量（r<0.8，至少3次实验中的2次）如下：ATP测定法，7种；WST-8测定法，2种；alamar蓝测定法，3种；HCIA细胞毒性测定法，0种。除HCIA测定法外，在3种测定法中相关性较差的化合物会导致细胞核和细胞大小增加。然而，无论细胞核和细胞大小如何，自动细胞计数器测得的细胞活力与HCIA测定法之间的相关性都很强。此外，自动细胞计数器和细胞毒性测定法获得的IC值之间的相关系数如下：ATP测定法，0.80；WST-8测定法，0.84；alamar蓝测定法，0.84；HCIA测定法，0.98。
综上所述，我们表明HCIA细胞毒性测定法产生的数据与自动细胞计数器相似，并且在测量细胞毒性方面高度准确。
