Sirenko Oksana, Hancock Michael K, Crittenden Carole, Hammer Matthew, Keating Sean, Carlson Coby B, Chandy Grischa
1 Molecular Devices, LLC , Sunnyvale, California.
2 Cellular Dynamics International-A Fujifilm Company , Madison, Wisconsin.
Assay Drug Dev Technol. 2017 Aug/Sep;15(6):280-296. doi: 10.1089/adt.2017.792.
Development of more complex, biologically relevant, and predictive cell-based assays for compound screening is a major challenge in drug discovery. The focus of this study was to establish high-throughput compatible three-dimensional (3D) cardiotoxicity assays using human induced pluripotent stem cell-derived cardiomyocytes. Using both high-content imaging and fast kinetic fluorescence imaging, the impact of various compounds on the beating rates and patterns of cardiac spheroids was monitored by changes in intracellular Ca levels with calcium-sensitive dyes. Advanced image analysis methods were implemented to provide multiparametric characterization of the Ca oscillation patterns. In addition, we used confocal imaging and 3D analysis methods to characterize compound effects on the morphology of 3D spheroids. This phenotypic assay allows for the characterization of parameters such as beating frequency, amplitude, peak width, rise and decay times, as well as cell viability and morphological characteristics. A set of 22 compounds, including a number of known cardioactive and cardiotoxic drugs, was assayed at different time points, and the calculated EC values for compound effects were compared between 3D and two-dimensional (2D) model systems. A significant concordance in the phenotypes was observed for compound effects between the two models, but essential differences in the concentration responses and time dependencies of the compound-induced effects were observed. Together, these results indicate that 3D cardiac spheroids constitute a functionally distinct biological model system from traditional flat 2D cultures. In conclusion, we have demonstrated that phenotypic assays using 3D model systems are enabled for screening and suitable for cardiotoxicity assessment in vitro.
开发更复杂、与生物学相关且具有预测性的基于细胞的化合物筛选检测方法是药物发现中的一项重大挑战。本研究的重点是利用人诱导多能干细胞衍生的心肌细胞建立高通量兼容的三维（3D）心脏毒性检测方法。通过使用高内涵成像和快速动力学荧光成像，利用钙敏染料监测细胞内钙水平的变化，来监测各种化合物对心脏球体搏动率和模式的影响。采用先进的图像分析方法对钙振荡模式进行多参数表征。此外，我们使用共聚焦成像和3D分析方法来表征化合物对3D球体形态的影响。这种表型检测方法能够对诸如搏动频率、幅度、峰值宽度、上升和衰减时间等参数以及细胞活力和形态特征进行表征。在不同时间点对一组22种化合物（包括一些已知的具有心脏活性和心脏毒性的药物）进行了检测，并比较了3D和二维（2D）模型系统中化合物效应的计算EC值。两种模型之间在化合物效应的表型上观察到显著的一致性，但在化合物诱导效应的浓度反应和时间依赖性方面观察到了本质差异。总之，这些结果表明3D心脏球体构成了一个与传统的二维平面培养在功能上不同的生物模型系统。总之，我们已经证明使用3D模型系统的表型检测方法可用于筛选且适用于体外心脏毒性评估。
