McPheeters Matthew T, Wang Yves T, Werdich Andreas A, Jenkins Michael W, Laurita Kenneth R
Pediatrics, Case Western Reserve University, Cleveland, Ohio, United States of America.
Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, United States of America.
PLoS One. 2017 Aug 24;12(8):e0183761. doi: 10.1371/journal.pone.0183761. eCollection 2017.
Human cardiac myocytes derived from pluripotent stem cells (hCM) have invigorated interest in genetic disease mechanisms and cardiac safety testing; however, the technology to fully assess electrophysiological function in an assay that is amenable to high throughput screening has lagged. We describe a fully contactless system using optical pacing with an infrared (IR) laser and multi-site high fidelity fluorescence imaging to assess multiple electrophysiological parameters from hCM monolayers in a standard 96-well plate. Simultaneous multi-site action potentials (FluoVolt) or Ca2+ transients (Fluo4-AM) were measured, from which high resolution maps of conduction velocity and action potential duration (APD) were obtained in a single well. Energy thresholds for optical pacing were determined for cell plating density, laser spot size, pulse width, and wavelength and found to be within ranges reported previously for reliable pacing. Action potentials measured using FluoVolt and a microelectrode exhibited the same morphology and rate of depolarization. Importantly, we show that this can be achieved accurately with minimal damage to hCM due to optical pacing or fluorescence excitation. Finally, using this assay we demonstrate that hCM exhibit reproducible changes in repolarization and impulse conduction velocity for Flecainide and Quinidine, two well described reference compounds. In conclusion, we demonstrate a high fidelity electrophysiological screening assay that incorporates optical pacing with IR light to control beating rate of hCM monolayers.
源自多能干细胞的人类心肌细胞（hCM）激发了人们对遗传疾病机制和心脏安全性测试的兴趣；然而，在适用于高通量筛选的检测方法中，全面评估电生理功能的技术一直滞后。我们描述了一种完全非接触式系统，该系统使用红外（IR）激光进行光学起搏，并结合多部位高保真荧光成像，以评估标准96孔板中hCM单层的多个电生理参数。同时测量多部位动作电位（FluoVolt）或Ca2+瞬变（Fluo4-AM），由此在单个孔中获得传导速度和动作电位持续时间（APD）的高分辨率图谱。确定了细胞接种密度、激光光斑大小、脉冲宽度和波长的光学起搏能量阈值，发现其在先前报道的可靠起搏范围内。使用FluoVolt和微电极测量的动作电位具有相同的形态和去极化速率。重要的是，我们表明，通过光学起搏或荧光激发对hCM造成的损伤最小，就可以准确实现这一点。最后，使用该检测方法，我们证明了hCM对氟卡尼和奎尼丁这两种充分描述的参考化合物表现出复极化和冲动传导速度的可重复变化。总之，我们展示了一种高保真电生理筛选检测方法，该方法结合了红外光光学起搏来控制hCM单层的跳动速率。
