Zlochiver Viviana, Kroboth Stacie L, Beal Christopher R, Cook Jonathan A, Joshi-Mukherjee Rosy
Aurora Research Institute, Advocate Aurora Health Care.
Aurora Research Institute, Advocate Aurora Health Care; Department of Biomedical Engineering, College of Engineering and Applied Science, University of Wisconsin-Milwaukee; Department of Medicine-Cardiovascular, School of Medicine, Johns Hopkins University;
J Vis Exp. 2019 Jul 15(149). doi: 10.3791/59906.
Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 µs. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.
心脏安全性筛查对于药物研发和治疗至关重要。因此，为了高效进行药物测试，迫切需要开发用于人诱导多能干细胞衍生心肌细胞（hiPSC-CM）制剂的新型高通量电生理方法。尽管多电极阵列（MEA）常用于可兴奋细胞的场电位测量，但Joshi-Mukherjee及其同事最近发表的一篇文章描述并验证了其在数天内对同一hiPSC-CM制剂进行复发性动作电位（AP）记录的应用。本文旨在提供详细的分步方法，用于接种心肌细胞以及通过电穿孔以1微秒的高精度和时间分辨率测量AP波形。这种方法解决了缺乏易于使用的方法来实现细胞内通路以进行高通量AP测量以进行可靠电生理研究的问题。讨论了在多孔MEA板上接种hiPSC-CM的详细工作流程和方法，并在相关处强调了关键步骤。此外，还报告了一个定制的MATLAB脚本用于快速数据处理、提取和分析，以全面研究波形分析，量化与心律失常和心脏毒性相关的各种AP持续时间参数在形态学上的细微差异。
