Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Summary

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

Abstract

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Introduction

Drug development is a long and expensive process. A study of new therapeutic drugs approved by the US Food and Drug Administration (FDA) between 2009 and 2018 reported that the estimated median cost of capitalized research and clinical trials was $985 million per product¹. Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market². Notably, cardiotoxicity is reported among multiple classes of therapeutic drugs³. Therefore, cardiac safety assessment is a crucial component during the drug development process. The current paradigm for cardiac safety assessment ....

Protocol

1. Preparation of media and solutions

1. Prepare hiPSC-CM maintenance medium by mixing a 10 mL bottle of 50x B27 supplement and 500 mL of RPMI 1640 medium. Store the medium at 4 °C and use it within a month. Equilibrate the medium to room temperature (RT) before use.
2. Prepare hiPSC-CM seeding medium by mixing 20 mL of serum replacement and 180 mL of hiPSC-CM maintenance medium (10% dilution, v/v). While freshly prepared seeding medium is preferred, it can be stored at 4 °C for no more than 2 weeks. Equilibrate the medium to RT before use.
3. Prepare extracellular matrix coating solution by thawing one bottle (10 mL) ....

Results

This protocol describes how to measure the contraction motion, field potential, action potential, and Ca²⁺ transient of hiPSC-CMs. A schematic diagram including the enzymatic digestion, cell seeding, maintenance, and functional assay conduction is shown in Figure 1. The formation of the hiPSC-CM monolayer is necessary for the contraction motion measurement (Figure 2B). A representative trace of the contraction-relaxation motion of hiPSC-CMs is shown i.......

Discussion

Human iPSC technology has emerged as a powerful platform for disease modeling and drug screening. Here, we describe a detailed protocol for measuring hiPSC-CM contractility, field potential, action potential, and Ca²⁺ transient. This protocol provides a comprehensive characterization of hiPSC-CM contractility and electrophysiology. These functional assays have been applied in multiple publications from our group^12,13,18

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm glass bottom dish with 20 mm micro-well #1.5 cover glass	Cellvis	D35-20-1.5-N	Patch clamp
50x B27 supplements	Life Technologies	17504-044	hiPSC-CM culture medium
6-well culture plate	E & K Scientific	EK-27160	hiPSC-CM culture
96-well flat clear bottom black polystyrene TC-treated microplates	Corning	3603	Contraction motion measurement
Accutase	Sigma-Aldrich	A6964	Enzymatic dissociation
Axion's Integrated Studio (AxIS)	Axion Biosystems		navigator software
Borosilicate glass capillaries	Harvard Apparatus	BF 100-50-10,	Patch clamp
CaCl2 1 M in H2O	Sigma-Aldrich	21115	Tyrode’s solution
Cell counting chamber slides	ThermoFisher Scientific	C10228	Cell counting
CytoView 48-well MEA plates	Axion Biosystems	M768-tMEA-48B	MEA
DMEM/F12	Gibco/Life Technologies	12634028	Extracellular matrix medium
DPBS, no calcium, no magnesium	Fisher Scientific	14-190-250
EGTA	Sigma-Aldrich	E3889	Intracellular pipette solution
EPC 10 USB patch clamp amplifier	Warner Instruments	89-5000	Patch clamp
Fura-2, AM, cell permeant	ThermoFisher Scientific	F1221	Ca2+ transient measurement
Glucose	Sigma-Aldrich	G8270	Tyrode’s solution
HEPES	Sigma-Aldrich	H3375	Tyrode’s solution
hiPSCs	Stanford Cardiovascular Institute iPSC Biobank
KCl	Sigma-Aldrich	529552	Tyrode’s solution
KnockOut Serum Replacement	ThermoFisher Scientific	10828-028	hiPSC-CM seeding medium
KOH 8 M	Sigma-Aldrich	P4494	Intracellular pipette solution
Lambda DG 4	Sutter Instrument Company		Ca2+ transient measurement; ultra-high-speed wavelength switching light source
Luna-FL automated fluorescence cell counter	WISBIOMED	LB-L20001	Cell counting
Maestro Pro MEA system	Axion Biosystems		MEA
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix	Corning	356231	Extracellular matrix medium
MgATP	Sigma-Aldrich	A9187	Intracellular pipette solution
MgCl2	Sigma-Aldrich	M8266	Tyrode’s solution
NaCl	Sigma-Aldrich	S9888	Tyrode’s solution
NaOH 10 M	Sigma-Aldrich	72068	Tyrode’s solution
NIS Elements AR
Pluronic F-127 (20% Solution in DMSO)	ThermoFisher Scientific	P3000MP	Ca2+ transient measurement
RPMI 1640 medium	Life Technologies	11875-119	hiPSC-CM culture medium
Sony SI8000 Cell Motion Imaging System	Sony Biotechnology		Contraction motion measurement
Sutter Micropipette puller	Sutter Instruments	P-97	Patch clamp
Trypan blue stain	Life Technologies	T10282	Cell counting