Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Summary

The analysis of changes in contractile function and cellular integrity of human iPSC-derived cardiomyocytes is of immense importance for nonclinical drug development. A hybrid 96-well cell analysis system addresses both parameters in a real-time and physiological manner for reliable, human-relevant results, necessary for a safe transition into clinical stages.

Abstract

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform.

The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm²) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling.

The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

Introduction

One of the major goals of modern drug development is the improvement of the bench-to-bedside success rate of new therapeutics in the drug discovery pipeline. Safety pharmacological testing of these new drugs often reveals adverse drug reactions on the cardiovascular system that accounts for almost one-quarter of the drug attrition rate at preclinical stages¹. The development and integration of new approach methodologies (NAMs) play a key role in the modernization of preclinical assessment, in particular core battery organs like the heart. Since these methodologies are animal-free approaches, the use of human-based cell models like cardiomyocyte....

Protocol

NOTE: The workflow for contractility and impedance/EFP measurement is given in Supplementary Figure 2.

1. Plate coating

1. Open the vacuum-sealed packaging and take out the 96-well plate. Handling procedures for 96-well plates of both modules are the same. Leave the contraction plate covered by the additionally supplied membrane guard until measurement in the contractility module.
2. Coat the flexible 96-well plates for seeding cardiomyocytes.
   1. Prepare a diluted EHS gel coating solution by transferring 2.75 mL of EHS gel ready-to-use solution in a sterile centrifuge tube. Then add....

Results

The effects of kinase inhibitor erlotinib on the contractility of hiPSC-CMs are shown in Figure 1. The cells were treated with concentrations ranging from 10 nM to 10 µM for 5 days and beat parameters were recorded daily. Erlotinib, an EGFR (epidermal growth factor receptor) and tyrosine kinase inhibitor with a comparably low risk of cardiotoxicity, had a minor dose and time-dependent effect on hiPSC-CMs only at concentrations in the micromolar range. At the lowest concentration (10 nM), erl.......

Discussion

The impedance/EFP/contractility hybrid system is a comprehensive methodology for high throughput safety pharmacological and toxicological assessment of cardiac liabilities for preclinical drug development. It provides a modern approach for preclinical safety testing without the use of animal models, but with higher throughput capabilities that significantly reduce time and costs. This system has the potential to be used as a complementary approach for the Langendorff Heart and other animal models for preclinical function.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Commercial human iPSC-derived cardiomyocytes	Fujifilm Cellular Dynamics International (FCDI)	R1059
Centrifuge (50 mL tubes)	Thermo Fisher Scientific	15878722
12-channel adjustable pipette (100-1250 μL)	Integra Biosciences	4634
DPBS with Ca2+ and Mg2+	GE Healthcare HyClone	SH304264.01
96 deep well plate	Thermo Fisher Scientific	A43075
EHS gel			Extracellular Matrix Gel
FLEXcyte 96/CardioExcyte hybrid device	Nanion Technologies	19 1004 1005	Hybrid cell analysis system
FLX-96 FLEXcyte Sensor Plates	Nanion Technologies	20 1010
Fibronectin stock solution (Optional to Geltrex)	Sigma Aldrich	F1141
Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix	ThermoFischer Scientific	A1569601
Human iPSC-derived cardiomyocytes plating and maintenance medium	FCDI	R1059
Incubator (37 °C, 5% CO2)	Thermo Fisher Scientific	51023121
Laminar Flow Hood	Thermo Fisher Scientific	51032678
NSP-96 CardioExcyte 96 Sensor Plates 2.0 mm transparent	Nanion Technologies	20 1011
Pipette tips (1250µL)	Integra Biosciences	94420813
Reagent Reservoir	Integra Biosciences	8096-11
Serological pipette (e.g. 25 mL)	Thermo Fisher Scientific	16440901
Single channel adjustable pipette (e.g. 100-1000 μL)	Eppendorf	3123000063
Vacuum aspiration system	Thermo Fisher Scientific	15567479
Optional: VIAFLO ASSIST	Integra Biosciences	4500	Lab automation Robot
Water bath (37 °C)	Thermo Fisher Scientific	15365877