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 cardiomyocytes (CMs) of induced pluripotent stem cell (iPSC) origin became the workhorse over the past decade for the modern assessment of safety pharmacological and toxicological issues². Widely used assay systems for such investigations are microelectrode array (MEA) and voltage-sensitive dye-based experimental approaches³.

Nevertheless, the claimed phenotypic and functional immaturity of this cell type puts obstacles in the way of an ideal human-based cell model, with the potential to reduce translational gaps between non-clinical and clinical studies⁴.

Tremendous research has been conducted over the years to understand the reason for the implied immature phenotype and to find ways to push the maturation process of human iPSC-CMs in vitro.

Lacking cardiac maturation cues such as prolonged cell culture times, an absence of other cell types in the vicinity, or a lack of hormonal stimulation was shown to affect the maturation process⁵. Also, the non-physiological environment of regular cell culture plates was identified as a significant cause that impedes the maturation of human iPSC-CMs, due to the missing physiological substrate stiffness of the native human heart^5,6.

Different assay systems with a focus on native physiological conditions were developed to tackle this issue, including 3D cell culture systems where cells are aligned three-dimensionally to resemble native cardiac architecture instead of typical two-dimensional cell cultures⁷. Although improved maturation is obtained with 3D assays, the need for a skilled workforce and the low throughput of these systems hampers an abundant use of this in the drug development process, since time and cost play a fundamental role in the assessment of new therapeutics on a financial level⁸.

Important readouts for safety pharmacological and toxicological assessment of new therapeutics are changes in functional and structural characteristics of human iPSC-CMs, since compound-induced adverse drug reactions of the cardiovascular system usually affect one or both of these properties^1,9. Well-known examples of such broad adverse reactions are anti-cancer drugs of the anthracycline family. Here, hazardous functional and adverse structural effects on the cardiovascular system are widely reported during and after cancer treatment in patients as well as with in vitro cell-based assays^10,11.

In the present study, we describe a comprehensive methodology for the assessment of both functional and structural compound side effects on hiPSC-CMs. The methodology includes the analysis of cardiomyocyte contractile force and impedance/Extracellular Field Potential (EFP) analysis. The contractile force is measured under physiological mechanical conditions, with the cells cultured on soft (33 kPa) silicone substrates, reflecting the mechanical environment of native human heart tissue.

The system is equipped with 96-well plates for high throughput analysis of human iPSC-CMs for preclinical cardiac safety pharmacological and toxicological studies, and thus provides an advantage to currently used 3D approaches like Langendorff heart or heart slices^12,13.

In detail, the hybrid system consists of two modules, either for the assessment of cardiac contractility under physiological conditions or the analysis of real-time cellular structural toxicity^6,14. Both modules work with specialized high throughput 96-well plates for fast and cost-effective data acquisition.

Without the need for a 3D construct, the contractility module employs special plates that contain flexible silicone membranes as the substrate for the cells instead of the stiff glass or plastic that regular cell culture plates usually consist of. The membranes reflect typical human biomechanical heart properties and therefore mimic in vivo conditions in a high throughput manner. While human iPSC-CMs often fail to display adult cardiomyocyte behavior regarding compound-induced positive inotropy in other cell-based assays¹⁴, a more adult-like reaction can be assessed when the cells are cultured on the plates of the contractility module. In previous studies, it has been demonstrated that iPSC-CMs exhibit positive inotropic effects upon treatment with compounds such as isoproterenol, S-Bay K8644, or omecamtiv mecarbil^6,15. Here, multiple contractility parameters can be assessed, such as primary parameters like the amplitude of contraction force (mN/mm²), beat duration, and beat rate, as well as secondary parameters of the contraction cycle like area under the curve, contraction, and relaxation slopes, beat rate variations, and arrhythmias (Supplementary Figure 1)¹⁶. Drug-induced changes in all parameters are assessed non-invasively by capacitive distance sensing. The raw data is analyzed subsequently by specialized software.

The structural toxicity module adds its unique impedance and EFP parameters as a readout for structural cellular toxicity and the analysis of electrophysiological properties^17,18. The electrical impedance spectroscopy technology reveals compound-induced changes in cell density or cell and monolayer integrity monitored in real-time, as shown with human iPSC-CMs treated with known cardiotoxic compounds¹³. With impedance readouts at different frequencies (1-100 kHz) it is possible to dissect a physiological response further, and thus revealing changes in membrane topography, cell-cell, or cell-matrix junctions is achievable. The additional EFP recording of human iPSC-CMs further enables the analysis of electrophysiological effects elicited by compound treatment, as was shown in the light of the CiPA study^17,19.

In the present study, human iPSC-CMs were employed, treated with epirubicin and doxorubicin, both well-described as cardiotoxic anthracyclines, and erlotinib, a tyrosine kinase inhibitor (TKI) with a rather low risk of cardiovascular toxicity. Chronic assessment with epirubicin, doxorubicin, and erlotinib was performed for 5 days. The result shows minor changes in contractility and base impedance when cells were treated with erlotinib, but a time and dose-dependent toxic decrease in contraction amplitude and base impedance when treated with epirubicin and doxorubicin respectively. Acute measurements were performed with calcium channel blocker nifedipine and show a decrease in contraction amplitude, field potential duration, and base impedance, demonstrating cardiotoxic side effects of this compound on functional as well as structural levels.

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 8.25 mL of DPBS with Ca²⁺ and Mg²⁺. Mix the solution carefully.
      NOTE: Optionally, fibronectin can also be used for coating the wells: prepare 13 mL of fibronectin coating solution in a sterile centrifuge tube by diluting 650 µL of fibronectin stock solution (1 µg/mL) in 13 mL of DPBS with Ca²⁺ and Mg²⁺, resulting in a 50 µg/mL working solution. Mix the solution carefully.
3. Transfer the coating solution into a sterile reagent reservoir placed in the lab automation robot.
4. Add 100 µL of the coating solution per well with the lab automation robot by using the program "ADD100µL". Place the lid back onto the 96-well plate and incubate for 3 h at 37 ˚C.
   ​NOTE: The program for the lab automation robot needs to be preset manually beforehand.

2. Seeding of human iPSC-derived cardiomyocytes into flexible 96-well plates (Day 0)

1. Thaw the cells according to manufacturer's guidelines.
2. Count the cells with a manual counting chamber and adjust the cells in the recommended plating medium according to the cell manufacturer instructions (e.g., 1 x 10⁵ cells/well), resulting in 11 x 10⁶ cells/11 mL for seeding an entire 96-well plate.
3. Remove the EHS gel solution from the wells with the lab automation robot using the program "REMOVE100µL". Remove the reagent reservoir containing the dispensed coating solution from the robot.
4. Transfer the cell suspension (11 mL total) into a sterile reagent reservoir placed in the lab automation robot and seed the cells with 100 µL/well using the program "CELLS_ADD100µL".
5. Immediately after cell seeding, transfer the flexible 96-well plate into the incubator (37 ˚C, 5% CO₂, humidity-controlled) and let the cells settle overnight.

3. Medium exchange of flexible 96-well plates (Day 1)

1. Warm at least 22 mL of cardiomyocyte maintenance medium per plate to 37 ˚C in a 50 mL centrifuge tube, 18-24 h after seeding the plates.
2. Transfer the fresh medium (at least 22 mL) into a sterile reagent reservoir and leave it right next to the lab automation robot. Place an empty reagent reservoir in the robot and perform medium removal with the program "REMOVE100µL". Afterward, exchange the reagent reservoir containing the waste medium with the reagent reservoir containing the fresh medium and dispense 200 µL of the fresh medium per well with the program "ADD100µL". Perform this step twice to reach 200 µL/well.
3. Immediately after medium exchange, transfer the plate back into the incubator.
4. Perform a medium exchange (200 µL/well) every other day until compound addition.

4. Final medium exchange before compound addition (Day 5-7)

1. Perform a final medium change 4-6 h before compound addition.
2. Warm at least 22 mL of assay buffer for one flexible 96-well plate. The assay buffer consists of maintenance medium or derivatives thereof (e.g., low/no serum media, phenol red free media, or other isotonic buffers).
3. Transfer the fresh medium into a sterile reagent reservoir and leave it right next to the lab automation robot. Place an empty reagent reservoir in the robot and perform medium removal. Afterward, exchange the reagent reservoir containing the waste medium with the reagent reservoir containing the fresh medium and dispense 200 µL/well of the fresh medium.
4. Immediately after medium exchange, transfer the flexible plate back into the incubator.

5. Compound addition and data recording (Day 5-7)

NOTE: An example measurement plan for the experiment is given in Supplementary Figure 3.

1. Prepare working solution per compound at 4x concentration in the laminar flow hood using a sterile regular 96-deep well plate. The compound solution is based on the assay buffer used in step 4. Transfer the 96-deep well plate containing the compound solution for at least 1 h into the incubator to adjust it to the same condition as the flexible plate.
   NOTE: The 1x concentration of each drug used for every experiment is provided in the figures and legends.
2. Transfer the plate to the respective measurement device 1 h before performing a baseline measurement.
3. Open Edit Protocol in the control software (part of the hybrid cell analysis system) and select the respective measurement mode contractility or impedance/EFP.
4. Define the sweep duration (length of one measurement; e.g., 30 s) and the repetition interval (time between measurements; e.g., 5 min) and save the protocol number.
5. Select Start protocol > Continue and fill in the requested fields.
6. Finally, select Start measurement. Perform a minimum of three baseline measurements (sweeps) in 5 min intervals shortly before compound addition.
   NOTE: Example data of a contractility baseline measurement using the contractility module before compound addition is depicted in Supplementary Figure 4
7. Remove 50 µL of the assay buffer from each well without removing the flexible 96-well plate from the measurement device.
8. Add 50 µL of the 4x concentrated compound solution into each well of the plate, according to the measurement plan.
9. Select Add region marker and define the compound plate layout and the volume of the compound solution after compound addition.
10. Finally, select Proceed with standard measurement or Proceed with measurement series according to the experimental plan.

6. Data analysis

1. With recording software, measure sweeps, whose length and repetition interval are defined by the user.
2. With analysis software, capture the shape of the signal by reading out parameters like amplitude, beat rate, pulse width, and so forth, automatically.
   NOTE: An averaged signal including the standard deviation, the so-called mean beat, is automatically calculated based on the data of one sweep. The user can define the contractility/IMP/EFP parameters that the software calculates and display.
3. With analysis software calculate the dose-response curve and IC50/EC50 for each compound.
   NOTE: The raw data and the analysis results generated with the analysis software can be easily exported in a variety of formats. Finally, the data reports are automatically generated to summarize and archive the experimental results. A comprehensive description of what and how an EFP signal is measured is discussed in ¹⁷.

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