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.

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.

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), erlotinib induced a statistically insignificant decrease in amplitude at the first measurement after compound application (1 h). In all subsequent measurements, no comparable effect was measured at this concentration. The transient decrease could be the result of a compound effect, but also result from a transient distortion of the beat pattern during the compound addition procedure, for example, of thermal or mechanical nature. Micromolar concentrations of erlotinib showed slight but significant time and dose-dependent cardiotoxic effects. The onset of the effect was seen from 96 h with 1 µM erlotinib, while 10 µM resulted in a significant decrease only 24 h after compound addition.

Chemotherapy agent epirubicin had both a time- and dose-dependent effect on hiPSC-CMs (Figure 2). The cells ceased beating within 24 h after the application of 10 µM epirubicin. With 1 µM, a drastic decrease in amplitude to 44% ± 2% of control after 24 h was followed by complete beat cessation until 48 h after compound addition. At 100 nM, a time-dependent decrease in beat amplitude over a period of 5 days was observed with a residual amplitude of 25% ± 3% on day 5. At the lowest concentration of 10 nM, the effect of epirubicin was only dose-dependent but not time-dependent, starting at the first measurement (1 h after compound addition). The amplitude fluctuated steadily between 60%-80% of control over the period of 5 days. The deviations in this group were larger compared to the higher concentrations. One possible reason could be the fact that this concentration had a measurable effect only on a portion of the wells.

Doxorubicin is another anthracycline class of medication, and the cell vitality of hiPSC-CMs was investigated by monitoring the base impedance over time. Figure 3 shows that a 24 h exposure to 300, 1, 3, and 10 µM doxorubicin decreases the cell viability in a concentration- and time-dependent manner.

Nifedpinie is a dihydropyridine calcium channel blocker that primarily blocks L-type calcium channels. Long-term monitoring over 24 h of cell viability reveals a time- and concentration-dependent decrease of base impedance which is used as a measure of toxicity (Figure 4A). Upon application of increasing nifedipine concentrations (3 nM, 10 nM, 30 nM, 100 nM), field potential recordings on hiPSC-CMs reveal a concentration-dependent shortening of the field duration normalized (FPD) as expected (Figure 4B,C). Nifedipine assessment regarding cardiac contractility also showed a significant concentration-dependent amplitude decrease upon acute measurement with 10 nM and 30 nM concentrations (Figure 5).

The results obtained with the hybrid cell analysis system demonstrate that three cardiac endpoints (contractility, structure, and electrophysiology) of hiPSC-CMs can be assessed using one system.

Figure 1: Contractility assessment of human iPSC-derived cardiomyocytes treated with erlotinib for 5 days. The x-axis shows time in hours, y-axis plots parameter amplitude in terms of percentage. Asterisks represent statistical significance with p < 0.05 (*) or p < 0.01 (**) (Wilcoxon Mann Whitney test, n = 4). Please click here to view a larger version of this figure.

Figure 2: Contractility assessment of human iPSC-derived cardiomyocytes treated with cardiotoxic anthracycline epirubicin over 5 days. The x-axis shows time in hours, y-axis plots parameter amplitude in terms of percentage. Asterisks represent statistical significance with p < 0.05 (*) or p < 0. 01 (**) (Wilcoxon Mann Whitney test, n = 4). Please click here to view a larger version of this figure.

Figure 3: Base impedance of human iPSC-derived cardiomyocytes after doxorubicin treatment. Time course of the base impedance of human iPSC-derived cardiomyocytes for 24 h exposure to 300, 1, 3, and 10 µM doxorubicin (n = 5).  Please click here to view a larger version of this figure.

Figure 4: Impedance and EFP recordings of human iPSC-derived cardiomyocytes. (A) Time course of the base impedance of human iPSC-derived cardiomyocytes for 24 h, upon exposure to 3, 10, 30, and 100 nM nifedipine (n = 5). (B) Time course of the field potential duration of human iPSC-derived cardiomyocytes for 1 h, upon exposure to 3, 10, 30, and 100 nM nifedipine (n = 5). (C) Electrode layout of the impedance/EFP plate. Please click here to view a larger version of this figure.

Figure 5: Contractility assessment (contractility module) of human iPSC-derived cardiomyocytes treated with nifedipine for 20 min. The x-axis shows time in min, Y-axis plots parameter amplitude in terms of percentage. Asterisks represent statistical significance with p < 0. 05 (*) or p < 0.01 (**) (Wilcoxon Mann Whitney test, n = 4). Please click here to view a larger version of this figure.

Supplementary Figure 1: Contractility parameters and raw data assessed with the contractility module. (Left) Parameters assessed with the contractility module. Parameters: Amplitude of contraction force (mN/mm²), beat duration, upstroke, and downstroke velocity, upstroke, and downstroke area under curve (AUC), beat rate, beat rate variations, arrhythmic events. (Right) Contractility raw data recordings of one well with untreated cardiomyocyte (A) beat rate, (B) beat rate variations, (C) early after contractions, and (D) arrhythmic events. Please click here to download this File.

Supplementary Figure 2: Workflow for contractility and impedance/EFP measurement. Please click here to download this File.

Supplementary Figure 3: Example layout of a measurement plan for a 96-well plate. Four compounds (highlighted in red, light green, brown, and dark green) with four concentrations and four replicates are depicted. Positive and negative controls (e.g., pre-culture conditions and DMSO) are highlighted in yellow. Backup cells are highlighted in blue. Please click here to download this File.

Supplementary Figure 4: Example data of a contractility baseline measurement using the contractility module before compound addition. In each chart the average of all contraction cycles of one sweep is depicted. To visualize the beat rate, two consecutive contractions are shown. Please click here to download this File.

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 functional and structural toxicity assessment.

As an animal-free approach, human iPSC-CMs are employed for the hybrid system²⁰. Here, the special flexible 96-well plates with the pro-maturation effect on human iPSC-CMs provide an important advantage in comparison to other cell-based assays^21,22, since throughput and a physiological environment are uniquely combined for more adult-like human cardiac risk assessment^6,15.

The most critical step in the protocol is the application of the compound, especially when acute effects are to be examined. Since the cells are cultured on soft substrates which conduct mechanical forces, excessive acceleration during plate handling and application of shear forces during pipetting can result in transient (5-10 min) alterations of the contraction parameters and should be avoided. In general, care should be taken during media replacements to avoid disruption of the membranes. The use of lab automation equipment is recommended.

Before the compound analysis is performed, a pre-culture step of 5-7 days is required, so that human iPSC-CMs, usually acquired in a cryo-preserved state, can recover from the thawing procedure and build a proper syncytium. For both modules, the contraction amplitude, as well as the beating rate, give insight into the ideal starting point of the measurement that usually occurs between days 5-7. The baseline measurement is a critical step to identify functional baseline characteristics used as inclusion criteria for the hiPSC-CMs studied³. The baseline values need to be recorded right before compound treatment so that changes in contraction behavior can be compared to non-treatment conditions (Supplementary Figure 3).

Accounting for variabilities and having defined baseline characteristics is key to successful measurements of hiPSC-CM and data interpretation. For this reason, best practice recommendations from Gintant et al., are followed using the hybrid cell analysis system; for instance, the baseline is to be recorded prior to the compound addition, and the baseline recording should fulfill certain prerequisites³.

Although the flexible 96-well plates are equipped with fragile membranes, a provided protector plate in combination with cautious handling will prevent damage. The membranes are pre-treated to enable stable attachment of cells to the silicone substrate, which has low intrinsic biocompatibility. The treatment is stable for at least 6 months. While the cells are constantly maintained at 37 ˚C, the mechanical properties of silicone materials are stable over a broad range of temperatures. Additionally, neither drugs nor solvents impact the silicone's properties at concentrations used in cell-based assays (e.g., DMSO concentrations remain below 0.1%).

The system was validated and optimized with a broad range of commercially available hiPSC-CMs. When using custom-made cells, testing standard extracellular matrix (ECM) proteins for optimal cell attachment is recommended (e.g., fibronectin, EHS matrix, and poly-L-lysine^6,15).

Electrophysiology, calcium signaling, and contractility are the three main cardiac endpoints being addressed in preclinical development. The hybrid system is currently limited to analyzing two of these cardiac endpoints-contractility and electrophysiology. Calcium signaling in cardiomyocytes cannot be analyzed directly but detected tangentially via contractility and electrophysiological properties.

Both the impedance/EFP as well as the contraction force measurement are performed with monolayers of cardiomyocytes. Despite the advantage of a physiological mechanical substrate stiffness during contraction measurement, the cells do not experience the three-dimensional environment of real human tissue. On the other hand, this allows the use of only a fraction of the expensive stem cell-derived cell models with standard laboratory equipment. Hence, this application promises to have a favorable relation of cost, robustness, and predictivity compared to in vivo/ex vivo or 3D models. Future applications of the hybrid system may include the analysis of other contractile cell types such as smooth muscle cells. In the regulation of cardiovascular homeostasis, these cells play a major role as counterparts of cardiomyocytes. The hybrid system also provides add-ons for specific projects, such as optical stimulation of human iPSC-CMs. For this purpose, a dedicated optical lid can be applied to both modules for the stimulation of human iPSC-CMs that were transfected with Channelrhodopsin-2 during preculture times.

Thus, the impedance/EFP/contractility hybrid system allows for modern preclinical safety and toxicity assessment by analyzing three different cardiac endpoints (contractility, structural changes, and electrophysiology) within one methodology. The advantage of addressing these endpoints with a human-based cardiac cell model on a high throughput level lifts this hybrid system beyond the current perspectives of preclinical cardiac risk assessment.