Name: hybrid cell analysis system to assess structural and contractile changes of human ipsc-derived cardiomyocytes for preclinical cardiac risk evaluation
Uploaded: 2022-10-20T13:30:00
Description: Watch this Scientific Journal Video about Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation at JoVE.

This work was supported by grants from the German Federal Ministry for Economic Affairs and Climate Action (ZIM) and from the German Federal Ministry of Education and Research (KMUinnovativ). We thank FUJIFILM Cellular Dynamics, Inc (Madison, WI, USA) for kindly providing cardiomyocytes and Ncardia B.V. (Leiden, The Netherlands) for kindly providing cardiomyocytes, used in this study.

NOTE: The workflow for contractility and impedance/EFP measurement is given in Supplementary Figure 2.
1. Plate coating
<ol>
	<li>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.</li>
	<li>Coat the flexible 96-well plates for seeding cardiomyocytes.
...

<ol>
	<li>Weaver, R. J., Valentin, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Today's+challenges+to+de-risk+and+predict+drug+safety+in+human+&amp;#34;mind-the-gap&amp;#34.">Today's challenges to de-risk and predict drug safety in human &amp;#34;mind-the-gap&amp;#34.</a> Toxicological Sciences. 167 (2), 307-321 (2019).</li><li>Burnett, S. D., Blanchette, A. D., Chiu, W. 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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...

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 heart5,6.
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 properties1,9. 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 assays10,11.
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 slices12,13.
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 toxicity6,14. 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 mecarbil6,15. 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 properties17,18. 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 study17,19.
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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Commercial human iPSC-derived cardiomyocytes&#160;</td>
			<td>Fujifilm Cellular Dynamics International (FCDI)</td>
			<td>R1059</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (50 mL tubes)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15878722</td>
			<td></td>
		</tr>
		<tr>
			<td>12-channel adjustable pipette (100-1250 &#956;L)</td>
			<td>Integra Biosciences</td>
			<td>4634</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS with Ca2+ and Mg2+</td>
			<td>GE Healthcare HyClone</td>
			<td>SH304264.01</td>
			<td></td>
		</tr>
		<tr>
			<td>96 deep well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A43075</td>
			<td></td>
		</tr>
		<tr>
			<td>EHS gel</td>
			<td></td>
			<td></td>
			<td>Extracellular Matrix Gel</td>
		</tr>
		<tr>
			<td>FLEXcyte 96/CardioExcyte hybrid device</td>
			<td>Nanion Technologies&#160;</td>
			<td>19 1004 1005</td>
			<td>Hybrid cell analysis system&#160;</td>
		</tr>
		<tr>
			<td>FLX-96 FLEXcyte Sensor Plates</td>
			<td>Nanion Technologies</td>
			<td>20 1010</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fibronectin stock solution (Optional to Geltrex)</td>
			<td>Sigma Aldrich</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix</td>
			<td>ThermoFischer Scientific</td>
			<td>A1569601</td>
			<td></td>
		</tr>
		<tr>
			<td>Human iPSC-derived cardiomyocytes plating and maintenance medium</td>
			<td>FCDI</td>
			<td>R1059</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator (37 &#176;C, 5% CO2)</td>
			<td>Thermo Fisher Scientific</td>
			<td>51023121</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td>Thermo Fisher Scientific</td>
			<td>51032678</td>
			<td></td>
		</tr>
		<tr>
			<td>NSP-96 CardioExcyte 96 Sensor Plates 2.0 mm transparent</td>
			<td>Nanion Technologies</td>
			<td>20 1011</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (1250&#181;L)</td>
			<td>Integra Biosciences</td>
			<td>94420813</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Reservoir</td>
			<td>Integra Biosciences</td>
			<td>8096-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette (e.g. 25 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>16440901</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel adjustable pipette (e.g. 100-1000 &#956;L)</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum aspiration system</td>
			<td>Thermo Fisher Scientific</td>
			<td>15567479</td>
			<td></td>
		</tr>
		<tr>
			<td>Optional: VIAFLO ASSIST</td>
			<td>Integra Biosciences</td>
			<td>4500</td>
			<td>Lab automation Robot</td>
		</tr>
		<tr>
			<td>Water bath (37 &#176;C)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15365877</td>
			<td></td>
		</tr>
	</tbody>
</table>

hybrid cell analysis system to assess structural and contractile changes of human ipsc-derived cardiomyocytes for preclinical cardiac risk evaluation

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.

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 &#181;M 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)...

Watch this Scientific Journal Video about Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation at JoVE.

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

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.

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...

The method can help answer preclinical cardiac safety and toxicity related question using real human-based data for a safe transition of new drug candidates into clinical stages. Instead of using a set of different in-vitro and in-vivo techniques, the main advantage of this hybrid system is the simultaneous assessment of human iPC-derived cardiomyocyte, electrophysiological and viability parameters, and the analysis of contractile properties under physiological conditions. This method can be applied for drug discovery as it covers electrophysiological, structural and contractile changes of cardiomyocytes and a higher throughput system that allows for the testing of 69 wells simultaneously.To begin plate coating, leave the bottom of the contraction plate covered by the additionally supplied membrane guard until measurement in the contractility module. For seeding cardiomyocytes, prepare a diluted EHS gel coating solution by transferring 2.75 milliliters of EHS gel ready to use solution and 8.25 milliliters of DPBS with calcium and magnesium in a sterile centrifuge tube. Then mix the solution.Remove the membrane guard from the contraction plate. Transfer the prepared coating solution into a sterile reagent reservoir in the lab automation robot. Use the program, add 100 microliters with the lab automation robot and add 100 microliters of the coating solution per well.Then place the lid back onto the 96-well plate and incubate for three hours at 37 degrees Celsius. After thawing and counting the cells, adjust the cells in the recommended plating medium according to the cell manufacturer instructions, resulting in 11 times 10 to the sixth cells per 11 milliliters for seeding an entire 96-well plate. Use the program remove 100 microliters to remove the EHS gel solution from the wells with the lab automation robot.Next, transfer the 11 milliliters of cell suspension into a sterile reagent reservoir placed in the lab automation robot, and seed the cells with 100 microliters of suspension per well. Using the program, cells add 100 microliters. Immediately after cell seeding, transfer the flexible 96-well plate into the humidity controlled incubator at 37 degrees Celsius, 5%carbon dioxide, and let the cells settle overnight.Warm 22 milliliters of cardiomyocyte maintenance medium to be added to each plate to 37 degrees Celsius in a 50 milliliter centrifuge tube. 18 to 24 hours after seeding the plates, transfer the fresh medium into a sterile reagent reservoir and leave it next to the lab automation robot. Then perform medium removal with the program, remove 100 microliters.Next place the reagent reservoir containing the fresh medium into the lab automation robot and dispense 200 microliters of the fresh medium per well with the program, add 100 microliters. Perform this step twice to reach 200 microliters per well. Immediately after medium exchange, transfer the plate into the incubator.Perform a medium exchange every other day until compound addition. Four to six hours before compound addition perform a final medium change by transferring 22 milliliters of fresh and warm assay buffer into a sterile reagent reservoir and leaving it next to the lab automation robot. Immediately after medium exchange transfer the flexible plate back into the incubator.One hour prior to baseline measurement transfer the plate to the respective measurement device. In the control software open Edit Protocol and select the respective Measurement Mode, Flex Site or Impedance EFP. Define the sweep duration or length of one measurement to 30 seconds and a repetition interval to 10 minutes before saving the protocol number.Then select Start Protocol and continue to fill in the requested fields. When the parameters are set, select Start measurement. Perform a minimum of three baseline measurements in five minute intervals shortly before compound addition.Remove 50 microliters of the assay buffer from each well without removing the flexible 96-well plate from the measurement device, followed by the addition of 50 microliters of the four times concentrated compound solution into each well of the plate according to the measurement plan. After compound addition, select add reagent marker to define the compound plate layout and the volume of the compound solution. Then select Proceed with Standard Measurement or proceed with Measurement Series according to the experimental plan.The Representative Analysis shows the effects of kinase inhibitor Erlotinib on the contractility of human iPSC-derived cardiomyocytes. At the lowest concentration, Erlotinib induced a statistically insignificant decrease in amplitude, whereas micromolar concentrations of Erlotinib showed time and dose-dependent cardiotoxic effects. The onset of the effect was seen from 96 hours with one micromolar Erlotinib, while treatment with 10 micromolar Erlotinib resulted in a significant decrease 24 hours after compound addition.After the application of 10 micromolar Epirubicin, cells ceased beating within 24 hours. With the application of one micromolar Epirubicin, a drastic decrease in amplitude after 24 hours was followed by complete beat cessation until 48 hours. At 100 nanomolar, a time-dependent decrease in beat amplitude was observed.At the lowest concentration of 10 nanomolar, the amplitude fluctuated steadily, confirming that the effect of Epirubicin was only dose-dependent. The treatment with Doxorubicin and Nifedipine for 24 hours reduce the cell viability in a concentration and time dependent manner. Upon application of increasing Nifedipine concentrations, field potential recordings on cells reveal a concentration dependent shortening of the field duration normalized.Nifedipine assessment regarding cardiac contractility also showed a significant concentration dependent amplitude decrease upon acute measurement with 10 nanomolar and 13 nanomolar concentrations. It is important to remember that the cells are generally sensitive to environmental parameters like temperature and pH changes as well as mechanical simulation which is especially supported on the contraction plates.

Research

JoVE Journal

Bioengineering

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Hybrid &#181;CT-FMT imaging and image analysis

Hybrid  &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...