This work was supported in part by an appointment to the Research Participation Program at the Center for Devices and Radiological Health administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. The authors thank Richard Gray, Trent Robertson, and Anna Avila for their suggestions and technical assistance. The study was funded through the U.S. Food and Drug Administration, Office of Science and Engineering Laboratories.

1. Preparation of the plates and media
NOTE: A typical extracellular matrix (ECM)-based hydrogel aliquot is ~200 &#181;L in a sterile 1.5 mL tube stored at &#8722;20 &#176;C.
<ol>
	<li>In a sterile tissue culture hood, prepare a sterile 6-well plate by transferring 2 mL of 0.1% gelatin (Table of Materials) to each well. Place the lid on the 6-well plate, and allow the coated plate to incubate at 37 &#176;C for a minimum of 1 h.</li>
	<li>One day before seeding the hiPSC-CMs on the flexible hydrogel substrate, thaw a hydrogel (Table of Materials) aliquot in the refrigerator on ice.</li>
	<li>Prepare the cardiomyocyte medium (Table of Materials) by mixing 500 mL of RPMI 1640, 10 mL of 50x B-27 Supplement, and 5 mL of penicillin-streptomycin9.</li>
</ol>
2. Seeding of cryopreserved hiPSC-CMs
<ol>
	<li>Two days before seeding the hiPSC-CMs on the flexible hydrogel substrate, pre-plate the hiPSC-CMs on 0.1% gelatin-coated sterile 6-well plates. Thaw the hiPSC-CMs using a standard thawing protocol9,25.</li>
	<li>Then, plate 1,500,000 total hiPSC-CMs (Table of Materials) per well according to the manufacturer&#39;s instructions (Figure 1A)26.</li>
	<li>Culture the hiPSC-CMs in standard cardiomyocyte medium for 2-4 days to allow the hiPSC-CMs to recover from the cryopreservation at 37 &#176;C and 5% CO2. Refresh the spent medium with 100% cardiomyocyte medium every 48 h.</li>
</ol>
3. Dissociation and counting of the pre-plated hiPSC-CMs
<ol>
	<li>Check the status of the hiPSC-CMs before dissociation. Evaluate the health of the hiPSC-CMs, ensuring viability and stable beating. 
	NOTE: The purity of the hiPSC-CM population is important (e.g., &#62;90% cardiac troponin T)7. A cardiomyocyte selection method (e.g., metabolic selection or sorting) is recommended to reduce the disruption of the hydrogel substrate by non-cardiomyocyte cells25,26.</li>
	<li>Wash the hiPSC-CMs 2x with 4 mL per well of D-PBS without CaCl2 or MgCl2 (Table of Materials). Aspirate the D-PBS, add 1 mL of room temperature dissociation reagent to each well, and then incubate for 15 min at 37 &#176;C.</li>
	<li>Add 10 mL of cardiomyocyte medium to a sterile 15 mL conical tube.</li>
	<li>Dissociate the hiPSC-CMs from the 6-well plate with a 1,000 &#181;L pipette (Figure 1B). Add the cell suspension to the 15 mL conical tube26.</li>
	<li>Rinse the well with 1 mL of fresh cardiomyocyte medium to collect any residual hiPSC-CMs, and add them to the 15 mL conical tube. Bring the final volume of the conical tube to 15 mL.</li>
	<li>Centrifuge for 5 min (200 &#215; g). Remove the supernatant up to the 1 mL mark. Resuspend the cells in cardiomyocyte medium to a final volume of 5 mL.</li>
	<li>Count the hiPSC-CMs with a manual or automated cell counter.</li>
	<li>Incubate the hiPSC-CM suspension at room temperature while the flexible hydrogel substrates are being prepared (30 min maximum).</li>
</ol>
4. Preparation of the flexible hydrogel substrates
<ol>
	<li>Prepare a 20 &#181;L pipette set at 1 &#181;L, 20 &#181;L pipette tips (e.g., 20 &#181;L), and a sterile 48-well glass bottom plate. Ensure that a stopwatch/timer is ready ahead of making the hydrogel substrates.</li>
	<li>In the sterile tissue culture hood, mix the ECM-based hydrogel substrate by gently tapping the tube, and immediately place it back on ice.</li>
	<li>Then, start the stopwatch/timer immediately before the first hydrogel substrate is plated-this is time zero.</li>
	<li>Pipette 1 &#181;L of the hydrogel substrate up and down ~3x to chill the pipette tip. Apply ~1 &#181;L of the undiluted hydrogel substrate horizontally to the bottom of each well of the 48-well plate (Figure 1C, Figure 2A, and Figure 3A), holding the pipette at a 45&#176; angle. Plate all the hydrogel substrates in the same orientation in each well (Figure 2 and Figure 3) to help identify the substrate when performing the experiments at 40x magnification7,9,17,27. 
	NOTE: Each ~1 &#181;L line is one hydrogel substrate (Figure 3). Typically, it takes ~5 min to prepare 48 wells. The use of a horizontally tilted microplate stand (e.g., 30&#176;) may enable a better view of the bottom of the well and the hydrogel substrate. Be sure to go the entire length of the well (i.e., from left to right). Short hydrogel substrates may be too thick, thus reducing the substrate stability. Do not allow the hydrogel substrate to dry in the pipette tip. If this occurs, quickly switch to a new tip, and immediately continue. Alternatively, chilled pipette tips can be used. The ECM-based hydrogel substrate may quickly polymerize when it is not on ice. The preparation of several wells at a time (e.g., 10) is suggested. Additionally, practicing in a mock 48-well plate is recommended.</li>
	<li>Place the lid on the 48-well plate, and allow the hydrogel substrates to incubate for 8-10 min at room temperature in the sterile tissue culture hood before adding the cells (Figure 2B). 
	NOTE: It is important to adhere to the suggested incubation times. An incubation time of more than 10 min will lead to stiff hydrogel substrates and no visible contractions. An incubation time of less than 8 min may lead to substrates that collapse.</li>
	<li>Immediately seed the hiPSC-CMs dropwise directly onto the hydrogel substrates, with ~30,000 viable hiPSC-CMs per well in a low medium volume of ~200 &#181;L of cardiomyocyte medium, using a 1,000 &#181;L pipette (Figure 2B and Figure 3B). 
	NOTE: This process halts the hydrogel substrate polymerization and ensures the hiPSC-CMs are on the substrate7,9,17,27.</li>
	<li>Place the lid on the plate, and allow the hiPSC-CMs to incubate undisturbed for 10-15 min at room temperature (Figure 2C) in the sterile tissue culture hood to enable the hiPSC-CMs to adhere to the hydrogel substrate.</li>
	<li>Gently add ~100 &#181;L of fresh cardiomyocyte medium to each well (final volume: ~300 &#181;L per well). Place the lid on the plate, and transfer to an incubator at 37 &#176;C, 5% CO2 for 2-4 days. Refresh the 100% medium every 24 h before the contraction experiments are performed. 
	NOTE: Visually inspect the hiPSC-CMs for the correct morphology; immediately after plating, the hiPSC-CMs should appear round (Figure 3B). By day 2 after seeding, a confluent 2D hiPSC-CM monolayer syncytium will be observed (Figure 3C) (Supplemental Video S1) with robust visible contraction (Supplemental Video S2).</li>
</ol>
5. Contraction recording and analysis
<ol>
	<li>Prepare the CCM assay medium, which is Tyrode&#39;s solution containing the following (in mmol/L): CaCl2 0.5, NaCl 134, KCl 5.4, MgCl2 1, glucose 10, and HEPES 10, with the pH adjusted to 7.4 with NaOH, and equilibrate to 37 &#176;C in a water bath. 
	NOTE: Submaximal extracellular calcium (0.5 mM) is used to enhance the CCM assay window.</li>
	<li>Turn on the microscope and environmental control chamber to equilibrate to 37 &#176;C and 5% CO2.</li>
	<li>Remove the cardiomyocyte medium from the 48-well plate, and gently rinse each well twice with 600 &#181;L of CCM assay medium.</li>
	<li>Add 300 &#181;L of CCM assay medium per well, and place the 48-well plate on the microscope in the environmental control chamber. Insert the electrodes, and equilibrate the cells for 5 min.</li>
	<li>Use video-based microscopy to record the contraction videos. Open the video recording software, and set a frame rate of 100 frames/s. Select a region of interest (ROI) near the center of the hiPSC-CM monolayer. 
	NOTE: Do not select an ROI near the edge of the hiPSC-CM monolayers as this may be unstable for contractile recordings.</li>
	<li>Then, field stimulate the cells with a commercial pulse generator (Table of Materials) to electrically pace the 2D hiPSC-CM monolayers. Pace the hiPSC-CMs at 1.5x threshold at 1 Hz with baseline pulse parameters (e.g., monophasic square wave pacing pulses with a 2 ms stimulus pulse duration of ~14 V/cm)17.</li>
	<li>Record the baseline, pacing only (i.e., before the CCM) (Figure 1D) contraction video for a minimum of five beats17,28.</li>
	<li>Then, stimulate the hiPSC-CM monolayer with an experimental electrical signal (Figure 1D). To follow this protocol, use the standard CCM stimulation parameters: two symmetrical biphasic pulses of 5.14 ms phase duration (20.56 ms total duration), ~28 V/cm (phase amplitude), zero interphase interval, and a 30 ms delay (i.e., time from the end of the pacing pulse to the beginning of the CCM pulse) (Figure 1D)29,30, and record the CCM-induced contraction video for a minimum of five beats.</li>
	<li>Turn off the CCM signal, stimulate with a baseline pacing pulse, and record a contraction video of the recovery period (i.e., after the CCM) for a minimum of five beats.</li>
	<li>Use standard contraction software to automatically analyze the contraction videos and quantify the key contractile properties (e.g., the contraction amplitude, contraction slope, relaxation slope, time to peak, time to baseline 90%, and contraction duration 50%)7,17,31,32.</li>
	<li>Use the 2D hiPSC-CMs on the flexible hydrogel substrate for contractile experiments from days 2-14 after plating on the substrate.</li>
</ol>

This article reflects the views of the authors and should not be construed to represent the U.S. Food and Drug Administration&#39;s views or policies. The mention of commercial products, their sources, or their use in connection with the material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services. The authors declare no competing interests for this work.

The protocol outlined herein describes a method to generate robustly contracting 2D hiPSC-CM monolayers on a flexible extracellular matrix (ECM)-based hydrogel substrate with commercial reagents7,17. The hiPSC-CMs seeded on the flexible hydrogel substrate remain viable and have enhanced contractile properties7. This technique relies on standard laboratory equipment and capabilities7. There are several critical steps in the protocol, including in relation to working with the ECM-based hydrogel substrate, that require careful attention to detail. One potential issue is the presence of serum in the medium. This may result in the hiPSC-CMs forming networks (e.g., endothelial/vascular networks) instead of a confluent monolayer sheet; hence, a serum-free medium is recommended during the establishment of the flexible hydrogel hiPSC-CM monolayers (i.e., day 0 to day 4). Likewise, preparing too many hydrogel substrates at one time may result in poor or uneven substrates due to operator fatigue. While it is important to work quickly, the integrity of each hydrogel substrate is critical. Similarly, one should carefully seed the hiPSC-CMs and change the medium; this should not be done forcefully. When changing the medium, it should be added gently from the top edge of the well so as not to disrupt the hydrogel substrate or the cells. As with standard 2D hiPSC-CM cultures (i.e., conventional tissue culture plastic or glass), plating at a low density will result in incomplete monolayer formation. It is important to visually inspect the hiPSC-CMs to confirm that they are on the hydrogel substrate and to use a timer to ensure accurate timing. Furthermore, culturing the 2D hiPSC-CM monolayers for more than 14 days on the hydrogel substrate may result in monolayer disruption, based on the ECM properties and instructions from the manufacturer of the substrate.
There are several limitations to the current method that have to be considered. First, the cells used in this protocol were from a commercial hiPSC-CMs provider, and those cells form a syncytium of electrically coupled cells. The syncytium contains a mixture of hiPSC-CMs from all three cardiac subtypes (i.e., ventricular, atrial, and nodal)17. Studies may benefit from a subtype-exclusive hiPSC-CM population (i.e., 100% ventricular or 100% atrial). Second, this method only used hiPSC-CMs, while non-myocytes, including cardiac fibroblasts, endothelial cells, and neurons, may enhance hiPSC-CM functionality22,36. Third, 2D hiPSC-CMs display several features of relatively immature cardiomyocytes, including spontaneous beating, amorphic morphology, and lack of an inotropic response8,37. Fourth, while this protocol produces robustly contracting 2D hiPSC-CM monolayers, it is likely that functionally enhanced 3D hiPSC-CM models such as engineered cardiac tissues (ECTs) will result in an enhanced CCM-induced contractile response under physiological calcium concentrations8,38. Finally, the protocol described here is designed for a 48-well format. However, with optimization and the inclusion of automation, this can be scaled to a high-throughput format (e.g., 96-well or 384-well plates).
The current gold standard for hiPSC-CM studies is conventional rigid 2D culture conditions (i.e., tissue culture plastic or glass). While useful for electrophysiology3 and calcium handling39 studies, the conventional methodology results in minimal contractile properties5,6,7. As a result, conventional rigid 2D culture conditions are not amenable to the assessment of CCM contractile effects8. Functionally enhanced 3D hiPSC-CM ECT methods38 are technically challenging, time-consuming, and require sophisticated equipment that is not readily available in every laboratory. In this protocol, we describe a simple methodology to generate robustly contracting 2D hiPSC-CM monolayers in a shorter timeframe than 3D ECT methods or long-term, conventional 2D methods7,40,41. Moreover, the reagents used here are commercially available, including the hydrogel substrate and the hiPSC-CMs, and both have considerable lot-to-lot consistency. While we used removable platinum wire electrodes (interelectrode distance: 2.0 mm, width: 1.0 mm), various electrode materials and configurations are amenable to CCM contractile assessments in vitro8,15,17,18,22. Likewise, there are multiple automated software available that enable the analysis of contraction videos7,31,32.
The majority of nonclinical methods to evaluate cardiac medical device contractility rely largely on costly in vivo animal models (e.g., dogs or pigs) and technically challenging papillary muscle strips (e.g., rabbits)18. This paper described a human in vitro model to evaluate the effects of cardiac electrophysiology medical device signals on contractility. This tool could reduce the dependence on animal studies and be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

As the United States population ages, the number of heart failure patients continues to rise, along with the direct medical costs1,2. There is a critical need to develop novel therapies to treat heart failure and innovative nonclinical methodologies to test such therapies. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been proposed as an in vitro tool to aid the therapeutic development process and have been used in regulatory submissions3,4. However, their widespread use has been limited for contractility studies due to the lack of robust contractile properties when plated in standard rigid 2D culture conditions (i.e., conventional tissue culture plastic or glass)5,6,7,8. We previously demonstrated the utility of plating isolated single hiPSC-CMs on a flexible hydrogel substrate to generate robust visible contractile properties9. We showed that isolated hiPSC-CMs have comparable contractile properties to those of freshly isolated adult rabbit ventricular cardiomyocytes. Moreover, we demonstrated the utility of this method for assessing the contractile responses to pharmacological agents7. Furthermore, other studies have applied this technology to mechanistic assessments for basic science and disease modeling10,11,12. Here, this methodology has been extended to 2D hiPSC-CM monolayers, and its utility in evaluating physiologically relevant cardiac contractility modulation (CCM) medical device electrical signals in vitro is demonstrated.
CCM is an intracardiac heart failure therapy in which non-excitatory electrophysiological signals are delivered to the myocardium during the absolute refractory period of the cardiac cycle13,14. Reproducible methods to evaluate CCM in human cardiac cell models are lacking. Previous work has employed various cardiac cell models to evaluate the CCM contractile response. We demonstrated in vitro that freshly isolated rabbit ventricular cardiomyocytes respond to CCM stimulation by a transient increase in calcium and contraction amplitude15. Another study in isolated canine ventricular cardiomyocytes demonstrated CCM-induced enhancement of the intracellular calcium transient amplitude16. However, the majority of CCM studies have used ex vivo and in vivo animal preparations. These studies are difficult to correlate with each other because they apply a variety of CCM pulse parameters and species17. One study in an isolated rabbit papillary model revealed increased CCM-induced contractility8,18, and an array of whole heart studies have demonstrated CCM-induced enhancement of contractile function19,20,21. These studies have provided important mechanistic insight. However, there is a lack of reproducible human models for in vitro cardiac EP contractile studies including CCM. Toward that end, we have developed several 2D and 3D hiPSC models and demonstrated CCM-induced enhancement of contractile properties in a parameter-dependent manner. Moreover, the CCM-induced inotropic effects have been found to be in part mediated by neuronal input and &#946;-adrenergic signaling8,17,22. Still, more needs to be known about the mechanisms of CCM therapy, and utilizing contracting human cardiomyocytes can assist in achieving this outcome. As such, there is a significant need to develop human nonclinical tools to evaluate novel CCM devices and signals, quicken the regulatory process, reduce the burden on animal models, and aid device developer decision-making8,17,23,24. It is important to develop easy, do-it-yourself protocols that can be transferred to any laboratory and that use standard equipment and low cell requirements to reduce the costs. This method elucidates the effects of CCM stimulation on human cardiomyocyte function and provides important insights on CCM safety or effectiveness17. Here, we describe the method for generating 2D hiPSC-CM monolayers on a flexible hydrogel substrate to produce a standardized nonclinical tool to quantify acute cardiac electrophysiology medical device (i.e., CCM) contractile responses in health and disease.

<table><tbody><tr><td>0.1% Gelatin</td><td>STEMCELL Technologies</td><td>7903</td><td>Pre-plating Culture Substrate</td></tr><tr><td>48-well Plate</td><td>MatTek&amp;nbsp;</td><td>P48G-1.5-6-F</td><td>Hydrogel Substrate hiPSC-CM Culture, Glass</td></tr><tr><td>6-well Plate</td><td>Thermofisher</td><td>140675</td><td>hiPSC-CM Culture, Plastic</td></tr><tr><td>B-27 Supplement, with insulin</td><td>Invitrogen</td><td>17504-044</td><td>Cardiomyocyte Media</td></tr><tr><td>Calcium Chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>c70-500</td><td>Tyrode&amp;rsquo;s solution</td></tr><tr><td>CellOPTIQ Platform and Software&amp;nbsp;</td><td>Clyde Biosciences</td><td></td><td>Contraction Recording and Analysis</td></tr><tr><td>Conical tube 15 mL</td><td>Corning&amp;nbsp;</td><td>352099</td><td>hiPSC-CM Dissociation</td></tr><tr><td>Digital CMOS Camera</td><td>Hamamatsu</td><td>C11440-42U30</td><td>Contraction Video Recording</td></tr><tr><td>D-PBS</td><td>Life Technologies</td><td>14190-144</td><td>Cell Wash</td></tr><tr><td>Environmental Control Chamber</td><td>OKOLAB INC</td><td>H201-K-FRAME</td><td>Environmental Regulation</td></tr><tr><td>Glucose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G8270-1kg</td><td>Tyrode&amp;rsquo;s solution</td></tr><tr><td>Hemocytometer</td><td>Fisher Scientific</td><td>22-600-107</td><td>hiPSC-CM Counting</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>Tyrode&amp;rsquo;s solution</td></tr><tr><td>iCell Cardiomyocytes Plating Medium</td><td>Fujifilm Cellular Dynamic, Inc.&amp;nbsp;</td><td>M1001</td><td>hiPSC-CM Plating Media</td></tr><tr><td>iCell Cardiomyocytes&lt;sup&gt;2&lt;/sup&gt;, 01434</td><td>Fujifilm Cellular Dynamic, Inc.&amp;nbsp;</td><td>R1017</td><td>hiPSC-CMs</td></tr><tr><td>Incubator (37 &amp;deg;C, 5% CO2)</td><td>Thermofisher</td><td>50116047</td><td>Maintain hiPSC-CMs</td></tr><tr><td>Inverted Microscope</td><td>Olympus</td><td>IX73</td><td>Imaging hiPSC-CMs</td></tr><tr><td>Magnesium Chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;)&amp;nbsp;</td><td>Fisher Scientific</td><td>m33-500</td><td>Tyrode&amp;rsquo;s solution</td></tr><tr><td>Matrigel Growth Factor Reduced Basement Membrane Matrix</td><td>Corning&amp;nbsp;</td><td>356230</td><td>Flexible Hydrogel Substrate</td></tr><tr><td>Microcentrifuge tubes 1.5 ml</td><td>Fisher Scientific</td><td>05-408-129</td><td>Hydrogel Substrate Aliquot</td></tr><tr><td>Model 4100 Isolated High Power Stimulator</td><td>AM-Systems</td><td>Model 4100&amp;nbsp;</td><td>Pulse Generator</td></tr><tr><td>MyCell Cardiomyocytes DCM LMNA L35P, 01016</td><td>Fujifilm Cellular Dynamic, Inc.&amp;nbsp;</td><td>R1153</td><td>DCM hiPSC-CMs</td></tr><tr><td>Pen-Strep</td><td>Invitrogen</td><td>15140-122</td><td>Cardiomyocyte Media</td></tr><tr><td>Pipette L-20</td><td>Rainin</td><td>17014392</td><td>Plating Hydrogel Substrate</td></tr><tr><td>Pipette P1000</td><td>Fisher Scientific</td><td>F123602G</td><td></td></tr><tr><td>Pipette tips, 1000 ul</td><td>Fisher Scientific</td><td>02-707-509</td><td></td></tr><tr><td>Pipette tips, 20 ul</td><td>Rainin</td><td>GPS-L10S</td><td>Making Hydrogel Substrate</td></tr><tr><td>Potassium Chloride (KCl)&amp;nbsp;</td><td>Fisher Scientific</td><td>P330-500</td><td>Tyrode&amp;rsquo;s solution</td></tr><tr><td>RPMI 1640, with glucose&amp;nbsp;</td><td>Invitrogen</td><td>11875</td><td>Cardiomyocyte Media</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>s641-212</td><td>Tyrode&amp;rsquo;s solution</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>221465</td><td>Tyrode&amp;rsquo;s solution</td></tr><tr><td>Stimulation Electrodes</td><td></td><td></td><td>Pacing and CCM Stimulation</td></tr><tr><td>Stopwatch/Timer</td><td>Fisher Scientific</td><td>02-261-840</td><td>Plating Hydrogel Substrate</td></tr><tr><td>Trypan Blue Stain</td><td>Life Technologies</td><td>T10282</td><td>hiPSC-CM Counting</td></tr><tr><td>TrypLE Express&amp;nbsp;</td><td>Life Technologies</td><td>12605-010</td><td>hiPSC-CM Dissociation</td></tr></tbody></table>

evaluation of cardiac contractility modulation therapy in 2d human stem cell-derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Described in this protocol is a simple, robust tool to generate visibly contracting 2D hiPSC-CM monolayers on a flexible hydrogel substrate. The measurement of the contractile properties is accomplished with video-based recording coupled with contractility analysis software. This enables the quantification of key parameters of cardiomyocyte contractility, including the contraction amplitude, contraction slope, relaxation slope, time to peak, time to baseline 90%, and contraction duration 50%. The model is used to characterize the baseline contractile properties of hiPSC-CMs (Figure 4) from various &#34;healthy&#34; donors and can be extended to the evaluation of cardiac electrophysiology medical device signals (i.e., CCM). The application of the standard CCM stimulation parameters (Figure 1D)29,30 resulted in enhanced contractile properties in vitro (Figure 5 and Table 1)17.
We further demonstrated that this method can be used to evaluate the effects of the modulation of extracellular calcium concentrations on human contractile properties with and without CCM stimulation (Figure 6)17. The expected baseline calcium dependence of contraction was observed7,17, as well as a CCM-induced increase in calcium sensitivity at the level of the cardiomyocyte monolayer. In addition, the pharmacological interrogation of the &#946;-adrenergic signaling pathway (Figure 7) revealed that the CCM-induced inotropic effects were in part mediated by &#946;-adrenergic signaling17. Moreover, this tool can be expanded to patient-specific disease cardiomyocytes, including those of dilated cardiomyopathy (DCM)33,34,35 (Figure 8), to understand the effect of CCM in the context of disease states; indeed, enhanced contractile amplitude and accelerated contraction and relaxation kinetics were observed at the CCM &#34;dose&#34; tested here (Figure 8). While we have a CCM-mimicking device in our laboratory, the methodology used here is not specific to that system and could be applied to other cardiac electrophysiology devices.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64848/64848fig01.jpg" /> 
Figure 1: Schematic summary of the 2D hiPSC-CM in vitro CCM model. (A)&#160;The&#160;hiPSC-CMs are pre-plated in monolayer format on gelatin (0.1%)-coated 6-well plates. (B) After 2 days in culture, the hiPSC-CMs are dissociated and prepared for plating on a flexible hydrogel substrate. (C) The isolated hiPSC-CMs are plated at a high density on hydrogel substrates arrayed in a 48-well format (left) and are assayed in (0.5 mM) extracellular calcium Tyrode&#39;s solution (right). (D) A commercial pulse generator and standard clinical CCM pulse parameters29,30 (right) are used to stimulate the hiPSC-CMs; the cardiac function is assessed by video-based analysis (left). (E) Representative contraction recordings before CCM (baseline: 5 V), during CCM (CCM: 10 V), and after CCM (recovery: 5 V). This figure has been reprinted from Feaster et al.17. Abbreviations: hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocyte; CCM = cardiac contractility modulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64848/64848fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64848/64848fig02.jpg" /> 
Figure 2: Schematic of the flexible hydrogel substrate plating and seeding. (A) Completely thawed, undiluted ECM-based hydrogel substrate is applied to a sterile 48-well plate (left panel), with 1 &#181;L of hydrogel substrate per well (right panel). (B) The hydrogel substrate is allowed to incubate at room temperature for 8-10 min (right panel), followed by plating the high-density hiPSC-CMs in a low medium volume (~200 &#181;L) (left panel). (C) After 10-15 min of incubation, medium is added to each well (left panel), and the plates are moved to a standard tissue culture incubator (right panel). Abbreviations: ECM = extracellular matrix, hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocyte; RT = room temperature. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64848/64848fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64848/64848fig03.jpg" /> 
Figure 3: Extracellular matrix-based hydrogel substrate. (A) Representative hydrogel substrate (no cells) in one well of a 48-well glass bottom plate immediately after the substrate is applied to the well. (B) Time 0 after the hiPSC-CMs are seeded. (C) Time 24 h after the hiPSC-CMs are seeded. This panel was reprinted from Feaster et al.17. The white arrows indicate the edge of the hydrogel substrate, 4x magnification. Scale bar = 1 mm. Abbreviation: hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocyte. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64848/64848fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64848/64848fig04.jpg" /> 
Figure 4: Characterization of the 2D hiPSC-CM monolayer contractile properties. (A) Representative contraction recording of the 2D hiPSC-CMs paced at 1 Hz (5 V). (B) Representative contraction traces depicting one contraction cycle. (C) Summary bar graphs. The data are mean &#177; SEM. n = 18. Abbreviation: hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocyte. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64848/64848fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64848/64848fig05.jpg" /> 
Figure 5: Acute effect of CCM on the 2D hiPSC-CM contractile properties. (A) Representative contraction recording for before CCM (5 V), during CCM (10 V), and after CCM (5 V). (B) Representative contraction traces of the immediate effects (i.e., last before-CCM beat, first CCM beat, and first after-CCM beat, indicated by +). (C) Summary bar graphs of the immediate effects. Percent change, data are mean &#177; SEM. n = 23. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001. This figure has been reprinted from Feaster et al.17. Abbreviations: hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocyte; CCM = cardiac contractility modulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64848/64848fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64848/64848fig06.jpg" /> 
Figure 6: Effect of extracellular calcium modulation on the CCM response. (A) Representative contraction traces of the immediate effects for each group before CCM (5 V), during CCM (10 V), and after CCM (5 V); the hiPSC-CMs were exposed to increasing concentrations of extracellular calcium (Cao) of 0.25-2 mM. (B-D) Transformed data (sigmoidal) to guide the eye demonstrating the effect of CCM on the calcium sensitivity of the contractile properties (i.e., the amplitude and kinetics) (hill slope = 1.0). n = 6-8 per group. This figure has been reprinted from Feaster et al.17. Abbreviations: hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocyte; CCM = cardiac contractility modulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64848/64848fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64848/64848fig07.jpg" /> 
Figure 7: Pharmacological challenge. Representative contraction traces for each group before CCM (5 V), during CCM (10 V), and after CCM (5V); the hiPSC-CMs were pretreated with (A) vehicle or (B) metoprolol (2 &#181;M). (C,D) Summary bar graphs for each condition. Percent change, data are mean &#177; SEM. n = 10 per group. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001. This figure has been reprinted from Feaster et al.17. Abbreviations: hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocyte; CCM = cardiac contractility modulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64848/64848fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64848/64848fig08.jpg" /> 
Figure 8: Acute effect of CCM on the contractile properties of diseased 2D hiPSC-CMs. (A) Representative contraction trace for DCM L35P, control baseline (before, 6 V), and DCM L35P plus CCM (10 V). (B) Summary bar graphs. Percent change, data are mean &#177; SEM. n = 3. *p &#60; 0.05, **p &#60; 0.01. Abbreviations: hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocyte; CCM = cardiac contractility modulation; DCM = dilated cardiomyopathy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64848/64848fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Video S1: Timelapse of the hiPSC-CMs on the extracellular matrix-based hydrogel. Two-dimensional hiPSC-CMs plated on the flexible hydrogel substrate; Time: 0-90 h; one well of a 48-well glass bottom plate; 4x magnification. The hiPSC-CMs form a horizontal monolayer syncytium (i.e., left to right). Scale bar = 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64848/Supplemental Video 1.mp4" target="_blank">Please click here to download this Video.</a>
Supplemental Video S2: hiPSC-CMs on the extracellular matrix-based hydrogel. Two-dimensional hiPSC-CMs plated on the flexible hydrogel substrate; Time: ~24 h; one well of a 48-well glass bottom plate; 4x magnification. The hiPSC-CMs form monolayer morphology and show robust contraction at ~24 h post plating. Scale bar = 1 mm. This video is from Feaster et al.17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64848/Supplemental Video 2.m4v" target="_blank">Please click here to download this Video.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>CCM</td>
			<td>After</td>
		</tr>
		<tr>
			<td>Amplitude&#160;</td>
			<td>16 &#177; 4%**</td>
			<td>4 &#177; 5%</td>
		</tr>
		<tr>
			<td>Time to Peak 50%&#160;</td>
			<td>-20 &#177; 9%*</td>
			<td>7 &#177; 5%</td>
		</tr>
		<tr>
			<td>Time to Peak 90%&#160;</td>
			<td>-22 &#177; 8%*</td>
			<td>6 &#177; 5%</td>
		</tr>
		<tr>
			<td>Time to Baseline 50%&#160;</td>
			<td>-8 &#177; 5%</td>
			<td>4 &#177; 4%</td>
		</tr>
		<tr>
			<td>Time to Baseline 90%&#160;</td>
			<td>-12 &#177; 6%*</td>
			<td>5 &#177; 5%</td>
		</tr>
		<tr>
			<td>Contraction Duration 10%&#160;</td>
			<td>-13 &#177; 6%</td>
			<td>3 &#177; 5%</td>
		</tr>
		<tr>
			<td>Contraction Duration 50%</td>
			<td>-6 &#177; 5 %</td>
			<td>3 &#177; 5%</td>
		</tr>
		<tr>
			<td>Contraction Duration 90%&#160;</td>
			<td>0 &#177; 5%</td>
			<td>3 &#177; 4%</td>
		</tr>
		<tr>
			<td>N</td>
			<td>23</td>
			<td>23</td>
		</tr>
	</tbody>
</table>
Table 1: Contractile properties. Percent change relative to before CCM (5 V); the data are mean &#177; SEM for all the beats in each group during CCM (10 V) and after CCM (5 V). n = 23. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001. This table has been reprinted from Feaster et al.17.

Watch this Scientific Journal Video about Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes at JoVE.com

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

evaluation-cardiac-contractility-modulation-therapy-2d-human-stem

Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.3K Views.  U.S. Food and Drug Administration. Office of Science and Engineering Laboratories bij de FDA ontwikkelt regelgevende wetenschappelijke hulpmiddelen om ontwikkelaars van medische hulpmiddelen en FDA-reviewers te helpen. Ons uiteindelijke doel is om de toegang van patiënten tot innovatieve, veilige en effectieve medische hulpmiddelen te versnellen door middel van de best beschikbare wetenschap. In dit protocol passen we geavanceerde geïnduceerde pluripotente stamceltechnologie toe op de beoordeling van medische hulpmiddelen di...

Video: Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Developmental Biology

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 replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

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 procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

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

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

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 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 ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

Medicine

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

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

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.

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.

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This paper describes a protocol for assessing changes in contractility in adult human primary ventricular cardiomyocytes utilizing the MyoBLAZER, a non-invasive optical method that preserves the normal physiology and pharmacology of the cells. This optical recording method continuously measures contractility transients from multiple cells in parallel, providing both medium-throughput and valuable information for each individual cell in the field of view, enabling the real-time tracking of drug effects. The cardiomyocyte contractions are induced by paced electrical field stimulation, and the acquired bright field images are fed to an image-processing software that measures the sarcomere shortening across multiple cardiomyocytes. This method rapidly generates different endpoints related to the kinetics of contraction and relaxation phases, and the resulting data can then be interpreted in relation to different concentrations of a test article. This method is also employed in the late stages of preclinical development to perform follow-up mechanistic studies to support ongoing clinical studies. Thus, the adult human primary cardiomyocyte-based model combined with the optical system for continuous contractility monitoring has the potential to contribute to a new era of in vitro cardiac data translatability in preclinical medical therapy development.

This protocol describes how to measure contractility in adult human primary cardiomyocytes from donor hearts with the MyoBLAZER system, a reliable platform for assessing drug-induced changes in contractility during preclinical development.

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

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