The authors have no acknowledgments.

1. Preparation of Cell Plates
NOTE: Prepare the cell plates 3&#8211;4 weeks before the measurement of drug effects.
<ol>
	<li>Day 0 of the experiment 
	NOTE: For planning purposes, the test compound application can be performed on any day between days 22&#8211;28 of the experiment.
	<ol>
		<li>Turn on the water bath at 37 &#176;C and warm 40 mL of plating medium (as supplied by the manufacturer). 
		NOTE: Use the cells with their supplier-provided matching culture and maintenance media.</li>
		<li>Thaw the hiPSC-CM by placing the cryotubes containing frozen cells in the water bath for 2 min.</li>
		<li>Transfer the 1 mL cell suspensions carefully to a 50 mL conical tube.</li>
		<li>Wash each cryotube with 1 mL of warm plating medium to retrieve the leftover cells and add the wash media to the same conical tube from step 1.1.3 containing cells.</li>
		<li>Mix carefully another 8 mL of warm plating medium into the cell suspension.</li>
		<li>Count live cells using the trypan blue exclusion method and a hematocytometer8. 
		NOTE: The requirement of 6 x 106 cells for one 384-well cell plate considers the possibility of up to 20% dead cells among the frozen cells, which is a maximum in our experience.</li>
		<li>Adjust the cell suspension to 5 x 105 live cells/mL with the warm plating medium.</li>
		<li>Distribute the cell suspension into two 384-well cell plates by adding 25 &#181;L to each well (about 12,500 cells per well). 
		NOTE: The cell plates do not need to be precoated with any matrix (see Discussion).</li>
		<li>Maintain the cell plates at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
	</ol>
	</li>
	<li>Day 1 of the experiment
	<ol>
		<li>Turn on the water bath at 37 &#176;C and warm 50 mL of maintenance medium supplemented with 1% v/v penicillin-streptomycin solution.</li>
		<li>Replace the plating medium with 50 &#181;L of warm maintenance medium in each well of the cell plates.</li>
	</ol>
	</li>
	<li>Days 2&#8211;21 (up to day 27) of the experiment 
	NOTE: The hiPSC-CMs do not need any passaging over this period, as they are non-dividing cells.
	<ol>
		<li>Renew half of the maintenance medium every 2&#8211;3 days and renew it entirely on days 7, 14, and 21. 
		NOTE: The fluorescence measurement can be performed between days 22 and 28 of the experiment. Longer durations have not been tried but may still be fine.</li>
	</ol>
	</li>
</ol>
2. Preparation of Instrument, Compound Plates, and Assay Plates
NOTE: Prepare the instrument, compound plates, and assay plates on the day of the measurement of drug effects, between days 22 and 28 of the experiment. Use a fluorescent plate reader equipped with temperature control and an appropriate fluorescence filter kit and excitation light source. For example, use a Fluo-3/Fluo-4/Fluo-8 fluorescence filter kit (excitation: 472 nm; emission: 520&#8211;560 nm) and a 150 W excitation light source unit.
<ol>
	<li>At least 2 h before the first cell plate is to be placed inside the plate reader for the measurement, turn on the reader and set the heating system at 37 &#176;C. 
	NOTE: This can also be done the evening before.</li>
	<li>Prepare the positive controls, forskolin, N6-cyclopentyl-adenosine, and E-4031 as stock solutions of 0.33 mM in DMSO, 10 mM in DMSO, and 3.33 mM in H2O, respectively. 
	NOTE: Stock solutions will be diluted 333-fold by the time they are added to the cells, and the aim is to achieve final test concentrations of 1 &#181;M forskolin, 30 &#181;M N6-cyclopentyl-adenosine, and 10 &#181;M E-4031, which produce reliable and reproducible effects.</li>
	<li>Prepare the 352 test compounds as DMSO stock solutions at 333-fold the planned test concentrations (e.g., 10 mM for a test at 30 &#181;M). 
	NOTE: The aim is to achieve a final concentration of 0.3% (v/v) DMSO in the cell plate after the compound application; this is the highest DMSO concentration that does not affect the rhythmic activity of the cardiomyocytes. The stock solutions can also be prepared the day before. A minimum of 5 &#181;L of stock solution is required for each duplicate measurement.</li>
	<li>Prepare the compound plates.
	<ol>
		<li>Warm 40 mL of maintenance medium supplemented with 1% v/v penicillin-streptomycin solution to room temperature.</li>
		<li>Distribute into two 384-well compound plates by adding 1.5 &#181;L of stock solution per well: i) the test compounds (two wells each), ii) the positive controls (forskolin, N6-cyclopentyl- adenosine, and E-4031, four wells each per compound plate), and iii) the negative control (DMSO, 20 wells per compound plate).</li>
		<li>Centrifuge the compound plates at 100 x g for 1 min.</li>
		<li>Add 37 &#181;L of maintenance medium at room temperature to each well (3.9% DMSO in 38.5 &#181;L at that stage).</li>
		<li>Centrifuge the compound plates at 100 x g for 1 min.</li>
		<li>Load the compound plates into the plate reader. 
		NOTE: The next steps should be performed as soon as possible to minimize evaporation in the compound plates.</li>
	</ol>
	</li>
	<li>Prepare the fluorescent dye.
	<ol>
		<li>Turn on the water bath at 37 &#176;C and warm 100 mL of maintenance medium supplemented with 1% v/v penicillin-streptomycin solution.</li>
		<li>Reconstitute the Ca-sensitive fluorescent dye and quencher with 10 mL of warm maintenance medium with antibiotics. 
		NOTE: This stock fluorescence medium contains the Ca-sensitive fluorescent dye, usually Fluo-4, and a quencher, and any leftover can be stored at 4 &#176;C for at least 5 days.</li>
	</ol>
	</li>
</ol>
3. Data Acquisition
NOTE: Perform the data acquisition on the day of the measurement of drug effects. Perform steps 3.1&#8211;.10&#160;entirely for the first cell plate, then repeat them for the second cell plate.
<ol>
	<li>Start the plate reader software (if needed) and load the pipet tips.</li>
	<li>Adjust the software settings to record a 2 min segment of Calcium waves with an acquisition frequency of at least 10 Hertz to define the baseline beating rate for each well.</li>
	<li>For one cell plate, dilute 3 mL of stock fluorescence medium into 12 mL of warm maintenance medium with antibiotics to make the fluorescence medium.</li>
	<li>Replace 20 &#181;L of medium in each well of the cell plate with 30 &#181;L of fluorescence medium.</li>
	<li>Pipette up and down 1x to mix.</li>
	<li>Place the cell plate into the plate reader as quickly as possible to let the hiPSC-CM acclimatize to the temperature.</li>
	<li>Wait for 60 min before starting the data acquisition.</li>
	<li>Start the data acquisition in the plate reader software to record a 2 min segment of Ca waves with an acquisition frequency of at least 10 Hz, to define the baseline beating rate for each well. 
	NOTE: For each recording sequence, make sure the cell plate is not transferred automatically out of the device after the data acquisition. This prevents the cell plate from cooling at room temperature. With some versions of plate reader software, it may be necessary to stop each recording before it ends completely to achieve this.</li>
	<li>Add the test and reference compounds with the plate reader robot by pipetting well-to-well 5 &#181;L from the compound plate into the cell plate (after addition, the DMSO concentration is 0.3% [v/v]).</li>
	<li>Start the data acquisition in the plate reader software to record 2 min segments of Ca waves starting about 5, 15, 30, 45, and 60 min after the compound addition (make sure each time that the cell plate stays in the device).</li>
</ol>
4. Data Analysis
NOTE: The data analysis can be performed any time after the measurement.
<ol>
	<li>Export the raw data for each recording period from the plate reader software as ASCII text files.</li>
	<li>Import the raw data into the data analysis software.</li>
	<li>For each well and all time points, extract the primary beating frequency. 
	NOTE: With the data analysis software listed in the Table of Materials, beating frequencies can be calculated with a custom analysis routine using the LombPeriodogram function, based on the Lomb&#8211;Scargle method of least-squares spectral analysis7. The periodogram should be calculated for a range of frequencies between 0.01 and 5 Hz. The primary frequency can be extracted from the periodogram using the PeakAutoFind routines. This analysis method provides more accurate measurements of beating frequencies than the method supplied as an option with the plate reader software. However, the latter can still be used if software customization is not an option.</li>
	<li>Export the primary beating frequencies for each recording period from the data analysis software as clipboard copies or as ASCII text files.</li>
	<li>Import the primary beating frequencies into a spreadsheet software by clipboard pasting or text file import. 
	NOTE: Steps 4.5&#8211;4.9 can be performed in any modern spreadsheet software.</li>
	<li>For each well, normalize to baseline the beating frequency at each time point after the drug application (5, 15, 30, 45, and 60 min) by dividing the primary beating frequency at that time point by the primary beating frequency at baseline.</li>
	<li>Calculate the mean DMSO effect at each time point after the drug application (5, 15, 30, 45, and 60 min) by averaging the normalized values for the 20 wells where DMSO was applied.</li>
	<li>For each well and each time point after the drug application (5, 15, 30, 45, and 60 min), subtract the mean DMSO effect (time-matched) for the same plate.</li>
	<li>Average the duplicate measurements. 
	NOTE: If a compound changes frequency or the software cannot find a primary frequency after the compound addition, a visual examination of the beating pattern can evaluate whether the compound produced arrhythmias.</li>
</ol>

<ol>
	<li>Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+risk+of+drug-induced+proarrhythmia+using+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Estimating the risk of drug-induced proarrhythmia using human induced pluripotent stem cell-derived cardiomyocytes.</a> Toxicological Sciences. 123, 281-289 (2011).</li><li>Jonsson, M. K., Wang, Q. D., Becker, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impedance-based+detection+of+beating+rhythm+and+proarrhythmic+effects+of+compounds+on+stem+cell-derived+cardiomyocytes.">Impedance-based detection of beating rhythm and proarrhythmic effects of compounds on stem cell-derived cardiomyocytes.</a> Assay and Drug Development Technologies. 9, 589-599 (2011).</li><li>Kattman, S. J., Koonce, C. H., Swanson, B. J., Anson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+and+their+derivatives:+a+renaissance+in+cardiovascular+translational+research.">Stem cells and their derivatives: a renaissance in cardiovascular translational research.</a> Journal of Cardiovascular Translational Research. 4 (1), 66-72 (2011).</li><li>Ma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+purity+human-induced+pluripotent+stem+cell-derived+cardiomyocytes:+electrophysiological+properties+of+action+potentials+and+ionic+currents.">High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents.</a> American Journal of Physiology Heart and Circulatory Physiology. 301 (5), H2006-H2017 (2011).</li><li>Sube, R., Ertel, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocytes+Derived+from+Human+Induced+Pluripotent+Stem+Cells:+An+In-Vitro+Model+to+Predict+Cardiac+Effects+of+Drugs.">Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells: An In-Vitro Model to Predict Cardiac Effects of Drugs.</a> Journal of Biomedical Science and Engineering. 10 (11), 23 (2017).</li><li>Sirenko, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+in+vitro+assessment+of+compound+effects+on+cardiomyocyte+physiology+using+iPSC+cells.">Multiparameter in vitro assessment of compound effects on cardiomyocyte physiology using iPSC cells.</a> Journal of Biomolecular Screening. 18, 39-53 (2013).</li><li>VanderPlas, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Lomb-Scargle+Periodogram.">Understanding the Lomb-Scargle Periodogram.</a> Cornell Univeristy Library. , (2017).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+Blue+Exclusion+Test+of+Cell+Viability.">Trypan Blue Exclusion Test of Cell Viability.</a> Current Protocols in Immunology. 111, (2015).</li><li>Ertel, E. A., Godfraind, T., Godfraind, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+channel+blockers+and+calcium+channels.">Calcium channel blockers and calcium channels.</a> Calcium Channel Blockers. , 11-80 (2004).</li><li>Sanguinetti, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+potassium+channels+by+antiarrhythmic+and+antihypertensive+drugs.">Modulation of potassium channels by antiarrhythmic and antihypertensive drugs.</a> Hypertension. 19, 228-236 (1992).</li><li>Duran-Riveroll, L. M., Cembella, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guanidinium+Toxins+and+Their+Interactions+with+Voltage-Gated+Sodium+Ion+Channels.">Guanidinium Toxins and Their Interactions with Voltage-Gated Sodium Ion Channels.</a> Marine Drugs. 15 (10), (2017).</li><li>Sharma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+of+tyrosine+kinase+inhibitor+cardiotoxicity+with+human+induced+pluripotent+stem+cells.">High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells.</a> Science Translational Medicine. 9 (377), (2017).</li><li>Puppala, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+gene+expression+profiling+in+human-induced+pluripotent+stem+cell--derived+cardiocytes+and+human+and+cynomolgus+heart+tissue.">Comparative gene expression profiling in human-induced pluripotent stem cell--derived cardiocytes and human and cynomolgus heart tissue.</a> Toxicological Sciences. 131 (1), 292-301 (2013).</li></ol>

The authors have nothing to disclose.

Two aspects are most critical for the successful recording of beating frequencies. The first is to exercise caution with cell plating and culture. In particular, it is important to try and avoid scratching the cell layer at the bottom of the wells when exchanging the medium. It is acceptable to touch the bottom of the wells with the pipettes, but the same angle should be used every time, thereby producing only a tiny scratch in the cell layer and not affecting the assay performance. The second critical aspect of obtaining reproducible homogeneous rhythms is to provide good temperature control at 37 &#176;C across the cell plate for the duration of the measurement. We could not obtain this homogeneity using devices other than the plate reader used here, but it may be feasible with special modifications to the temperature regulation: it would make the protocol presented here more broadly usable beyond a single brand of plate reader. To achieve temperature stability for the duration of the experiment with the device used here, it was necessary to stop each recording before it ended; otherwise, the robot would eject the measured cell plate. This technical issue may disappear with the next release of the plate reader software, but it remains critical for now. If a cell plate is mistakenly transferred outside the plate reader, it must be loaded back inside as fast as possible. Nevertheless, the quality of the experiment will deteriorate, because temperature changes affect the beating rate extremely rapidly.
Some other aspects, which have not been tested thoroughly, may be less important. For example, hiPSC-CM manufacturers recommend coating cell-culture plates before seeding the cells, but in this specific assay, coating was not used, because the cells adhere quite easily on various surfaces, and it is very difficult to properly coat 384-well plates. Yet, cell plate coating may still be allowable, or it may even improve the assay quality. We also never tested whether solvents other than DMSO would be acceptable, but it is expected from experience with other recording technologies that similar concentrations of EtOH or MeOH would also be tolerable. We generally use hiPSC-CMs from the same manufacturer, and cells from only one additional supplier were tested, which appeared to work in a similar manner. Likewise, we have used only a small number of different batches of hiPSC-CMs that were selected by prechecking them to verify that they behaved similarly to the initial batch. One or two batches were deemed inappropriate because their syncytia had poor stability or reproducibility under the culture conditions used here. Otherwise, the pharmacology appeared very similar across batches when testing a limited panel of &#34;typical&#34; compounds (forskolin, N6-cyclopentyl- adenosine, and E-4031, as well as endothelin, isoproterenol, amlodipine, and ponesimod). We only used hiPSC-CM derived from healthy donors. It may be worthwhile to assess whether hiPSC-CM derived from patients with heart disease would provide different results, although no difference between healthy donors and patients was observed when evaluating the cardiotoxicity of tyrosine kinase inhibitors12. Finally, we normally wait 22&#8211;28 days in culture before measuring drug effects: in our experience with impedance recordings of the same cells, a steady-state for slow impedance (an indicator of cell layer stability) and fast impedance (an indicator of beating frequency) is reached after 12&#8211;15 days in culture. However, we decided to wait 22&#8211;28 days, because this is the time when the expression profile of cardiac channels and maturation markers has stabilized13. It was not examined whether comparable results would be obtained if the cells were used earlier or later.
The protocol described here uses a very straightforward measurement of the spontaneous beating rate of hiPSC-CM to evaluate potential drug effects on human cardiac electrophysiology. Its main advantages over other methodologies are that i) it is amenable to a high-throughput screening environment, ii) it records the activity of the cardiomyocytes and the effects of drugs at physiological temperatures, and iii) it does not require electrophysiological expertise for the execution or for an assessment of results.
In a validation study performed with many drugs approved for human use, we showed that the assay reacts to drugs used in human medicine as predicted by existing clinical data5. Because this method considers all potential effects on cardiac rhythm, it complements the Comprehensive in vitro Proarrhythmic assay (CiPA) initiative14 that specifically assesses pro-arrhythmic potential.
In the future, this method could provide a further understanding of the mode-of-action of drugs shown to affect the spontaneous beating rate. It is likely that additional mechanistic information is present in the fluorescence recordings of Ca transients (e.g., in their amplitude or shape). If the fluorescence recordings are performed at higher acquisition rates (e.g., 30 Hz), these parameters are easily extracted in addition to beating rate, and it may be interesting to correlate changes in these parameters with the known effects of clinically used drugs.

The present protocol depicts a method to measure drug effects on the spontaneous beating frequency of syncytia of hiPSC-CM at physiologically relevant rhythms. Human-induced pluripotent stem cells can differentiate into functional cardiomyocytes that establish spontaneously beating syncytia in vitro1,2,3,4. These hiPSC-CM can be obtained in large numbers through commercial providers or through production in the laboratory, and they are a useful source of cells to generate models of human cardiac physiology and pharmacology. In particular, they can be used to predict or to characterize cardiac effects that may occur when a drug is administered to humans5.
The beating frequency of hiPSC-CM syncytia can be measured under physiological conditions using microelectrode arrays or impedance sensing1: these noninvasive techniques provide very detailed information on drug effects, but they are rather low-throughput and they do not enable testing large compound libraries within realistic time and budget constraints. A more effective system can be elaborated using a 384-well fluorescence imaging plate reader and a Ca-sensitive dye6, but classical plate readers are hindered by suboptimal temperature control and acquisition frequency. These limitations are illustrated by unphysiological beating rates (~15 bpm, compared to 35&#8211;55 bpm in controlled environments1) and poor Ca signal resolution (an acquisition frequency of 8 Hz is at the lower limit to record rates that can reach 120 bpm under stimulated conditions, and it cannot extract information such as slope or duration). The method described here combines the recording of beating patterns at physiological rhythms and at sufficient resolution to preclude these concerns.
On the positive side, this method is simple, reliable, and high throughput, which allows the rapid testing of large numbers of compounds at reasonable costs. On the negative side, this method requires a fast plate reader with effective temperature control, which is an expensive investment, and it provides little mechanistic information on observed drug effects, which may necessitate further testing with more detailed methods.
Approximatively 6 x 106 hiPSC-CM are needed for one measurement in one 384-well cell plate. hiPSC-CM are usually supplied commercially as frozen aliquots of ~4 x 106 cells in 1 mL. Therefore, it is convenient to prepare two cell plates with three frozen aliquots. In most cases, due to the low variability of this assay, it is sufficient to perform duplicate measurements of the test compounds and, in each cell plate, quadruplicate measurements of the positive controls (forskolin, N6-cyclopentyl-adenosine, and E-4031), and 20-plicate measurements of the negative control (DMSO alone). Therefore, a maximum of 352 compound/concentration pairs can be evaluated with two 384-well cell plates. The following protocol considers such an experiment performed with 352 test compounds, two cell plates, and 12 million cells supplied as three frozen aliquots; it can be easily scaled-up if additional data points are needed.

<table><tbody><tr><td>FDSS7000 fluorescent plate reader</td><td>Hamamatsu Photonics&lt;br/&gt; (Massy, France)</td><td>not a catalog item</td><td></td></tr><tr><td>FDSS7000 Fluo-3/Fluo-4/Fluo-8 fluorescence filter kit</td><td>Hamamatsu Photonics&lt;br/&gt; (Massy, France)</td><td></td><td>installed initially&lt;br/&gt; in the device</td></tr><tr><td>FDSS7000 temperature control</td><td>Hamamatsu Photonics&lt;br/&gt; (Massy, France)</td><td>A10118-09</td><td></td></tr><tr><td>FDSS7000 150-W Excitation Light Source Unit</td><td>Hamamatsu Photonics&lt;br/&gt; (Massy, France)</td><td>C11653-11</td><td></td></tr><tr><td>FDSS7000 pipet tips for 384-well plates</td><td>Hamamatsu Photonics&lt;br/&gt; (Massy, France)</td><td>A8687-62</td><td></td></tr><tr><td>Waveform Analysis Software for Cardiomyocytes</td><td>Hamamatsu Photonics&lt;br/&gt; (Massy, France)</td><td>U8524-12</td><td>optional alternative to the Igor Pro software</td></tr><tr><td>Igor Pro data analysis software</td><td>Wavemetrics&lt;br/&gt; (Portland, Oregon, USA)</td><td>Latest version</td><td></td></tr><tr><td>iCell-Cardiomyocytes Kit</td><td>Cellular Dynamics International (Madison, WI)</td><td>R1106</td><td>includes cells, plating and maintenance media</td></tr><tr><td>Cor.4U Cardiomyocyte Kit</td><td>Ncardia Germany&lt;br/&gt; (Cologne, Germany)</td><td>Ax-B-HC02-4M</td><td>includes cells, plating and maintenance media&lt;br/&gt; alternative to the iCell-Cardiomyocytes</td></tr><tr><td>384-well cell culture plates</td><td>Greiner-Bio-One (Frickenhausen, Germany)</td><td>781091</td><td></td></tr><tr><td>384-well compound plates</td><td>Greiner-Bio-One (Frickenhausen, Germany)</td><td>781280</td><td></td></tr><tr><td>FLIPR Calcium 4 Assay Kit</td><td>Molecular Devices&lt;br/&gt; (Sunnyvale, California)</td><td>R8142</td><td></td></tr><tr><td>Forskolin</td><td>Sigma-Aldrich&lt;br/&gt; (Buchs, Switzerland)</td><td>F6886</td><td></td></tr><tr><td>N6-cyclopentyl-adenosine</td><td>Sigma-Aldrich&lt;br/&gt; (Buchs, Switzerland)</td><td>C8031</td><td></td></tr><tr><td>E-4031</td><td>Enzo Life Sciences&lt;br/&gt; (Lausen, Switzerland)</td><td>BML-KC158</td><td></td></tr><tr><td>Penicillin-Streptomycin solution</td><td>Gibco/ThermoFisher&lt;br/&gt; (Reinach, Switzerland)</td><td>15140-122</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Gibco/ThermoFisher&lt;br/&gt; (Reinach, Switzerland)</td><td>15250061</td><td></td></tr></tbody></table>

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.

Figure 1 shows a representative recording of Ca waves at baseline across one 384-well plate. In most cases, all wells reveal a regular beating rate with little variability across the plate (mean &#177; SD = 40.1 &#177; 3.5 bpm; range = 34&#8211;54 bpm). Very occasionally (less than 1 out of 10 experiments), a few wells (less than 5) have arrhythmia or absence of rhythm. In three 384-well plates, we have also tried cardiomyocytes from another supplier (see Table of Materials) and obtained very similar baseline values for the spontaneous beating.
Blockers of cardiac ion channels affect the rhythm of the cardiomyocytes in a reproducible manner5. Amlodipine, a blocker of the slow Ca channels9, accelerates the rhythm more than 100% in a concentration-dependent manner up to 1 &#181;M (Figure 2A). The effect is well developed within the first 5 min, but it still increases approximatively 50% within the next 30 min before it stabilizes. Above 1 &#181;M, amlodipine arrests the beating (dashed lines), as can be expected from the fact that Ca cycling is critical to the spontaneous contractions. Other selective dihydropyridine Ca channel blockers produce very similar effects.
E-4031, a blocker of the rapid delayed rectifier K channels10, decelerates the rhythm about 65&#8211;70% in a concentration-dependent manner up to 10 &#181;M (Figure 2B). The effect develops within the first 5 min and it does not vary within the next 60 min. Other selective IKr blockers produce similar effects, although the maximal reduction in rate can vary (e.g., 35&#8211;40% for cisapride, 70% for E-4031, and 90% - 95% for dofetilide). The basis for this disparity is not known.
Tetrodotoxin, a blocker of the fast Na channels11, decelerates the rhythm about 15% in a concentration-dependent manner, up to 1&#8211;30 &#181;M (Figure 2C). The effect is well developed within the first 5 min and it does not vary much within the next 60 min. However, above 30 &#181;M, tetrodotoxin arrests the beating within the first 5 min, whereas after 30 min, it arrests it above 1 &#181;M. Other Na channel blockers, such as lidocaine or mexiletine, produce similar all-or-nothing effects.
Bepridil, a mixed blocker of cardiac Na, Ca, and K channels9, also produces an all-or-nothing effect (Figure 2D). This suggests that in this preparation, a block of Na channels is the primary action of bepridil. An acceleration due to Ca channel block is not seen, and the more progressive slowing due to IKr block appears masked by the Na channel effect.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58290/58290fig1.jpg" /> 
Figure 1: Representative baseline Ca waves across a 384-well plate. (A) All wells across the plate demonstrate a regular beating rate with little variability. This image is captured directly from the plate reader software. Each trace is 10 s in duration. The fluorescence intensity is arbitrary (relative light units derived from the video camera greyscale). (B) This blow-up of one 10 s trace shows the low noise of the signal. <a href="https://www.jove.com/files/ftp_upload/58290/58290fig1large.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/58290/58290fig2.jpg" /> 
Figure 2: Representative effects of cardiac ion channel blockers. These panels show concentration-response curves with (A) the selective Ca channel blocker amlodipine, (B) the selective IKr blocker E-4031, (C) the selective Na channel blocker TTX, and (D) the non-selective channel blocker bepridil. <a href="https://www.jove.com/files/ftp_upload/58290/58290fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates at JoVE.com

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 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 ...

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6.2K Views.  Idorsia Pharmaceuticals Ltd. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van hartveiligheid farmacologie. Heeft een nieuw menselijk geneesmiddel bijvoorbeeld het risico om het normale hartritme aan te passen? Het belangrijkste voordeel van deze techniek is dat het grote aantallen verbindingen snel en tegen een gematigde prijs kan evalueren.Het aantonen van de procedure zal Camille Forny zijn, een onderzoeksmedewerker in mijn laboratorium. Om voor te bereiden op de meting van d...

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

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

Immunology and Infection

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Biochemistry

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Bioengineering

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 ...

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.

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 ...

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

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 ...

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

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