Name: contractions of human-ipsc-derived cardiomyocyte syncytia measured with a ca-sensitive fluorescent dye in temperature-controlled 384-well plates
Uploaded: 2018-10-18T13:30:00
Duration: 7 min 42 s
Description: 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

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 
			(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 
			(Massy, France)</td>
			<td></td>
			<td>installed initially 
			in the device</td>
		</tr>
		<tr>
			<td>FDSS7000 temperature control</td>
			<td>Hamamatsu Photonics 
			(Massy, France)</td>
			<td>A10118-09</td>
			<td></td>
		</tr>
		<tr>
			<td>FDSS7000 150-W Excitation Light Source Unit</td>
			<td>Hamamatsu Photonics 
			(Massy, France)</td>
			<td>C11653-11</td>
			<td></td>
		</tr>
		<tr>
			<td>FDSS7000 pipet tips for 384-well plates</td>
			<td>Hamamatsu Photonics 
			(Massy, France)</td>
			<td>A8687-62</td>
			<td></td>
		</tr>
		<tr>
			<td>Waveform Analysis Software for Cardiomyocytes</td>
			<td>Hamamatsu Photonics 
			(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 
			(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 
			(Cologne, Germany)</td>
			<td>Ax-B-HC02-4M</td>
			<td>includes cells, plating and maintenance media 
			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 
			(Sunnyvale, California)</td>
			<td>R8142</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma-Aldrich 
			(Buchs, Switzerland)</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>N6-cyclopentyl-adenosine</td>
			<td>Sigma-Aldrich 
			(Buchs, Switzerland)</td>
			<td>C8031</td>
			<td></td>
		</tr>
		<tr>
			<td>E-4031</td>
			<td>Enzo Life Sciences 
			(Lausen, Switzerland)</td>
			<td>BML-KC158</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Gibco/ThermoFisher 
			(Reinach, Switzerland)</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco/ThermoFisher 
			(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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Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Shrinkage of Dental Composite in Simulated Cavity Measured with Digital Image Correlation

Polymerization shrinkage of dental resin composites can lead to restoration debonding or cracked tooth tissues in composite-restored teeth. In order to understand where and how shrinkage strain and stress develop in such restored teeth, Digital Image Correlation (DIC) was used to provide a comprehensive view of the displacement and strain distributions within model restorations that had undergone polymerization shrinkage.
Specimens with model cavities were made of cylindrical glass rods with both diameter and length being 10 mm. The dimensions of the mesial-occlusal-distal (MOD) cavity prepared in each specimen measured 3 mm and 2 mm in width and depth, respectively. After filling the cavity with resin composite, the surface under observation was sprayed with first a thin layer of white paint and then fine black charcoal powder to create high-contrast speckles. Pictures of that surface were then taken before curing and 5 min after. Finally, the two pictures were correlated using DIC software to calculate the displacement and strain distributions.
The resin composite shrunk vertically towards the bottom of the cavity, with the top center portion of the restoration having the largest downward displacement. At the same time, it shrunk horizontally towards its vertical midline. Shrinkage of the composite stretched the material in the vicinity of the &#8220;tooth-restoration&#8221; interface, resulting in cuspal deflections and high tensile strains around the restoration. Material close to the cavity walls or floor had direct strains mostly in the directions perpendicular to the interfaces. Summation of the two direct strain components showed a relatively uniform distribution around the restoration and its magnitude equaled approximately to the volumetric shrinkage strain of the material.

In order to understand the spatial development of polymerization shrinkage stress in dental resin-composite restorations, Digital Image Correlation was used to provide full-field displacement/strain measurement of restored model glass cavities by correlating images of the restoration taken before and after polymerization.

Polymerization shrinkage of dental resin composites can lead to restoration debonding or cracked tooth tissues in composite-restored teeth. In order to ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular (LV) filling compared with a continuous linear jet. Vortex ring development is most often quantified with vortex formation time (VFT), a dimensionless parameter based on fluid ejection from a rigid tube. Our group is interested in factors that affect LV filling efficiency during cardiac surgery. In this report, we describe how to use standard two-dimensional (2D) and Doppler transesophageal echocardiography (TEE) to noninvasively derive the variables needed to calculate VFT. We calculate atrial filling fraction (&#946;) from velocity-time integrals of trans-mitral early LV filling and atrial systole blood flow velocity waveforms measured in the mid-esophageal four-chamber TEE view. Stroke volume (SV) is calculated as the product of the diameter of the LV outflow track measured in the mid-esophageal long axis TEE view and the velocity-time integral of blood flow through the outflow track determined in the deep transgastric view using pulse-wave Doppler. Finally, mitral valve diameter (D) is determined as the average of major and minor axis lengths measured in orthogonal mid-esophageal bicommissural and long axis imaging planes, respectively. VFT is then calculated as 4 &#215; (1-&#946;) &#215; SV/(&#960;D3). We have used this technique to analyze VFT in several groups of patients with differing cardiac abnormalities. We discuss our application of this technique and its potential limitations and also review our results to date. Noninvasive measurement of VFT using TEE is straightforward in anesthetized patients undergoing cardiac surgery. The technique may allow cardiac anesthesiologists and surgeons to assess the impact of pathological conditions and surgical interventions on LV filling efficiency in real time.

We describe a protocol to measure vortex formation time, an index of left ventricular filling efficiency, using standard transesophageal echocardiography techniques in patients undergoing cardiac surgery. We apply this technique to analyze vortex formation time in several groups of patients with differing cardiac pathologies.

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Intrathecal Application of a Fluorescent Dye for the Identification of Cerebrospinal Fluid Leaks in Cochlear Malformation

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such as meningitis. A method is presented here using intrathecal administered fluorescein in a clinical case of bilateral congenital ear malformation. A fluorescent dye is administered intrathecally to achieve intraoperative visualization of CSF leaks. The dye is applied 20 min before surgery, and concentration of 5% is used. Per every 10 kg of body weight, 0.1 mL of the fluid is applied intrathecally. The fluorescein is visualized using a fully digital microscope. The origin of the fluid leak is identified in the stapes footplate. During primary surgery, it is sealed, and cochlea implantation is performed for hearing restoration. In this specific case, 6 weeks later, the implant was explanted due to acute meningitis, and the electrode array was left as a spacer. Postoperatively, in the aural smear, &#946;-transferrin was detected. During a revision mastoidectomy, dislocated coverage of the leak was found. The stapes was removed and oval window sealed. Five days after revision surgery, no &#946;-transferrin was detected in the aural smear. During the revision of cochlea implantation 6 months later, intact coverage of the oval niche was observed. Thus, intrathecal fluorescein application proves to be a reliable tool for the detection of CSF leaks. It facilitates the orientation in malformations and complicated or unknown surgical situs. In the literature, its use is described for CSF fistulas in endonasal surgery but is rarely described in skull base and mastoid surgeries. The method has been used successfully in several cases with CSF leaks, and the results confirm the feasibility of safely accessing the origin of the leak.

Intrathecally applied fluorescein is used to achieve intraoperative visualization of CSF leaks. This protocol describes a lumbar puncture, the application of 5% fluorescein, and intraoperative visualization using a fully digital microscope.

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such ...

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...