The following protocol describes the development of human-induced pluripotent stem cell-derived cardiomyocyte networks on multiwell MEA plates to reversibly electroporate the cell membranes for action potential measurements. Action potential parameters can then be used to generate those response curves to test compounds for electrophysiology. Multiwell MEA coding and cell plating are the most challenging steps that need special attention.
One must perform these steps diligently and swiftly to prevent droplets spreading and drying out. With practice, this can be easily overcome. We developed a custom graphical user interface for single segmentation, quality assurance and parameter extraction.
Our robust MATLAB workflow was implemented to rapidly reduce large volumes of raw experimental data into an unbiased grouping of action potential waveforms. Begin by thawing human-induced pluripotent stem cell-derived cardiomyocytes according to manuscript directions. Gently suspend the cells using a transfer pipette to dissociate cell clumps.
Then carefully dispense two milliliters of cell suspension into each well of a substrate-coated six-well plate and place the plate in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide. Cells should adhere to the gel tracts by 24 hours and beat spontaneously at 48 hours post-plating. Two days prior to plating add 0.5 milliliter of human iPSC cardiomyocyte culture medium to each well of a 24-well multi-electrode array, or MEA plate, and perform a baseline recording to verify signal-to-noise ratio as a quality check of the MEAs.
Then, aspirate the media, rinse the wells with sterile water, and sterilize under a UV light in a laminar flow hood overnight. On the next day, add 0.1 milliliter of FBS to each well for hydrophilic treatment of MEA surfaces and incubate the plate at room temperature for 30 minutes. Then aspirate the FBS, and rinse each well twice with 0.5 milliliters of sterile water.
Leave the plate to dry in the laminar flow hood overnight. On the following day, pipette five microliters of working fibronectin dilution and carefully dispense the droplet in the center of each well to cover all 12 electrodes. Immediately place the plate on a raised surface inside a humidifying chamber containing enough sterile water to cover the entire dish surface.
Place the chamber with the plate in the cell culture incubator for three hours and then proceed with cell plating. When ready to plate the cardiomyocytes, adjust the cell density to 6, 000 cells per microliter by diluting them in human iPSC cardiomyocyte thawing medium. Use gentle flicking to keep the cells from sedimenting.
Bring the MEA plate into the laminar flow hood and carefully remove the fibronectin drop with a P10 pipette without touching the electrodes. Immediately dispense a five-microliter cell droplet to the center of the well making sure to cover all 12 electrodes. Do this one well at a time to prevent fibronectin from drying.
When finished plating, place the MEA plate back in the loosely covered humidifying chamber and return it to the cell culture incubator for three hours. After the incubation, use a P200 pipette to carefully add 200 microliters of human iPSC cardiomyocyte thawing medium to each well without disturbing the cells. Place the MEA plate back in the cell culture incubator and replace culture medium 24 hours after plating.
When ready to acquire signal from the cardiomyocytes, initiate the acquisition software and insert the MEA plate according to manuscript directions. Click the Explore button to visualize the signal in all wells and verify the signal quality in steady state conditions. Take notes on electrodes with field potential, or FP, signals in the millivolt range.
Then click the same button to stop the exploration. No data has been recorded up to this point. Click the Go button to start recording.
The electrodes in each well will show FP signals in the raw data window. After 30 seconds of recording, click on the Stimulate button and allow electroporation to take place on the selected sites for 30 seconds. Then click on the same button to stop simulation and continue recording for 60 seconds.
Use the MATLAB-based custom software to segment and extract various field potential and action potential data parameters. First, run the waveform analysis code and click on File and select Process.h5. Find and select the previously created mwd.
h5 file. Click on the Save Directory button to change the storage location of the output files. Then create a signal processing queue by selecting the electrode and well combinations of interest and then clicking the Queue button.
Repeat this step to add more electrode and well combinations to the queue. If cells were treated with drugs, the queue can be edited by clicking directly on Med Name, Med Concentration. Once the queue has been finalized, click the Initialize Waveforms button, which will start the preliminary processing where the signals are identified and extracted for segmentation.
When processing is finished, click on the Zoom In button, and select the action potential, or AP, area of interest. Click the Keep button, and review the panels. The peaks and troughs are detected for every waveform, and the normalized APs are superimposed.
When finished, click the Keep button to move on to the next trace in the queue, and repeat the process for the rest of the electrode and well combination signals. The viability and plating density of the post-thawed cardiomyocytes is critical for multiwell MEA culture. Proper plating results in a healthy monolayer culture with spontaneous beating at 48 hours, while poor cell viability results in cultures with a high percentage of non-myocyte populations.
Cell droplet dispersion affects culture density and can even lead to cell death, so precise cell placement is important. The cells cultured on MEAs are subjected to a quality check for electrical activity 48 hours after plating. If 50%of the electrodes within a network and 70%of the total networks do not produce FP signals, then the culture is suboptimal.
Electroporation mediated AP recordings can be obtained multiple times 48 hours post MEA plating. Multiple electroporations of the same cell site at zero, 24, 48, 72, and 96 hours have no significant effect on the AP shape over time. Additionally, no correlation between FP and post electroporation AP amplitudes from the same cell site has been observed.
A significant advantage of this method is that the multiwell MEA plate can be reused multiple times. To demonstrate the reliability of this array, 3, 815 AP waveforms are pulled from three restoration batches, and AP duration data is extracted to examine repeatability of the results. When of interest, it is possible to perform additional assays, such as gene expression, calcium transient measurements and patch clamp to investigate the behavior of specific ionic currents.
This technique paves the way for researchers to enhance electrophysiological maturation as well as to screen chronic dose effects on cardiomyocytes.
