Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

1. Preparation of Solutions and Reagents

1. Prepare hPSC-CM culture medium using low-insulin, bovine serum albumin, polyvinylalcohol, essential lipids (LI-BPEL) medium^34,35,36 by combining the reagents described in Table 1. Filter the medium through a 0.22 µm pore filter and store at 4 °C for up to 2 weeks.
2. Prepare human recombinant fibronectin stock solution by reconstituting 1 mg of human recombinant fibronectin in 5 mL of sterile distilled water to reach 200 µg/mL concentration, aliquot, and store at -80 °C.
3. Prepare 1% (w/v) enzyme detergent solution (see Table of Materials), to help removal of residual cells from the microarray chip, by dissolving 1 g of enzyme detergent powder in 100 mL of warm (~40-45 °C) deionized water. Allow the solution to cool down and store at 4 °C for up to one year.

2. Sterilization of MEA Chips (Figure 1A)

NOTE: Several different configurations of the MEAs are available, with single- or multi-well formats. The protocol described here uses the single chamber MEA containing 60 recording electrodes in an 8 x 8 grid arrangement (see Table of Materials). The electrode diameter is 30 µm and the distance between electrodes is 200 µm. One reference electrode is also present.

1. Rinse the chip thoroughly with deionized water.
2. Put the MEA chips inside a glass Petri dish that can be autoclaved. Wrap the dish in aluminum foil.
3. Sterilize the chips in a laboratory pressure cooker for 6 min. Allow the dishes to cool down before opening.
   NOTE: Alternatively, the chips can be sterilized by immersing them in 1 mL of 80% (v/v) ethanol at RT for 15-30 min.
4. Place the chips in a cell culture hood and expose the surface to UV light for approximately 30 min.

3. Coating of MEA Chips (Figure 1B and 1C)

1. Place the clean chips inside a standard 10 cm Ø plastic sterile Petri dish.
2. Add 8 mL sterile distilled water to the Petri dish to form a humidified chamber, which will prevent the small volume of culture medium on the chip to dry when placed in the incubator.
3. Use custom-made polytetrafluoroethylene (PTFE) rings (Figure 1B) to ensure plating of the cardiomyocytes in the center of the chip, where the electrode array is located. (The PTFE rings are previously stored in 80% ethanol.) In the culture hood, remove the rings from the ethanol, place them in a sterile Petri dish without a lid and allow the rings to dry in the hood.
4. Place a dry ring inside one MEA chip using flame-sterilized tweezers (Figure 1C).
   NOTE: Alternatively use 80% ethanol to wash the tweezers and allow to dry in the tissue-culture hood before using them.
5. Thaw one aliquot of human recombinant fibronectin stock (200 µg/mL in distilled water) and dilute in PBS with Ca²⁺ and Mg²⁺ to obtain a working solution of 40 µg/mL. Coat the electrodes by adding 50 µL of 40 µg/mL fibronectin inside the ring.
   NOTE: Coating using human recombinant fibronectin ensures optimal hPSC-CM attachment. However, other types of coating proteins such as bovine fibronectin or extracellular matrix proteins mixtures can be used.
6. Close the lid of the 10 cm Ø plastic Petri dish and carefully transfer the dish containing the MEA chip into the incubator. Incubate at 37 °C for at least 1 h, or at 4 °C O/N.
7. Transfer the dish containing the MEA chip to the cell-culture hood. Before using the MEA chip, aspirate the 50 µL of fibronectin using a P200 pipette or a vacuum system in the hood, without displacing the PTFE ring. Use plastic tips for this step. Ensure that no solid objects (e.g., pipette tips) touch the inside of the dish as this can damage the electrodes.
   NOTE: This will extend the life of MEA chips.
8. Gently add 950 µL of LI-BPEL medium (see Table 1 and Table of Materials), ensuring that it is evenly distributed and that the ring does not float. Return the dish to the incubator.

4. hPSC-CMs Dissociation and Plating (Figure 2)

NOTE: The protocol here described makes use of hPSC-CMs that were differentiated in a monolayer culture using cytokines³⁴ at ~18 days after starting the differentiation. However, it has been proven to be suitable for any 2D and 3D hPSC-CM culture. When using differentiated cultures at earlier or later time points, adjusting the incubation time of the dissociation enzyme (see Table of Materials) might be necessary. The following volumes are meant for a single well of a 12-well plate format (3.8 cm²).

1. Aspirate the medium from the hPSC-CMs culture well.
2. While working under a tissue culture hood, add 1-2 mL/well of PBS without Ca²⁺/Mg²⁺ to wash the culture. Aspirate the PBS.
3. Add 500 µL/well of the dissociation enzyme. Incubate for 5 min at 37 °C.
4. Add 1 mL LI-BPEL/well to dilute the enzyme. Gently detach the monolayer of hPSC-CMs by gently scratching using a P1000 pipette. Collect the cell suspension in a 15 mL tube.
5. Rinse the well with 1 mL LI-BPEL to collect all the remaining cells and cell clumps.
6. Add another 2-3 mL of LI-BPEL to reach a final volume of 5-6 mL and gently pipette up and down 3-5x with a 5 mL pipette to dissociate cell clumps.
   NOTE: Dissociation into single cells at this point is not necessary, since it will affect cell survival. Presence of small clusters will ensure higher cell viability.
7. Centrifuge the cells at RT for 3 min at 300 x g.
8. Remove the supernatant, trying to remove most of the surplus fluid but without dislodging the cell pellet.
9. Resuspend the cell pellet in 250 µL LI-BPEL (using a P1000 and pipetting extremely gently).
10. Distribute ~50 µL of cell suspension per MEA (up to 5 MEAs can be prepared from one well of a 12-well plate) by pipetting the cell suspension directly into the center of the PTFE ring, on top of the electrode array.
    NOTE: At this stage cells are difficult to count due to the presence of cell clusters and the total number of cells per MEA can substantially vary, depending on the hPSC-CM source used. While plating, ensure that the cloud of dissociated cells covers the area of the electrodes.
11. Carefully transfer the MEAs to the incubator at 37 °C and allow the cells to attach O/N

5. Ring Removal and Medium Refreshment (Figure 3)

1. At 1 day after plating, carefully remove the ring in a sterile environment using sterile tweezers (Figure 3A-3B).
2. Rinse the ring in 80% (v/v) ethanol and store it in a 50 mL tube containing fresh 80% (v/v) ethanol.
3. Gently remove 500 µL of medium from the MEA chip and add 500 µL of fresh LI-BPEL.
4. Transfer the MEAs to the incubator at 37 °C.
   NOTE: The cells should start beating 1-7 days after ring removal and medium change (Figure 3C).
5. Measure electrical activity of hPSC-CMs on MEAs 1-7 days after ring removal.

6. Check Signal Quality (Figure 4)

1. Switch on the computer and launch the software suite linked to the MEA set up: TCX-Control, MC_MEA Select and MC_Rack. Set the temperature to 37 °C in TCX-Control to record measurements at physiological temperature.
2. Remove the dish containing the MEA chip from the incubator. Open the lid, take out the MEA chip, and place it on a tissue to absorb residual water.
3. Carefully wipe the external contacts of the plate with a tissue and clean them using a cotton swab moistened with 100% (v/v) ethanol to remove any residual water or debris, which might cause signal noise.
4. Transfer the MEA plate to the heated (37 °C) recording head-stage to detect the spontaneous activity (e.g., hardware: see Table of Materials; software: MC_Rack).
   1. Open MC_Rack: Click 'Edit' → 'Add MC_Card' to create a new protocol. Use the drop down menu 'Edit' to add different Recorder and Display windows to the protocol.
      NOTE: A sampling frequency of at least 10 kHz is recommended. The protocol we use contains one Longterm Display Tool, with a full layout of the whole MEA chip, and one Spike Sorter, essential to capture drug effect in real time. The Spike Cutout is tuned with a 'Pre Trigger' of 20 ms, a 'Post Trigger' of 800 ms and a 'Dead Time' of 2 ms. The protocol can be saved as '.rck' file and reloaded before starting the experiments.
5. Start the protocol in play mode by clicking on the 'play' button.
   NOTE: At this point it is possible to reload the protocol saved under step 6.5. In MC_Rack, load the protocol (.rck file extension) by clicking 'File' → 'Open'.
6. If the signals exhibit clearly visible R peaks and T peaks, wait 10-15 min to conclude the adaptation phase. Examples of good and bad quality traces are shown in Figure 4.
   NOTE: If no T-peak can be detected in any of the electrodes, do not proceed with the experiment. Usually this is the result of poor electrical activity of the hPSC-CMs or poor attachment of the cells to the electrodes.

7. Start Experiment and Recording

1. Click 'record' and then 'play', and acquire data for 10 min under baseline conditions to determine the steady state. Annotate the electrodes that have the best signal so that they can be easily identified and exported later for the analysis.
2. For drug-response assessment, add increasing concentrations of drug at every 10 min. As an example, add the hERG blocker E4031 at a final concentration of 1 μM. For this, remove 100 μL of medium and add the same volume of 10 μM E4031 dissolved in the medium.
   NOTE: As previously demonstrated by Cavero and colleagues³¹, a wise choice of the volume in which the drugs are dissolved is important, since it can profoundly alter the drug response curve.
3. Repeat step 7.2 for all the other drug concentrations of interest.
4. Click 'stop' to conclude the recordings at the end of the protocol.

8. MEA Cleaning for Reuse

1. Once the experiment recording is finished, gently remove the medium with a P1000 pipette. Do not to touch the inside of the dish as this can damage the electrodes. Discard according to local safety rules.
2. Rinse the MEA chips with deionized water using a wash bottle, and repeat the wash step 3-4x.
   NOTE: At this point it is not necessary that the cells are completely detached from the chip.
3. Add 1 mL of enzyme detergent solution 1% (v/v) into each well and incubate O/N at 4 °C to allow cell detachment and cell removal.
4. One day later, rinse the MEA chips thoroughly with deionized water to remove enzyme detergent solution and residual cells and add 1 mL deionized water. Clean MEA chips can be stored immersed in deionized water at 4 °C.

9. Data Export

1. Open the MC_Data Tool software linked to the MEA set up.
2. Click 'File' → 'Open MCD'.
3. Click 'Tools' → 'Convert MCD to ABF'.
4. Select the electrodes with the best recording signals to be exported for the analysis. Choose the directory in which to save the exported files and click 'Save'. The export procedure may take several min depending on the number of the electrodes exported and the total size of the recording files.

10. Data Analysis

1. Download and install an electrophysiology data acquisition and analysis program (e.g., pClamp). Once complete, launch the analysis software (e.g., Clampfit).
2. RR Interval calculation (Figure 5).
   1. Place two vertical cursors to define the region of interest on the trace. Select 'Event detection' → 'Threshold Search'. The horizontal cursor should cross all the events that must be quantified, as shown in Figure 5A.
   2. Click 'OK' and then 'Accept the Entire Category'. The software will then search through the trace for all the suitable events. Blue marks will be placed above the events.
   3. Adjust the sensitivity of the automatic selection by tuning the parameters in the main event detection window.
   4. Once all the events have been automatically identified, go to the result window (Window → Results) and copy the column entitled "Intervent Interval", which contains the frequency data (Figure 5B).
3. QT Interval calculation (Figures 6-9):
   1. Place one cursor right before and one after a single FP at the steady state condition. Select 'Event Detection' → 'Create Template' (Figure 6).
   2. Check that the FP is correctly identified and click 'Add'. The template will be moved to the bottom panel.
   3. Save the template as a '.atf' file. In this way, a template trace has been created that will be searched by the software throughout the entire recording (Figure 7). Create one template for each condition, since a drug effect might alter the shape of the FP. If necessary, filter the trace slightly to include a maximum of 10.000 points within the two cursors.
   4. Once the template has been saved with '.atf' extension, select 'Event Detection' → 'Template Search' and load the template.
   5. Adjust the "Template match threshold" to correctly identify all the FP in the selected interval.
   6. Once all the events have been correctly identified, save them in a new '.abf' file.
4. Open the file with analysis software and automatically calculate the 'Time of Peak' for both the Q/R and the T peaks (Figures 8, 9). If traces are very noisy, apply a filter. Calculate the QT interval by subtracting the Q/R value from the T value in a spreadsheet editor of choice.