This method enables to record intracellular alike action potentials on traditional MEA'S, thereby allowing a better description of the AP shape and a more sensitive classification of pro-arrhythmic potentials. Compared to typical field potential recordings the intracellular alike actual potential shape provides more insight into the precise authorization of the underlying ion channels. When applied to cardiomyocytes derived from clinical patients through stem cell technology, the method can contribute to causal diagnosis, and personalization of C R P and, cardiac diseases like arrhythmias Our technician, Karin Gebhardt will demonstrate the cardiomyocyte cultivation.
To begin coat the electrode fields of the previously autoclaved MEA's, Dropwise with fibronectin using a 10 microliter pipette under the laminar flow hood. To reduce the osmotic shock, transfer the plating solution dropwise for 90 seconds into the 50 milliliter tube containing thawd cardiomyocytes. Gently add eight milliliters of plating medium into the tube and mix the cell suspension carefully.
Using a 10 milliliter pipette, remove the supernatant, ensuring not to discard the pellet and adjust the cell number to final concentration. Before cell seating remove the applied coating solution from the MEA electrode area using a 10 microliter pipette. To avoid drying of the coating seed the cells immediately by adding four microliters of the cells dropwise onto the electrode fields for both, six well MEA and one well M E A.Now, allow the cells to adhere to the wells for one hour at 37 degrees Celsius, and 5%carbon dioxide.
Under the laminar flow hood, add 200 microliters and one milliliter of sterile plating medium heated to 37 degrees Celsius for six Well MEA, and single well MEA respectively. For MEA recordings, place the MEA system on top of the device with the MEA chip holder centered over the objective hole. Then allow the laser to focus on the electrodes by positioning the MEA setup, so that the objective is directly under the hole of the MEA system.
To allow the cells to recover from the mechanical disturbance, transfer the MEA chip with the cultivated cells from the incubator to the MEA setup 15 minutes before the recording. Next, clean the contact pads and pins carefully, using an isopropanol swab to decrease the noise levels. Place the MEA carefully in the MEA setup, and position the six well MEA chip with the serial number visible on the right hand side or a single well MEA chip with the reference electrode to the left.
Now, set the MEA system integrated heating to 38 degrees Celsius. Close the lid of the device as the integrated safety switch only allows the laser to be activated If the lid is closed over the MEA chip. Using the MEA config program set the high pass and low pass MEA system filter to 0.1 hertz or less and 3, 500 hertz respectively.
Use the MC rack software or any alternative software for recording. Adjust the input range according to the experiment. Ensuring the signal does not saturate the amplifier and the sampling rate.
Use the long-term display function of the software to check the recording. After inserting the MEA chip into the MEA setup, and setting up the software initialize the laser mechanics using the FB ALP software. Now, click on the initialization button.
At the end of initialization the virtual laser point will be in well D for a six well MEA and at the bottom left for a single well MEA respectively. Then using the control key with a mouse click move the virtual laser point into the middle of the D five electrode and adjust the focus by holding the control key and scrolling with the mouse wheel. Alternatively, use the auto focus function.
Press the button set P one, and the virtual laser point will automatically move into the well F.The system is now aligned. Adjust the laser power and process time according to the experimental requirements. To enable the laser, click on the laser off button which will appear as laser on, Switch to the recording software.
Choose a file name and click on the red recording button followed by the play button on top of the window to record the measurement. Before opening the cells with the laser record a baseline for 60 seconds. Switch back to the initialization software and deactivate electrodes to be excluded by the laser on the virtual map on the right side of the software window.
To start the laser use Alt mouse click, and select the center electrode of this well. This initiates the laser to open the cells on each electrode of this well automatically, repeat for each well to open all the previously activated electrodes of this selected well. Dissolve millimolar concentration of Nifedipine E-40 31, and Dofetilide in DMSO first, and then in complete medium to 10 times the desired concentration.
Never exceed a final DMSO concentration of 0.1%in the well. Record the baseline activity for 60 seconds. Start the laser induced peration as demonstrated earlier, resulting in a transformation of the FPS into liAPs.
Apply all the drugs as single concentration per well. Remove 20 microliters and 100 microliters of medium per well from six well and single well MEA's respectively. Add 20 microliters or 100 microliters of the drug stock solution to be measured to the well.
Depending on the MEA type, and carefully pipette up and down two to three times. Allow the compounds to wash in for 300 seconds. During this time, the liAP shape may transform into the FP shape.
Again, induced laser induced peration and record possible compound induced defects on the liAP for an additional 60 seconds. The addition of nifedipine reduced the plateau phase of the liAPs in a concentration dependent manner, and thereby shortened the entire liAP. This shortening was comparable to the field potentials of cardiomyocytes from unmanipulated electrodes.
E-40 31 inhibited the repolarization of relevant KV one point 11 potassium channels and led to the arrhythmic behavior of cardiomyocytes at increased concentrations. Also, E-40 31 increased the liAP duration in a concentration dependent manner. At the end of the liAP small positive voltage deflections were observed at 0.1 micromolar which became more prominent with higher concentrations, indicating a transient new depolarization called EAD's.
At the highest concentration of 0.1 micromolar, these EAD's escalated over time into ectopic beats. Which are premature action potentials. EAD and ectopic beats are key indicators of pro arrhythmic activity.
And in the end, the electrical activity results in arrhythmic beating. The concentration response relationships correlated with FP's and liAP recordings. Further, in the presence of dofetilide, both FP and liAP displayed durations of approximately two seconds within the same well, the FP waveform presented regular repolarization deflections.
While in the liAP EAD's are detectable. It's important to adjust the focus before each melomen as an out of focus microscope image could affect the opening of the cells. Also, action potential is taken shortly after opterperation should be used for analysis.
This technique is more sensitive in detecting prior arrhythmic effects compared to standard MEA, allowing more precisely the detection of adverse compound effects.
