다중 전극 배열 (MEAs)을 이용한 인간 다 능성 줄기 세포 유래 심근 세포 (hPSC-CMs)의 전기 생리 학적 분석

The overall goal of this procedure is to dissociate cardiomyocytes derived from human pluripotent stem cells and plate them on multi-electrode arrays to measure and quantify the duration and the frequency of their typical electrical signal also called field potential. This method can help answer key questions in the field, such as what are the electrophysiological differences between cardiomyocytes carrying mutations and controls. The main advantage of this technique is that it allows drug screening on human cardiomyocytes derived from patient cells.

Visual demonstration of this method is critical. As the dissociation and plating steps are essential for successful electrical signal recording. Generally individuals new to this method will struggle because the identification of good or bad quality electrical signals is not trivial and the analysis of free potential requires experience.

To begin this procedure place the clean chips in a standard 10 centimeter diameter plastic sterile Petri dish. Next add eight milliliters of sterile distilled water to the Petri dish to form a humidified chamber which will prevent the small volume of culture medium on the chip from drawing when placed in the incubator. Then use custom made PolyTetraFluoroEthylene rings to ensure plating of the cardiomyocytes in the center of the chip where the electrode array is located.

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. Then place a dry ring inside an MEA chip using flame sterilized tweezers.

Coat the electrodes by adding 50 microliters of 40 micrograms per milliliter fibronectin inside the ring. After that cover the Petri dish with the lid and carefully transfer the dish containing the MEA chip into an incubator. Following that transfer the dish containing the MEA chip to the cell culture hood.

Before using the MEA chip, aspirate 50 microliters of fibronectin using a P200 pipette or a vacuum system in the hood without displacing the PTFE ring. Ensure that no solid objects touch the electrodes by keeping the tip angled. Then gently add 950 microliters of LI-BPEL medium ensuring that it is evenly distributed and that the ring does not float.

Subsequently return the dish to the incubator. This is the initial culture of the beating cardiomyocytes. In a tissue culture hood, aspirate the medium from the hPSC-CMs culture well.

Add one to two milliliters of PBS without calcium and magnesium to each well to wash the culture and aspirate the PBS. Afterward add 500 micro liters of the disassociation enzyme to each well. Incubate the sample for 5 minutes at 37 degrees Celsius.

After five minutes add one milliliter of LI-BPEL to each well to dilute the enzyme. Detach the monolayer of hPSC-CMs by gently scratching them with a P1000 pipette. Then collect the cell suspension in a 15 milliliter tube.

Next rinse the well with one milliliter of LI-BPEL to collect all the remaining cells and cell clumps. Add another two to three milliliters of LI-BPEL to reach a final volume of around five to six millimeters and gently pipette up and down three to five times with a five milliliter pipette to dissociate cell clumps. Following that, centrifuge the cells at room temperature for three minutes at 300 times g.

After that remove the supernatant without dislodging the cell pellet. Then re-suspend the cell pellet in 250 microliters of LI-BPEL. Subsequently distribute 50 microliters of cell suspension per MEA by pipeting the cell suspension directly into the center of the PTFE ring on top of the electrode array.

Carefully transfer the MEAs to the incubator at 37 degrees celsius and allow the cells to attach overnight. One day after plating, carefully remove the ring in a sterile environment using sterile tweezers. Rinse the ring in 80 percent ethanol.

And store it in a 50 milliliter tube containing 80 percent fresh ethanol. Next gently remove 500 micro liters of medium from the MEA chip. And add 500 micro liters of fresh LI-BPEL to it.

These are the beating cardiomyocytes after ring removal. Afterward transfer the MEAs to the incubator at 37 degrees Celsius. Then measure the electrical activity of hPSC-CMs on MEAs starting from one day after ring removal and up to one week.

Now launch the software suite linked to the MEA setup. Next set the temperature to 37 degrees Celsius in TCX-Control to record the measurements. After that remove the dish containing the MEA chip from the incubator.

Take out the MEA chip from the dish and place it on a tissue to absorb the residual water. Carefully wipe the external context of the plate with a tissue and clean it using a cotton swab moistened with 100 percent ethanol to remove any residual water or debris which might cause signal noise. Then transfer the MEA plate to a 37 degrees Celsius recording head stage to detect spontaneous activity.

Open MC Rack and click edit to load the MEA hardware. To create a new protocol, use the drop down menu edit to add different recorder and display windows to the protocol. Subsequently start the protocol in play mode by clicking on the play button.

If the signals exhibit visible R peaks and T peaks clearly wait 10 to 15 minutes to conclude the adaptation phase. To start the experiment, click record and then play and acquire data for 10 minutes under the baseline conditions to determine steady state. Annotate the electrodes that had the best signal so that they can be easily identified and exported later for analysis.

For a drug response assessment, add increasing concentrations of drug every 10 minutes. Click stop to conclude the recording at the end of the protocol. Here are the representative traces recorded with an MEA showing a good quality trace with RQ and T peaks clearly visible with high signal to noise ratio.

A bad quality trace without clearly visible RQ and T peaks And a noisy trace with RQ and T peaks clearly visible but with low signal to noise ratio. And here are the representative examples of good quality field potential traces with different morphologies that can be recorded during MEA experiments using hPSC-CMs. The shaded area represents the QT interval measured during the analysis.

Since the field potential at the MEA resembles the first derivative of the action potential, the integral of the field potential traces have been calculated as theoretical demonstrations of the T waves that are close to the complete action potential re-polarizations. While attempting this procedure, it's important to remember to go for the highest number of electrodes to ensure reliable and consistent signals. Following this procedure, other methods like quanto tibial time PCR or single cell patch clamp can be used in order to answer additional questions like specific gene expression related to electrical phenotype or specific ion-current densities.

After its development this technique paved the way for researchers in the field of stem cells, to explore cardiac arrhythmias and in-vitro pharmacology using cardiomyocytes derived from human pluripotent stem cells. After watching this video you should have a good understanding of how to dissociate and plate human pluripotent stem cell derived cardiomyocytes on the MEAs for electrophysiological measurements and drug screening. Do not forget that working with drugs can be extremely hazardous and precautions such as standard GLP should always be taken while performing this procedure.