Human-induced pluripotent stem cell-derived cardiomyocytes represent a powerful tool for studying mutation mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. There's a need for a platform in which contractility and calcium handling of human-induced pluripotent stem cell-derived cardiomyocytes can be measured in a user-friendly and reproducible manner. This method enables researchers to study contractility of human induced pluripotent stem cell-derived cardiomyocytes by pixel correlation and measuring intercellular calcium transience simultaneously by loading the cells with calcium sensitive FloA-PhoR.
By using this platform, it's possible to perform per measurements in a well-preserved temperature environment on different plate formats and to access the data instantly. This method enables the study of cellular patho mechanisms and evaluates the effect of compounds and the functionality of human IPSC derived cardiomyocytes that can be helpful in the preclinical drug screening process. To begin, switch on the optics based measurement system, microscope light emitting diode source, and the computer.
Start the program on the computer and open a new file by clicking New under the file in the top left of the screen. Click on collect, choose experiments, and then select IPSC and calcium measurements. Next, replace the human IPSC cardiomyocyte culture medium with 0.5 milliliters per well of tyrode solution in a 24 well plate.
Replace the plate lid with the climate control lid and place the plate inside the optic space measurement system in the device, adjusted to 37 degrees Celsius temperature. Click on open cell finder under the toolbar and wait for a new screen to appear. Select the plate format under chamber type and well at the top of the screen.
Select a well and choose an area within the well to observe synchronized contraction and relaxation. Press start on the bottom left of the screen. Then click add measurement to measure the area.
Measure approximately 10 areas per well in a 24 well plate. For only baseline measurements, continue the measurement from the next well. For testing the compounds, press complete once the areas within a well are measured.
Open the optics based measurement instrument and add the desired stressor or compound to the well. After incubating at the desired time period, click on automatic measurement to perform paired measurements in the same areas of the well where the baseline measurements were performed. Press complete after all the measurements are performed.
Choose the next well from the top right of the screen and continue measurements for all the required wells. After measuring all the required wells, click stop and save the file to the desired location. Dissolved the Fura-2 AM aliquots in tyrode solution to get a final concentration of one micromolar Fura-2 AM to add to the cells.
Aspirate the medium and replace it with tyrode solution containing Fura-2 AM.Incubate for 15 minutes at 37 degrees Celsius in an incubator or the optics based measurement system. Remove the tyrode solution containing Fura-2 AM and wash the wells two times with fresh tyrode solution. Finally, incubate the cells in tyrode solution for another five minutes at 37 degrees Celsius to allow desterification of Fura-2 AM.Place the plate inside the optics based measurement system to proceed with the contractility measurements.
Select a well. Choose an area within the well to observe synchronized contraction and relaxation, and then click start and add measurement. In addition to the contractility traces, real time ratio metric calcium transient appears on the screen.
To analyze the data, click Cytosolver on the desktop. Click on import from the file menu and select the files to be analyzed. After the analysis, observe the accepted and rejected transients on the screen.
Click export on the top left of the screen and enable average transient data. Export to Excel file and then click export. Inspect the analyzed data presented in a spreadsheet.
Turn off the computer and all the other instruments. Contractility measurements were analyzed with the Cytosolver program and the parameters of resting/beating frequency, time to peak contractility, and time to baseline contractility of the control human IPSC cardiomyocytes were reported. The application of 500 nanomolar iso, a non-selective beta adrenergic receptor agonist, significantly increased the beating frequency in all wells.
Iso also increased the kinetics, evidenced by a decrease in time to peak contractility and time to baseline contractility. Like contractility, the calcium measurements were also analyzed and the parameters were reported.
