This method can help answer key questions in the field of cardiac safety pharmacology. For example, does a new human medicine carry a risk of modifying the normal cardiac rhythm? The main advantage of this technique is that it can evaluate large numbers of compounds rapidly and at a moderate cost.
Demonstrating the procedure will be Camille Forny, a research associate in my laboratory. To prepare for the measurement of the contractions of syncytia of human-induced pluripotent stem cell-derived cardiomyocytes, or HIPSCCMs, start by placing CryoTubes containing frozen cells in a 37 degree Celsius water bath for two minutes. Transfer the entire thawed celled suspension from each tube to one single, fresh 50 milliliter conical tube.
To retrieve the leftover cells, wash each tube with one milliliter of 37 degree Celsius prewarmed plating medium and add this medium to the 50 milliliter conical tube containing thawed cells. Add eight milliliters of warm plating medium to the tube and carefully mix the suspension. Use trypan blue exclusion method and a hemocytometer to count live cells.
By adding warm plating medium, adjust the concentration to 50, 000 cells per milliliter. To distribute the cells into two new 384-well cell culture plates, add 25 microliters to each well, which will yield about 12, 500 cells per well. Incubate the cells at 37 degrees Celsius in 384-well plates in humidified atmosphere with 5%carbon dioxide for a total of three to four weeks.
The following day, replace the plating medium with 50 microliters of warm maintenance medium. Afterwards, replace half of the maintenance medium every two to three days. On days seven, 14, and 21, replace the medium entirely.
Once the cells have been maintained in culture for three to four weeks, they have formed spontaneously contracting syncytia, and the effect of compounds on these contractions can be measured. On the day of the assay, at least two hours before the first cell plate is to be placed inside the plate reader for the measurement, turn on the reader and set the heating system at 37 degrees Celsius. To prepare the compound plates, warm to room temperature 40 milliliters of maintenance medium supplemented with 1%volume-by-volume penicillin streptomycin.
While the medium is warming up, distribute the test compounds and the controls into two 384-well compound plates by adding 1.5 microliters of stock solution per well. Centrifuge the compound plates at 100 G for one minute. Add 37 microliters of maintenance medium to each well and centrifuge the plates again at 100 G for one minute.
To prepare the fluorescent dye, warm 100 milliliters of maintenance medium supplemented with 1%volume-by-volume penicillin streptomycin in a 37 degree Celsius water bath. Then, add 10 milliliters of the warmed medium to the mix of calcium-sensitive fluorescent dye and quencher to reconstitute them as a stock fluorescence medium. Start the plate reader software.
Prepare the software to record a two minute segment of calcium waves with an acquisition frequency of at least 10 hertz. To make fluorescence medium for each cell plate, dilute three milliliters of stock fluorescence medium with 12 milliliters of warm maintenance medium with antibiotics. Replace 20 microliters of medium in each well of the cell plate with 30 microliters of fluorescence medium and mix by pipetting up and down once.
Immediately after that, place the plate into a plate reader in order to let the HIPSCCMs adjust to the reader temperature for 60 minutes. Then, start the plate reader software. Record a two minute segment of calcium waves with an acquisition frequency of at least 10 hertz to define the baseline beating rate for each well.
It is important to make sure the cell plate is not transferred out of the device after data acquisition. With some software versions, you must stop the recording before it ends completely. Use the plate reader robot to pipette five microliters of the test and reference compounds well-to-well from the compound plate into the cell plate.
After that, start the data acquisition in the plate reader software to record two minute segments of calcium waves starting at five, 15, 30, 45, and 60 minutes after the compound addition. To start data analysis, export the raw data for each recording period from the plate reader software in ASCII text files. And then import them into data analysis software.
From the data analysis software, export the primary beating frequencies for each recording period as clipboard copies. Extract the primary beating frequency for each well and every time point. Finally, import the beating frequencies into a spreadsheet software by clipboard pasting and continue the data analysis as described in the text protocol.
Recording of calcium waves at baseline across one 384-well plate shows that in most cases all wells reveal a regular beating rate with little variability across the plate. When blockers of cardiac ion channels are added, they affect the rhythm of the cardiomyocytes. Amlodipine, a blocker of the slow calcium channels, accelerates the rhythm more than 100%in a concentration dependent manner up to one micromolar.
And above that, amlodipine arrests the beating. E-4031, a blocker of the rapid delayed rectifier potassium channels decelerates the rhythm about 65 to 70%in a concentration dependent manner up to 10 micromolar. Tetrodotoxin, a blocker of the fast sodium channels decelerates the rhythm about 15%in a concentration dependent manner up to one to 30 micromolar, whereas above 30 micromolar, tetrodotoxin arrests the beating within the first five minutes.
Bepridil, a mixed blocker of cardiac sodium, calcium, and potassium channels also produces an all-or-nothing effect, suggesting that sodium channel block is the primary action of bepridil. While attempting this procedure, it's important to remember to be extremely gentle when pipetting in the cell culture plate. These tissues are very fragile and easily damaged if touched by pipettes.
Don't forget that working with human cells and active drugs can be hazardous, and precautions such as wearing safety gloves and glasses should always be taken while performing this procedure.
