The method can help answer preclinical cardiac safety and toxicity related question using real human-based data for a safe transition of new drug candidates into clinical stages. Instead of using a set of different in-vitro and in-vivo techniques, the main advantage of this hybrid system is the simultaneous assessment of human iPC-derived cardiomyocyte, electrophysiological and viability parameters, and the analysis of contractile properties under physiological conditions. This method can be applied for drug discovery as it covers electrophysiological, structural and contractile changes of cardiomyocytes and a higher throughput system that allows for the testing of 69 wells simultaneously.
To begin plate coating, leave the bottom of the contraction plate covered by the additionally supplied membrane guard until measurement in the contractility module. For seeding cardiomyocytes, prepare a diluted EHS gel coating solution by transferring 2.75 milliliters of EHS gel ready to use solution and 8.25 milliliters of DPBS with calcium and magnesium in a sterile centrifuge tube. Then mix the solution.
Remove the membrane guard from the contraction plate. Transfer the prepared coating solution into a sterile reagent reservoir in the lab automation robot. Use the program, add 100 microliters with the lab automation robot and add 100 microliters of the coating solution per well.
Then place the lid back onto the 96-well plate and incubate for three hours at 37 degrees Celsius. After thawing and counting the cells, adjust the cells in the recommended plating medium according to the cell manufacturer instructions, resulting in 11 times 10 to the sixth cells per 11 milliliters for seeding an entire 96-well plate. Use the program remove 100 microliters to remove the EHS gel solution from the wells with the lab automation robot.
Next, transfer the 11 milliliters of cell suspension into a sterile reagent reservoir placed in the lab automation robot, and seed the cells with 100 microliters of suspension per well. Using the program, cells add 100 microliters. Immediately after cell seeding, transfer the flexible 96-well plate into the humidity controlled incubator at 37 degrees Celsius, 5%carbon dioxide, and let the cells settle overnight.
Warm 22 milliliters of cardiomyocyte maintenance medium to be added to each plate to 37 degrees Celsius in a 50 milliliter centrifuge tube. 18 to 24 hours after seeding the plates, transfer the fresh medium into a sterile reagent reservoir and leave it next to the lab automation robot. Then perform medium removal with the program, remove 100 microliters.
Next place the reagent reservoir containing the fresh medium into the lab automation robot and dispense 200 microliters of the fresh medium per well with the program, add 100 microliters. Perform this step twice to reach 200 microliters per well. Immediately after medium exchange, transfer the plate into the incubator.
Perform a medium exchange every other day until compound addition. Four to six hours before compound addition perform a final medium change by transferring 22 milliliters of fresh and warm assay buffer into a sterile reagent reservoir and leaving it next to the lab automation robot. Immediately after medium exchange transfer the flexible plate back into the incubator.
One hour prior to baseline measurement transfer the plate to the respective measurement device. In the control software open Edit Protocol and select the respective Measurement Mode, Flex Site or Impedance EFP. Define the sweep duration or length of one measurement to 30 seconds and a repetition interval to 10 minutes before saving the protocol number.
Then select Start Protocol and continue to fill in the requested fields. When the parameters are set, select Start measurement. Perform a minimum of three baseline measurements in five minute intervals shortly before compound addition.
Remove 50 microliters of the assay buffer from each well without removing the flexible 96-well plate from the measurement device, followed by the addition of 50 microliters of the four times concentrated compound solution into each well of the plate according to the measurement plan. After compound addition, select add reagent marker to define the compound plate layout and the volume of the compound solution. Then select Proceed with Standard Measurement or proceed with Measurement Series according to the experimental plan.
The Representative Analysis shows the effects of kinase inhibitor Erlotinib on the contractility of human iPSC-derived cardiomyocytes. At the lowest concentration, Erlotinib induced a statistically insignificant decrease in amplitude, whereas micromolar concentrations of Erlotinib showed time and dose-dependent cardiotoxic effects. The onset of the effect was seen from 96 hours with one micromolar Erlotinib, while treatment with 10 micromolar Erlotinib resulted in a significant decrease 24 hours after compound addition.
After the application of 10 micromolar Epirubicin, cells ceased beating within 24 hours. With the application of one micromolar Epirubicin, a drastic decrease in amplitude after 24 hours was followed by complete beat cessation until 48 hours. At 100 nanomolar, a time-dependent decrease in beat amplitude was observed.
At the lowest concentration of 10 nanomolar, the amplitude fluctuated steadily, confirming that the effect of Epirubicin was only dose-dependent. The treatment with Doxorubicin and Nifedipine for 24 hours reduce the cell viability in a concentration and time dependent manner. Upon application of increasing Nifedipine concentrations, field potential recordings on cells reveal a concentration dependent shortening of the field duration normalized.
Nifedipine assessment regarding cardiac contractility also showed a significant concentration dependent amplitude decrease upon acute measurement with 10 nanomolar and 13 nanomolar concentrations. It is important to remember that the cells are generally sensitive to environmental parameters like temperature and pH changes as well as mechanical simulation which is especially supported on the contraction plates.
