Cardiac safety assessment is a crucial component during the drug development process. Human iPSC derived cardiomyocytes have proven to be a reliable, efficient, and human based model for pre-clinical cardiac safety assessment. We have established methodologies to comprehensively investigate the functional characteristic of human iPSC derived cardiomyocytes, which are of really important disease modeling, drug induced cardio toxicity screening and precision medicine.
The growing incorporation of human iPSC derived cardiomyocytes into the standard pre-clinical safety evaluations process has the potential to improve the accuracy of toxicity prediction of a candidate's compounds. Begin by culturing the human iPSC derived cardiomyocytes for at least 10 days before taking the measurement. Turn on the temperature controller and set it at 37 degrees celsius.
Turn on the microscope, camera, and carbon dioxide supply. Fill the water jacket of the plate holder with an appropriate amount of sterile deionized water. Place the 96 well plate on the plate holder, and incubate the cells for at least five minutes.
Open the view software, then set the camera frame rate to 75 frames per second, and the video or image resolution to 1024 x 1024 pixels. Apply the auto focus and auto brightness functions, record videos and images of human iPSC derived cardiomyocytes. Carefully place an eight microliter droplet of extra cellular matrix coating solution over the recording electrode area of each well of the 48 well microelectrode array plates.
Then add three milliliters of sterile deionized water to the area surrounding the wells to prevent coating solution evaporation. Using a 200 microliter pipette tip, remove most of the extra cellular matrix medium from the well surface within the same single row or column without touching the electrodes. Seed 50, 000 cells per well in an eight microliter droplet of seeding medium over the recording electrode area.
Repeat until all wells have been plated with cells. Then incubate the microelectrode array plates in a humidified cell culture incubator. Slowly add 150 microliters of seeding medium to each well of the microelectrode array plates.
On day 10 post plating, check the density of spontaneous beating human iPSC CM monolayer under a microscope. Place the 48 well microelectrode array plate in the recording instrument. Ensure that the temperature is 37 degrees celsius and carbon dioxide is 5%Open the navigator software.
In the experimental setup window, select cardiac real time configuration and field potential recordings. Apply spontaneous or paste beating configurations. In beat detection parameters, set detection threshold to 300 micro volts, minimum beating period to 250 milliseconds, and maximum beating period to five seconds.
Select polynomial regression for field potential duration calculation. Check whether the baseline electrical activity signal is mature and stable, and acquire the baseline cardiac activities for one to three minutes. Perform the recording 10 days post plating.
Replace the human iPSC CM maintenance medium with tyrode's solution, and allow the cells to acclimate for 15 minutes. Insert the temperature sensors in the chamber, and put the 35 millimeter dish inside. Adjust the temperature to 37 degrees celsius.
Pull the micro pipettes from borosilicate glass capillaries using a micro pipette puller. Fill the micro pipettes with the intracellular solution, and insert them into the holder connected to the head stage of the patch clamp amplifier. Open the acquisition software, load the action potential recording protocol, and select the voltage clamp configuration.
Insert the micro pipette into the bath solution, and select appropriate single human iPSC CMs. Adjust and lower the position of the micro pipette next to the selected cell using a micromanipulator. When the pipette tip is inserted into the bath solution, check for pipette resistance on the oscilloscope monitor.
Position the pipette close to the cell, and the current pulses will decrease slightly to reflect the increasing seal resistance. Apply gentle suction with negative pressure to increase the resistance. This will form a gigaseal.
Then apply the seal function. Apply additional suction to break the membrane to get a whole cell recording configuration. At this point, switch from voltage clamp mode to current clamp mode, or no current injection using the amplifier dialogue.
Press the record button to generate and save the action potential recording files. Perform the calcium transient measurement at least 10 days post plating. Prepare fresh tyrode's solution and Fura-2 AM loading solution.
Aspirate the human iPSC CM maintenance medium, and wash the chambers with tyrode's solution twice. Apply 100 microliters of Fura-2 AM loading solution and incubate for 10 minutes at room temperature. Replace the Fura-2 AM loading solution with tyrode's solution and incubate for at least five minutes for complete deesterification of Fura-2 AM.Add a drop of immersion oil on the 40x oil immersion objective of an inverted epifluorescence microscope.
Place the cell chamber in the stage adaptor of the microscope. Install the temperature controller wires to the bath chamber, and set it to 37 degrees celsius. Install the electrical field stimulation electrodes, and set the pulses at 0.5 hertz with a 20 millisecond duration.
Turn on the ultra high speed wavelength switching light source, and set it to 340 nanometers and 380 nanometers fast switching mode. Apply an exposure time of 20 milliseconds for both wavelengths, and a recording time of 20 seconds. Record the video reflecting real time changes of calcium transient, export the data to a spreadsheet for analysis.
Human iPSC derived cardiomyocytes monolayer was used for the contraction motion measurement. Analysis of a trace of the contraction relaxation motion using the analyzer software detected the contractions start, contraction peak, contraction end, relaxation peak, and relaxation end. Full coverage of the human iPSC derived cardiomyocytes monolayer was observed on all the electrodes within the microelectrode array plates.
The mature waveforms recorded by each electrode reflected the cardiac field potential with identifiable characteristics. After analysis, beat period, field potential duration, and spike amplitude were obtained. Whole cell patch clamp recordings in current clamping mode recorded the spontaneous action potentials of single cardiomyocytes.
After analysis, several parameters, including the amplitude, max diastolic potential, and action potential duration were obtained. After the analysis of calcium imaging, F340 by F380 ratio over time was obtained, and raw traces of stimulated calcium transients were plotted. In addition, parameters such as amplitude, diastolic calcium, and calcium decay tau were obtained.
It is important to use the human iPSC derived cardiomyocytes that are in good beating conditions, as well as to ensure the high purity of the human iPSC derived cardiomyocytes. This protocol includes four specific techniques to study human iPSC derived cardiomyocyte functionality, other configuration, or automatic whether to study cardiac ionic currents that play a crucial role in cardiac
