Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Office of Science and Engineering Laboratories at the FDA is developing regulatory science tools to assist medical device developers and FDA reviewers. Our ultimate goal is to accelerate patient access to innovative, safe, and effective medical devices through the best available science. In this protocol, we apply cutting edge induced pluripotent stem cell technology to the assessment of medical devices that are used to treat life-threatening cardiac conditions.

This simple tool enables reproducible evaluation of cardiac electrophysiology devices in the dish using standard laboratory equipment. It can also be used with patient-specific cells derived from donors with various heart diseases. To begin, plate the thawed human iPSC derived cardiomyocytes on 0.1%gelatin coated sterile six-well plates at least two days prior to seeding the cardiomyocytes on the flexible hydrogel substrate.

Culture the human iPSC derived cardiomyocytes in standard cardiomyocyte medium for two to four days at 37 degrees Celsius and 5%carbon dioxide to allow them to recover from the cryo-preservation. Refresh the spent medium with 100%cardiomyocyte medium every 48 hours. Check the status of the human iPSC derived cardiomyocytes before dissociation by evaluating the health of the cells, ensuring viability and stable beating.

Wash the cardiomyocytes with four milliliters per well of DPBS without calcium chloride or magnesium chloride. Add one milliliter of room temperature dissociation reagent to each well and incubate for 15 minutes at 37 degrees Celsius. Next, add 10 milliliters of cardiomyocyte medium to a sterile 15 milliliter conical tube.

Dissociate the cardiomyocytes from the six-well plates with a 1, 000 microliter pipette and add the cell suspension to the conical tube. Rinse the wells with one milliliter of fresh cardiomyocyte medium to collect any residual cardiomyocytes and add them to the conical tube. Then bring the final volume of the conical tube to 15 milliliters with the medium.

Centrifuge the tube at 200 G for five minutes and remove the supernatant up to the one milliliter mark. Resuspend the cells in the cardiomyocyte medium to a final volume of five milliliters and count the cardiomyocytes with a manual or automated cell counter. Next, incubate the cardiomyocyte cell suspension for about 30 minutes at room temperature while the flexible hydrogel substrates are prepared.

Ready a 20 microliter pipette set at one microliter, pipette tips, a sterile 48-well glass bottom plate, and a stopwatch timer in the tissue culture hood. Mix the extracellular matrix or ECM-based hydrogel substrate by gently tapping the tube and immediately placing it back on ice. Start the stopwatch timer immediately before plating the first hydrogel substrate and mark this as time zero.

Pipette one microliter of the hydrogel substrate up and down approximately three times to chill the pipette tip. Then apply approximately one microliter of the undiluted hydrogel substrate horizontally to the bottom of each well in the 48-well plate, holding the pipette at a 45 degree angle. Plate all the hydrogel substrates in the same orientation in each well to help identify the substrate when performing the experiments at 40X magnification.

Place the lid on the 48-well plate and allow the hydrogel substrates to incubate for eight to 10 minutes at room temperature in the tissue culture hood before adding the cells. After the incubation, immediately seed the cardiomyocytes dropwise directly onto the hydrogel substrates with approximately 30, 000 viable cardiomyocytes per well in a low medium volume of approximately 200 microliters of cardiomyocyte medium. After closing the lid, allow the cardiomyocytes to incubate undisturbed for 10 to 15 minutes at room temperature in the hood to enable the cells to adhere to the hydrogel substrate.

Gently add 100 microliters of fresh cardiomyocyte medium to each well. Place the lid closed plate on an incubator at 37 degrees Celsius and 5%carbon dioxide for two to four days. Turn on the microscope and environmental control chamber to equilibrate to 37 degrees Celsius and 5%carbon dioxide.

Remove the cardiomyocyte medium from the 48-well plate and gently rinse each well twice with 600 microliters of the cardiac contractility modulation or CCM assay medium. Then add 300 microliters of CCM assay medium per well and place the 48-well plate on the microscope in the environmental control chamber. Insert the electrodes and equilibrate the cells for five minutes.

To record the contraction videos using video-based microscopy, open the video recording software and set a frame rate of 100 frames per second. Then select a region of interest or ROI near the center of the hiPSC-CM monolayer. Then field stimulate the cells with a commercial pulse generator to electrically pace the 2D cardiomyocytes monolayers.

Place the cardiomyocyte at 1.5 times threshold at one hertz with baseline pulse parameters. For example, monophasic square wave pacing pulses with a two millisecond stimulus pulse duration of five volts. Record the baseline pacing only contraction video before the CCM for a minimum of five beats.

Then stimulate the cardiomyocytes monolayer with an experimental electrical signal of 30 milliseconds delay, two symmetrical biphasic pulses of 5.14 milliseconds phase duration, with 20.56 milliseconds total duration, 10 volts phase amplitude, and zero interphase interval. Record the CCM-induced contraction video for a minimum of five beats. Turn off the CCM signal, stimulate with a baseline pacing pulse and record a contraction video of the recovery period after the CCM for a minimum of five beats.

Use a standard contraction software to analyze the contraction videos automatically and quantify the key contractile properties like contraction amplitude, contraction slope, relaxation slope, time to peak, time to baseline 90%and contraction duration 50%The 2D human iPSC derived cardiomyocytes monolayer contractual properties were characterized and the key parameters of cardiomyocyte contractility were quantified. The application of the standard CCM stimulation parameters resulted in enhanced contractile properties in vitro. The effects of extracellular calcium concentrations modulation on human contractile properties with and without CCM stimulation were evaluated.

The expected baseline calcium dependence of contraction was observed, as well as a CCM-induced increase in calcium sensitivity at the level of the cardiomyocyte monolayer. In addition, the pharmacological interrogation of the beta androgenic signaling pathway revealed that the CCM-induced inotropic effects were in part mediated by beta adrenergic signaling. Moreover, this tool can be expanded to patient-specific disease cardiomyocytes, including those of dilated cardiomyopathy to understand the effect of CCM in the context of disease states.

Here, we focus primarily on evaluating human contractile properties. However, using this tool, one could also evaluate other cardiac excitation contraction coupling readouts including action potentials and calcium handling. This is the first regulatory science tool to combine induced pluripotent stem cell by technology with assessment of cardiac devices.

This alternative method paves the way for medical devices to be assessed in a dish and has the potential to reduce the need for animal and human subject testing.