This protocol describes a workflow for the generation, maintenance, and optical analysis of cardiac spheroids. These cardiac spheroids are essential to fill the gap in current in vitro disease models. This technique allows researchers to create next-generation, high-throughput screening and functional storage of cardiac spheroids.
Begin with adding one milliliter per centimeter square of sterile cardiac detachment solution to each well of a culture plate containing confluent human-induced pluripotent stem cell-derived cardiomyocytes. Incubate the plate at 37 degrees Celsius for 15 minutes. Confirm the detachment of cells by observing white and round-shaped cells under 4x magnification of a bright-field microscope.
Using a five-milliliter pipette, mechanically dissociate the cells by flushing them with two milliliters of warm basal medium to prepare a single cell suspension. Transfer the cell suspension to a 15-milliliter conical tube, and centrifuge for three minutes at 300g. Then aspirate the supernatant, and resuspend the cells in one milliliter of hiPSC-CM replating media.
Use a 1, 000-microliter pipette tip to dissociate the cell pellet. After three or four mixes, as the solution appears homogenous, load it into a cassette to count the cells, and transfer the appropriate amount of cells in 100 microliters of replating medium to each well of an ultra-low attachment, round-bottom, 96-well plate. Place the cardiac spheroids plate on an orbital shaker in the incubator at 70 rpm with a 37 degrees Celsius temperature, 5%carbon dioxide, 21%oxygen, and 90%humidity for 24 hours.
To avoid spheroid rupture, aspirate only 50 microliters of medium from each well, and add 100 microliters of RPMI plus B27 medium per well for the first 48 hours. Next, aspirate 100 microliters of the medium from each well, and add 100 microliters of the maturation medium per well. Maintain the cells in the maturation medium, and refresh the medium every two to three days.
Place the spheroid plate on ice for 10 minutes for pre-cooling before centrifuging the plate for three minutes at 70g. Remove the medium till 50 microliters remain in the well. Then add 200 microliters of ice-cold hiPSC freezing medium per well.
Seal the plate with a plate-sealing film. To prepare the silicon mold, mix the components of the silicon elastomer kit in a 10-to-1 ratio, and add the solution inside the bottom part of the well plate. De-bubble the solution using a vacuum pump for 15 to 20 minutes.
Place the mold into an oven, and cure it at 60 degrees Celsius for eight hours to obtain a semi-flexible elastomer that is peeled off the plate. Place the sealed plate carefully into a silicone mold, ensuring uniform heat exchange between the well plate and the freezer. Freeze the plate at 80 degrees Celsius for a minimum of four hours in the prepared silicone mold before transferring the plate to a liquid nitrogen tank or a 150 degrees Celsius freezer for long-term storage.
To ensure a quick thawing of cardiac spheroids, collect one cell plate with cardiac spheroids at a time from the liquid nitrogen, and place it in the incubator for 15 minutes at 37 degrees Celsius, having 5%carbon dioxide, 21%oxygen, and 90%humidity. Remove the sealing film off the plate, and aspirate 150 microliters of each well before adding 200 microliters of a warm basal medium to each well. Centrifuge the plate at 70g for three minutes, and repeat the warm basal medium wash.
Once done, remove 150 microliters of the medium, and add 200 microliters of cardiomyocytes thawing medium in each well. Then repeat the RPMI wash as mentioned during cardiac spheroid generation, followed by maintaining the cells in the maturation media. After one week of the culture, the thawed cardiac spheroids are optimal for calcium handling optical imaging.
Aspirate 150 microliters of each well. Treat the thawed cardiac spheroids with 100 microliters of Cal-520 AM calcium dye medium per well in dark conditions, and incubate the plate for 60 minutes. Prepare a calcium acquisition and analysis system by powering the microscope and ensuring the environmental control option is on.
Adjust the camera and framing aperture dimensions to minimize the background area. To measure the calcium signal, using a 488-nanometer laser, set the contrast to a black background with a bright green signal during calcium release. Record a video with a consistent stream of 2 to 10 peaks within 10 seconds.
Record a 10-seconds video, and scan across the 96-well plate, initially moving to the left, then downward in a zigzag fashion to cover the whole plate. After acquiring the calcium transients, analyze the data using fluorescence traces analysis software. The cardiac spheroids acquired a 3D structure by day one post-seeding, which could be cultured for up to six weeks.
The majority of three-week-old cardiac spheroids expressed regular sarcomere organization with alpha actinin and troponin T.High levels of alpha actinin in day zero and three-week-old cardiac spheroids indicated a constant and highly pure cellular composition during culturing. An increased expression of the cardiac genes, desmosomes, and mitochondria were observed in spheroids than hiPSC-CMs cultured in 2D for 90 days. The beating percentage of cardiac spheroids was similar in the first three weeks post-generation and dropped significantly in week six due to cardiac spheroids deterioration.
The beating rate was significantly reduced at week three compared and dropped at week six as a result of deterioration. Calcium transient parameters indicated a higher peak value at week two, while the rise time, decay time, and the calcium transient duration at 90%of decay were significantly increased at week three, showing that hiPSC-CM-derived spheroids were functionally optimal at weeks two and three post-generation. The spheroid size increased with the number of cells used for seeding.
In larger size, old spheroids, the beating was significantly higher. A similar and significantly higher beating rate was shown by 5k, 10k, and 20k spheroids compared to 2.5k spheroids. Cryopreservation did not affect cell viability within the cardiac spheroids.
Similar expression of sarcomeric proteins in the thawed and fresh age-matched spheroids indicated cryopreservation efficiency. There was no significant change in the beating rate of thawed or fresh cardiac spheroids. No significant changes were observed in rise time, decay time, and the CTD90 of thawed or fresh CS.Next to disease modeling and high-throughput drug screenings, these spheroids can also be used for bioreactors of extracellular vesicle production and extracellular vesicle-related therapies.
Also, the biobanking of these spheroids can be used as a novel cardiomyocyte supply for regenerative strategies. Accumulating evidence suggests that biobanks of patient-derived stem cell models for cystic fibrosis, cancer, and cardiac spheroids have great potential for unraveling molecular mechanisms for drug screenings and for personalized medicine.
