The protocol combines the power of generating human cardiovascular cell types using induced pluripotent stem cells and a versatile technique to generate beating cardiac spheroids in less than 72 hours. These cardiac spheroids can be used for disease modeling and drug testing. One of the main advantages of this facile technique is that one could generate nearly 1000 beating cardiac spheroids in a standard 12-well plate.
And most importantly, each and every cardiac spheroid can be assayed for its contractile function using imaging based technique. After melting agarose in the microwave, place it in the biosafety cabinet and allow it to cool for three minutes. Pipette 700 microliters of the molten agarose and add it to the silicone micromold of a nine by nine array, taking care to avoid generating bubbles.
Carefully place the mold on the pre-cold ice block to accelerate the agarose duration. Once the agarose becomes translucent, carefully bend the edges of the micromold to losen the agarose replica and gently peel the replica from all sides to detach it from the silicone micromold. Transfer the agarose microtissue tray containing 81 circular recesses into a sterile 12-well plate, then add two milliliters of PBS to the agarose microtissue tray, and inspect it under the microscope for any trapped bubbles or irregularly shaped wells.
Submerge the agarose tray in two milliliters of 70%ethanol overnight, followed by UV treatment in the biosafety cabinet for one hour. Remove the 70%ethanol and wash twice with distilled water, and once with two milliliters of PBS. Once the induced pluripotent stem cell derived cardiomyocytes, cardiac fibroblasts, and endothelial cells are counted after trypsinization and neutralization, place the cell suspensions on the ice.
In a new tube, mix the induced pluripotent stem cell derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. Then, remove the PBS from the well and the cell seeding chamber without touching the recesses, and add 200 microliters of the cell suspension dropwise. Allow the cells to settle at 37 degrees Celsius in a carbon dioxide incubator for two hours.
Add micro tissue fabrication medium surrounding the agarose mold to cover the surface of the inner chamber. After 24 hours, observe for self-assembled and compact spheroids in the circular recesses. Change the medium every two days to maintain the spheroids.
Typically, after 48 hours, spontaneous beating of the spheroids can be observed. Culture the individual cell types separately on basement membrane matrix medium, or gelatin coated chamber slides. Gently flush the cardiac microtissues out of the circular recesses, and collect them in a 15 milliliter chronicle tube.
Aspirate the medium and rinse the cells or the microtissues, with one milliliter of PBS. Then, fix the cells or the microtissues with fixation buffer containing 4.2%PFA and incubate at room temperature. Aspirate the PFA and incubate the cells or the microtissues with one milliliter of permeabilizing solution.
Then, aspirate the solution and rinse with two to three milliliters of PBS. Add 500 to 1000 microliters of blocking solution to the cells and incubate for at least one hour for the chamber slides, and for three to four hours for the microtissues. Incubate the samples with conjugated antibodies in the blocking solution for one hour for the chamber slides, and overnight at four degrees Celsius for the cardiac microtissues.
Wash the chamber slides three times with 500 microliters of 0.1%Tween 20, with five minute durations between each wash, then perform a final wash with PBS. Wash the cardiac micro tissues five times with two milliliters of 0.1%Tween 20, with a 20 minute duration between each wash. Then, perform a final wash for an additional 20 minutes.
Incubate the cells or the microtissues with DAPI prior to confocal microscopy, then carefully transfer the cardiac microtissues to a 35 millimeter glass bottom dish, and add PBS to submerge the microtissue. Flush the microtissues out of the circular recesses with medium one using a wide or one milliliter pipette tip into a 15 milliliter chronical tube, and allow them to settle. Then, aspirate the medium and rinse with one milliliter PBS.
Add 200 to 300 microliters of the enzyme digestion buffer and incubate for 10 minutes at 37 degrees Celsius. Then mix the microtissues gently for one minute, and repeat the incubation. After the incubation, mix the microtissues vigorously with a regular one milliliter pipette tip to digest it into single cells.
Obtain a turbid cell suspension, and immediately neutralize the cell suspension with five milliliters of medium containing 5%FBS. Strain the cell suspension through a 40 micrometer cell strainer, and count the total number of cells. Centrifuge the single-cell suspension at 300 times G for five minutes at four degrees Celsius.
Aspirate the supernatant and resuspend the cells in annexin binding buffer with FITC Annexin V and propidium iodide, or TO-PRO-3 dead cell exclusion dye, and incubate for 10 minutes on ice. After the incubation, add 300 microliters of the annexin binding buffer to the cell suspension and transfer it to a round bottom FACS tube for flow cytometry analysis. Purified induced pluripotent stem cell-derived cardiomyocytes, endothelial cells and cardiac fibroblasts were visualized using phase-contrast microscopy.
The purity of different cell types was determined using immuno staining and flow cytometry. Troponin T was used as a marker for cardiomyocytes, and over 90%purity was achieved. The platelet endothelial cell adhesion molecule CD31 and vimentin were used as markers for endothelial cells and cardiac fibroblasts, and indicated purity over 98 and 96%respectively.
Immunofluorescence staining revealed that the cardiomyocytes were the heaviest and occupied the center of the microtissues, whereas the endothelial cells were interspersed throughout the microtissues, and the cardiac fibroblasts predominantly occupied the periphery. The digestion of the microtissues using this pace one and Liberase TL resulted in highly viable cell proportions with fewer apoptotic cells after two weeks in culture. The one hour exposure of the cardiac microtissues to a high concentration of doxorubicin induced dose dependent cardiotoxicity.
The micro tissues contractility, and the vectors movement generated a pseudo heat map which illustrated the average contraction profile across the microtissue. The contractile motion of the cardiac microtissues generated positive peaks that were measured as the contraction velocity, the relaxation velocity, and the beat rate, which was calculated as time the between two contraction cycles. The contractility of the cardiac microtissues was consistent over four weeks in culture.
Cells heating is one of the most important steps in this particular protocol, where one must take care to carefully dispense the cell suspension dropwise into the agarose array for even distribution within the microwells, and to prevent overflow. Using the optimized radiation protocol of these microtissues, one could subject these to high throughput sequencing technologies, such as single cell RNA-seq or single cell ATAC-seq to understand cell specific gene expression patterns resulting due to different treatment conditions.
