One of the major challenges in engineering a human heart tissue includes its vascularization. Our laboratory has developed an in vitro model of the human heart by co-culturing cells found in vivo. These include cardiac myocytes, cardiac fibroblasts and cardiac endothelial cells.
All together, these cells allow the recapitulation of the human heart microenvironment including its molecular, cellular, and extra cellular components. We use them in our lab to test different drugs for drug toxicity essays. These include doxorubicin, a well-known cardiotoxic drug that affects the human hearts even years following its treatment.
We culture cardiac fibroblasts, endothelial cells and myocytes separately before forming spheroids. In order to grow cardiomyocytes, we collect the vial from minus 80 degrees and we warm it at 37 degrees for four minutes in a water bath. We then spray the cryo vial with 70%ethanol and move it to a bio-safety cabinet.
We gently collect the cell suspension and transfer it to a 15 milliliter Falcon tube. We collect one milliliter of prewarm plating medium, rinse the cryo vial once and then add it to the 50 milliliter Falcon tube, one drop at a time every four seconds. While add in the plating medium, gently shake the Falcon tube.
With a cell pipette, collect seven milliliter of plating medium and gently add it to the 15 milliliter Falcon tube. Once added the final volume of plating medium to the Falcon tube, transfer the cell suspension to the fibronectin coded tissue flasks. Finally, move cells to an incubator.
After two days, we collect the tissue flasks from the incubator and we check cell confluence under the microscope. At this point, we move the flask to a bio-safety cabinet and we remove the plating medium. We gently rinse cells with the same plating medium from the tissue flask four or five times.
We replace it with fresh maintenance medium and move them to an incubator for one to two additional days. On the day we create hangings of cultures, we collect all the three cell types from the incubator. We chip in cells by removing the media.
We rinse them with three milliliters of PBS. And finally we add the chips into the cells. Flasks are then moved to an incubator, and incubated for up to five minutes.
In order to generate cardiac spheroids, we co-culture 20, 000 cells per hanging drop. These include 10, 000 cardiomyocytes, a thousand cardiac fibroblasts and 5, 000 cardiac interior cells. These are co-cultured in hanging drop 384 well plates.
They're plated using our liquid handling system. We also add PBS on the sidewalls of the hanging drop plates to prevent the cultures from drying out in incubator. Carefully move the hanging drop plate containing cells to the incubator.
Between three to four days, spheroid will form. We collect the hanging drop plate from the incubator and we check that spheroids are forming within each well. As shown in these images, we have a set of spheroids within the center of each well.
Once formed, we collect spheroids and embed them in collagen gels. A pipette tip is cut at inside so to prevent spheroid damage. Spheroids are collected and transferred to a 50 milliliter Falcon tube where they're pulled together.
We spin down the spheroid suspension at 300 G for five minutes to separate them from the medium. We use dark walled 96 well plates to image spheroids, the media is removed or replaced with a collagen gel in a Falcon tube. Sodium hydroxide is added to the spheroid gel suspension before they're plated in the dark wall plate.
100 microliter of spheroid suspension is added into each well and incubated for 30 minutes at 37 degrees. Drugs, including doxorubicin and other agents are added to the spheroid plate and incubated for 24 hours. The following day, we prepared a solution containing calcium AM and ethidium homodimer, labeling live under the cells respectively.
This solution is prepared in adding the dime to this filter plate. Spheroids are then incubated at 37 degrees for one hour. We use a plate reader to measure the fluorescence of calcium AM and their the ethidium homodimer in the plate.
These measurements are then used by our software to perform a statistical analysis. We measure the contractile activity of spheroids treated with the different drugs using an ion optic system. Spheroids are moved to the stage between two electrodes.
These allows to both control and measure the contractile activity by field potential. In this way, we can test different voltages and frequency ranges to stimulate spheroids. A spheroid at the time is imaged, and used for those measurements.
Spheroids that did not receive doxorubicin will be able to construct following the electrical stimulation. On the contrary, doxorubicin treated spheroids will not contract. To visualize the endothelial cell network form within each spheroid, we fixed them with a 4%paraformaldehyde solution for one hour at room temperature.
We moved the plate to a shaker that allows the gentle permeation of the solution through the gel. The PFA is removed and rinsed three times with a PBS solution containing 0.01%sodium oxide. We brought mobilization of course by adding a 0.02%solution of Triton X in PVSA for 20 minutes at room temperature.
The blocking solution contains 3%BSA in PBC, which is then added to the plate for one hour at room temperature. A solution containing the primary antibody against CD 31. A marker for endothelial is added to the plate that is then incubated overnight at four degrees.
The following day, the primary antibody solution is removed and the plate rinsed three times with a PBS solution containing 0.01%sodium oxide. The secondary antibody solution containing the hex stain as well is added to the plate that is then incubated overnight at four degrees. Finally, the second antibody solution is removed and the plate rinsed three times with a PBS solution containing 0.01%sodium oxide.
After staying with primary and secondary antibodies, the plate can be imaged using our confocal microscope. A proper vascular network is critical for cardiac cell survival and function. Cardiac spheroids present an endothelial cell network that better recapitulates the one present in the human heart compared to monolayer cultures of cardiac cells.
Given the unique features, cardiac spheroids represent advanced tools for in-vitro testing for cardiovascular research.
