Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Every 10 minutes, someone in Australia has a heart attack. This can leave them with damaged heart muscle, and that can lead to heart failure. We have found that we can convert stem cells, from blood or skin, into beating heart cells.

Then, if we put these reprogrammed cells, through our 3D bioprinter, using bioink, we believe that we can make heart patches with cells matching people's own cells. A deeply anesthetized mouse, using ketamine xylazine, is intubated using a 20 gauge catheter. After intubation of the tracker, the mouse is carefully placed on a heating pad, then connected to the ventilator that automatically sets the target volume based on the mouse weight.

Under a light anesthesia using 2%iso fluoride, the mouse just is clean with iodine and ethanol. A left Lateral thoracotomy is performed to expose the heart. A retractor is placed between the third and the fourth rib.

The pericardium is cut carefully. The LAD identified under the microscope. For the LAD ligation step, it is important to avoid the suture cutting through the LAD.

Therefore, a reinforcing piece of 3.06 silk suture is placed on the heart as shown, running in the same direction and just on top of the LAD. Using a 7.06 silk suture, the LAD is isolated and permanently ligated. This allows the blanching of the heart which is receiving a heart attack.

This will lead to remodeling and the left ventricle will fail over time. A 3D bioprinted heart patch is generated in the lab using a 3D bioprinter. This allows the layer by layer, deposition of cardiac cells, using a combination of cells and hydrogels.

You can see one floating in this video and now in this image. Here is an image of the patch appearance when imaged by epifluorescence light microscopy in a well of a six well plate. The patch has been stained with Hoechst stain for cell nuclei, and this blue stain is also highlighting the autofluorescent hydrogels.

This is an intact algenate gelatin patch suitable for transplantation, similar to the one shown in the video. With our method, most patches started to disintegrate between 14 and 28 days, in culture, as shown here in this image of another patch, which has disintegrated. We found that the optimal time to transplant patches was between day seven and day 14 after bioprinting.

This was when patches containing cardiac cells started to beat, showing a degree of tissue maturation, but before patches started to disintegrate. The patch is brought to the surgical room, and slowly and carefully placed on top of the heart in part of the mouse. Carefully is also moved to the area of the infer.

The retractor is slowly removed. Finally, the third and fourth ribs are closed together using a 6.0 prolene suture. Together with a muscle, the ribs are closed, followed by the closure of the skin.

After closure of the chest, the mouse is injected with Antisedan and furosemide. Slowly, the mouse will regain independent breathing activity, and will respond to toe pinch. The mouse is closely monitored, and once it awakes from the anesthetic, it is placed to its own cage.

This image shows, with the adopted area, where the bioprinted patch will sit on top of the mouse heart. In conclusion, we have shown that 3D bioprinted algenate gelatin patches can be transplanted by our method, onto the epicardium, in a mouse model of myocardial infarction. In the bioprinting phase, algenate gelatin hydrogels have excellent printability due to their rheological properties, allowing for extrusion without damaging cells, but also having the biomechanical strength to retain their structure after 3D bioprinting, and during transplantation.

Our method is likely to be widely feasible and also suited to testing multiple groups of 3D bioprinted patches. For instance, with different cellular contents.