Creazione di tessuto cardiaco che mostra l'integrazione meccanica degli spheroidi usando la biopittura 3D

The purpose of this video is to demonstrate 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Firstly, cardiomyocytes, endothelial cells and fibroblasts are isolated, counted and mixed at desired cell ratios. They're then distributed using a multi-channel pipette and to individual wells in ultra low attachment 96 well plates.

The cells in the 96 well plates are incubated for three days, after three days, they form spheroids that beat spontaneously. Confocal microscopy reveals the expression of connects in 43, which is fluorescently labeled green in Troponin T positive cardiomyocytes, which are fluorescently labeled far red within the spheroids. The 96 well plates containing the spheroids are then loaded into the 3D bio-printer.

The 3D bio-printer starts by transferring the 96 well plate to a location just beside the needle array using a moving platform. The 3D bio-printer then inspects all the spheroids in the 96 well plate to obtain relevant data regarding each spheroid such as its diameter and its shape. This is a close up view of the 3D bioprinting process.

The spheroid is picked up from the 96 well plate on the left by a nozzle using vacuum suction and placed a specific location in a needle array on the right. This process is repeated until the 3D bioprinting process is complete. Until all the spheroids and 96 well plate have been bioprinted, then the 3D bioprinter will store the exising 96 well plate and load a new plate, using the moving platform.

This is a timelapse video showing an aerial view of the 3D bioprinting of a nine by nine patch at 100 times speed, the needles in the needle array are arranged in a group formation and spheroids are loaded onto each needle from above. After three days under sterile conditions, the 3D bioprinter cardiac patch is very carefully removed from the needle array by gently sliding the piece of hard plastic in the needle array under the patch upwards. Lubrication with phosphate buffered saline or PBS, maybe applied to the needles during this step as needed, for smoother and easier removal.

The 3D bioprinted cardiac patch is very fragile, immediately after removal from the needle array, so due care must be taken in order not to damage it. The 3D bioprinted cardiac patch is then transferred into a 35 millimeter tissue culture dish containing appropriate cell media. Immediately after removal from the needle array, it is noted that the patch is already beating, with the needle holes changing in size with each beat.

The 3D bioprinted cardiac patch is allowed to mature, during which time, the tissue voids caused by the needle array are filled up by surrounding tissue. The 3D bioprinting cardiac patch continues to beat spontaneously. In conclusion, it is feasible to 3D bioprint cardiac tissue without the use of biomaterials.

3D bioprinted cardiac patches exhibit mechanical integration of component spheroids. 3D bioprinting of cardiac tissue has promising clinical applications and cardiac tissue regeneration and has 3D models of heart tissue, replication in predictive pharmacology and toxicology and development of cell and genome directed therapies.