316Engineered extracellular vesicle-mediated delivery of miR-199a-3p increases the viability of 3D-printed cardiac patches.

In recent years, extrusion-based three-dimensional (3D) bioprinting is employed for engineering cardiac patches (CP) due to its ability to assemble complex structures from hydrogel-based bioinks. However, the cell viability in such CPs is low due to shear forces applied on the cells in the bioink, inducing cellular apoptosis. Herein, we investigated whether the incorporation of extracellular vesicles (EVs) in the bioink, engineered to continually deliver the cell survival factor miR-199a-3p would increase the viability within the CP. EVs from THP-1-derived activated macrophages (MΦ) were isolated and characterized by nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo-TEM), and Western blot analysis. MiR-199a-3p mimic was loaded into EVs by electroporation after optimization of applied voltage and pulses. Functionality of the engineered EVs was assessed in neonatal rat cardiomyocyte (NRCM) monolayers using immunostaining for the proliferation markers ki67 and Aurora B kinase. To examine the effect of engineered EVs on 3D-bioprinted CP viability, the EVs were added to the bioink, consisting of alginate-RGD, gelatin, and NRCM. Metabolic activity and expression levels of activated-caspase 3 for apoptosis of the 3D-bioprinted CP were evaluated after 5 days. Electroporation (850 V with 5 pulses) was found to be optimal for miR loading; miR-199a-3p levels in EVs increased fivefold compared to simple incubation, with a loading efficiency of 21.0%. EV size and integrity were maintained under these conditions. Cellular uptake of engineered EVs by NRCM was validated, as 58% of cTnT