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

Abstract

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⁺ cells internalized EVs after 24 h. The engineered EVs induced CM proliferation, increasing the ratio of cell-cycle re-entry of cTnT⁺ cells by 30% (Ki67) and midbodies+ cell ratio by twofold (Aurora B) compared with the controls. The inclusion of engineered EVs in bioink yielded CP with threefold greater cell viability compared to bioink with no EVs. The prolonged effect of EVs was evident as the CP exhibited elevated metabolic activities after 5 days, with less apoptotic cells compared to CP with no EVs. The addition of miR-199a-3p-loaded EVs to the bioink improved the viability of 3D-printed CP and is expected to contribute to their integration in vivo.

Keywords: 3D bioprinting; Cardiac patch; Cardiomyocytes; Extracellular vesicles; Tissue Engineering; miRNA.

Figure 1

Characterization of macrophage-derived EVs. (A) Representative size distribution plot of EVs isolated from activated macrophages. (B) Cryo-transmission electron micrographs of EVs (black arrows; scale bar: 200 nm). (C) Immunoblots of EVs and cell lysate fractions for the EVs marker TSG101 and ER protein calnexin (n = 4).

Figure 2

Electroporation optimization for EV loading with miRNA. (A and B) Relative expression levels of cel-miR-39 (A) and miR-199a-3p (B) in EVs following application of different electroporation settings, compared with naïve EVs (nEVs) and EVs incubated with free miRNA (iEVs), determined by RT-PCR. Data represent mean ± SEM (n = 3) *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001; Tukey’s test, one-way ANOVA. (C–F) Electroporation effect on EVs. (C) Representative particle concentration (left) and normalized concentration (right) size distributions for iEVs and EVs after electroporation in 850 V and 5 pulses, measured by NTA. (D) Representative cryo-transmission electron micrographs of EVs after electroporation at 850V (left) and incubation with miRNA (right); scale bar: 200 nm. (E) Size distribution of electroporated EVs (EP-EVs, blue), iEVs (orange) and nEVs (green), measured according to cryo-transmission electron microscopy.

Figure 3

Neonatal rat cardiomyocytes uptake MΦs-EVs. (A) Optimization of CFSE-labeled MΦs-EVs (green) uptake by NRCM, stained for F-actin (red) and nuclei (blue). Cells were incubated with 4 × 10⁹ EVs/ml or 8 × 10⁹ EVs/ml for 12 h and 24 h. EV-positive cells are indicated by white arrows. Scale bar: 50 µm. (B) Representative images of CFSE-labeled MΦ-EVs found inside NRCMs. Cells were incubated for 24 h without EVs (top panel) or with 6 × 10⁹ EVs/mL (bottom panel). Scale bar: 50 µm. (C) High-magnification images of XY (top) and reconstructed YZ (bottom) planes, 24 h after adding MΦs-EVs (i) and filtered CFSE dye (ii), showing internalization of the labeled EVs into NRCM. Nuclei (blue); Cardiac troponin (red) and EVs (green). Scale bar: 10 µm.

Figure 4

Electroporated EVs induce NRCM proliferation and cytokinesis. (A) Representative images of Ki67+ (red) and cTnT+ (green) NRCM (white arrows) to demonstrate increased Ki67 staining 48 h after exposure to miR-199a-3p-electroporated EVs. Scale bar: 50 μm. (B) Quantification of Ki67⁺ cTnT+ NRCM 24 and 48 h from exposure to EVs. Data are mean ± SEM., n = 3–4, *p < 0.05, Tukey’s multiple comparisons test, two-way ANOVA. Quantification was based on counting of Ki67⁺ cTnT+ co-stained cells relative to total cTnT⁺ cells. (C) Representative images of Aurora kinase B⁺ (red) cTnT⁺ (red) NRCM (white arrows) to demonstrate increased cytokinesis 48 h after exposure to miR-199a-3p-electroporated EVs. Scale bar: 50 μm. (B) Quantification of midbodies⁺ cTnT⁺ NRCM 48 h from exposure to EVs. Data are mean ± SEM., n = 3–4, *p < 0.05, Tukey’s multiple comparisons test, one-way ANOVA. Quantification was based on counting of midbodies+ cTnT⁺ co-stained cells relative to total cTnT+ cells.

Figure 5

Engineered MΦs-EVs present pro-angiogenic potential. (A–C) CFSE-labeled MΦs-EVs (green) uptake by HUVECs. (A) Representative images of CFSE-labeled MΦs-EVs found inside HUVECs (white arrows), incubated for 24 h with engineered EVs. Nuclei (blue); F-Actin (red) and EVs (green). Scale bar: 50 µm. (B) Flow cytometric analysis of EV uptake by HUVEC cells (green), compared with non-treated cells (gray) and filtered CFSE dye (orange). (C) Quantification of cellular uptake, according to flow cytometry. (D–F) HUVECs tube formation evaluation. (D) Representative fluorescently labeled HUVECs tube formation following 24 h with engineered EVs (right), naïve EVs (middle) or without treatment (left). (E) Quantification of total vessel length, normalized to positive control. (F) Quantification of the number of junctions. Data are mean ± SEM, n = 3, *P < 0.05, Tukey’s multiple comparisons test, one-way ANOVA.

Figure 6

Engineered EVs improve cell survival within 3D-bioprinted cardiac patches (CPs). (A) Schematic description of the 3D bioprinting procedure. (B) 3D-bioprinted CPs. (i) Images of the 3D-bioprinted CP (1 cm in diameter, 2 mm high). (B, ii–iii) Confocal images of XY (ii) and reconstructed YZ (iii) planes, 24 h post-3D bioprinting of fluorescently labeled cells. (C and D) Expression levels of the cardiac-specific proteins inside the CPs, after 5 days in culture. (C) Flow cytometric analysis of cTnT⁺ (top) and sarcomeric α-Actinin⁺ (bottom) of NRCM co-printed with engineered EVs (green) or without EVs (red), compared with secondary antibody control (gray). (D) Relative mean fluorescent intensity (MFI) of cardiac-specific proteins. (E and F) Cell viability analysis within the 3D-bioprinted CPs, following 5 days in culture. (E) Relative specific metabolic activity of CP residing cells. (F) DNA content in the CPs relative to day 1 post-printing, quantified by Hoechst. (G) Flow cytometric analysis of relative cellular viability within the 3D-bioprinted CPs, following 5 days in culture. (H) Quantification of activated caspase 3⁺ α-Actinin⁺ NRCM, 5 days post-printing, shows decreased apoptosis among CM co-printed with engineered EVs (mean ± SEM, n = 3).