Page 328 - IJB-9-2

International Journal of Bioprinting Engineered EVs increase viability of 3D printed cardiomyocytes

2.9. Tube formation assays were assessed at different shear rates between 0.1 and 10 s .
−1
HUVECs of passages 5 were resuspended in growth The measuring device was equipped with a temperature
medium without vascular endothelial growth factor control unit (Peltier plate, ±0.05°C) operated at 28°C
(VEGF), supplemented with either miR-199a-3p- (Figure S2 in Supplementary File)
engineered EVs (6 × 10 EVs/mL) or controls (naïve MΦ-
9
EVs or PBS), and then seeded onto Matrigel (Corning)- 2.10.3. 3D bioprinting and cardiac patches
coated, 48-well plates at a concentration of 4 × 10 cells/ maintenance
4
cm for 18 h. For positive control, HUVECs resuspended Prior to bioprinting, the bioink was deposited into a
2
in growth medium supplemented with 20 ng/mL VEGF sterilized 30 mL printer cylindrical cartilage sealed with
were used. After 18 h, the cells were washed in PBS and a fit plunger, using a Luer-to-Luer connector. The printer
labeled with 5 µM CFSE (in PBS) for 20 min, and then cartilage was then sealed and centrifuged for 1 min
washed in growth medium. Image acquisition was carried at 150 ×g to remove remaining air bubbles. A sterile
using a fluorescent microscope. The total number of 25-gauge needle tip was connected to the cartilage, and
junctions and cumulative vessel length in each image were a cap connecting the print head to the barrel was added.
quantified using Angiotool software (version 0.6a, NIH/ The barrel was put in the low-temperature head of the
NCI, USA) and normalized to the values of the positive bioprinter (EnvisionTEC 3D-bioplotter Developer Series,
[39]
control. Germany), set to 28°C. The bioink solution was allowed
to reach the printing head temperature for 30 min before
2.10. Cardiac patches 3D bioprinting bioprinting began.
2.10.1. Bioink preparation Three-dimensional bioprinting was performed using
Alginate (LVG) was covalently modified with RGD peptide the freeform reversible embedding of suspended hydrogel
as previously described . RGD-modified alginate solution (FRESH) approach, as previously described . The cardiac
[40]
[41]
(LVG-RGD) was prepared by dissolving lyophilized patches were printed onto a 12-well plate filled with FRESH
alginate-RGD in double-distilled water under stirring for gelatin supporting bath at room temperature, using applied
2 h. Fifteen percent gelatin stock solution was prepared by pressure of 0.5 bar and a printing velocity of 12 mm/s.
dissolving bovine gelatin (Type B) in Dulbecco’s Modified Patches were 1 cm in diameter and 2 mm thick, printed
Eagle Medium (DMEM) under stirring for 1 h at 37°C. in 14 layers, and fabricated using an infill pattern of 90°
Crosslinking of alginate was achieved by mixing 1.8 mL grids with 0.6 mm spacing (center-to-center) and a 30%
of 2.5% w/v LVG-RGD with 0.3 mL of 3% w/v D-gluconic overlap between printed layers. Patches were printed in
acid salt solution, using a homogenizer to equally triplicates. Computer-aided design models of the patches
distribute the calcium ions throughout the solution. The and grids printed were created using SOLIDWORKS
mixture was further stirred at 37°C until used. Next, software and imported as STL files to the printing control
0.6 mL of gelatin stock solution was added to the cross- system through the Bioplotter RP software. Following
linked alginate, allowed to mix for 10 min before being bioprinting, patches were incubated for 1 h at 37°C with
transferred to a sterile syringe. Following gelatin addition, 5% CO to free the 3D-printed constructs from the gelatin
2
0.3 mL of isolated NRCM, either mixed with 1.2 × 10 support bath. The patches were then washed and incubated
11
engineered EVs/mL solution or PBS, were loaded into an for 10 min at 37°C in DMEM supplemented with 0.1 M
additional sterile syringe, which was then immediately CaCl to further crosslink the 3D-bioprinted constructs.
2
connected to the polymer-loaded syringe through a Luer- Following incubation, the cardiac patches were incubated
to-Luer connector. The solutions were mixed by gently in DMEM growth medium supplemented with 20% FBS
pushing the pistons back and forth for 1 min, until the in new 12-well plate. Medium was changed daily for each
solutions were homogenously mixed. The final bioink cardiac patch until analysis.
solution consisted of 1.5% LVG-RGD (w/v), 0.3% (w/v)
D-gluconic acid salt, 3% Gelatin, 1 × 10 NRCM/mL, and 2.11. Mechanical stiffness of 3D-bioprinted CPs
7
6 × 10 EVs/mL. The elastic modulus (Young’s modulus) of the
10
3D-bioprinted CPs was analyzed by an Instron 4505
2.10.2. Bioink rheological characterization mechanical tester (courtesy of Prof. Ronit Bitton, Ben-
The viscoelastic properties of bioink were analyzed using Gurion University of The Negev, Israel) equipped with a
a stress control rheometer (TA Instruments, model AR 100 N load cell. The crosshead speed was set to 5 mm/min,
2000), operated in the cone-plate mode with a cone angle and load was applied until the specimens were compressed
of 1° and a 60-mm diameter. Storage (G′) and loss (G″) to approximately 100% of the original thickness. The
moduli were measured in a frequency range of 0.1–10 Hz, elastic modulus was calculated as the slope of the initial
while the apparent viscosities (Pa*s) of the bioink solutions linear portion of the stress–strain curve (n = 4).

Volume 9 Issue 2 (2023) 320 https://doi.org/10.18063/ijb.v9i2.670