Page 326 - IJB-9-2

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

LVG; >65% guluronic acid monomer content) were from through a 0.22-µm filter, followed by two-step ultrafiltration,
FMC Biopolymers (Drammen, Norway). All reagents were using 100-kDa MWCO Amicon™ centrifugal filters (Sigma-
of analytical grade. Merck), according to manufacturer’s protocol.

2.2. Cell culture 2.4.2. Nanoparticle tracking analysis (NTA) of size and
Human monocytic THP-1 cells (ATCC® TIB-202) concentration
were cultured in RPMI 1640 supplemented with 2 mM Size distribution and concentration of isolated EVs were
L-glutamine, 1 mM sodium pyruvate, penicillin (100 U/mL), measured using NanoSight NS300 system (Malvern, UK). EV
and streptomycin (100 µg/mL). THP-1 monocytes were samples were diluted in PBS until individual nanoparticles
seeded at a density of 0.3 × 10 cells/mL and differentiated could be tracked. The samples were captured for 60 s at
6
into MΦs by incubation with 100 ng/mL phorbol room temperature. NTA software was used to measure
12-myristate 13-acetate in serum-free RPMI medium for particle concentration (particles/mL) and size distribution
2 days followed by a 24-h incubation in RPMI culture (in nanometers). For each sample, five measurements were
medium. MΦ activation was performed by 24-h incubation taken, and the mean value was determined.
with serum-free RPMI medium, supplemented with 2.4.3. Cryo-transmitting electron
20 ng/mL human recombinant interferon gamma (IFN-γ; microscopy (Cryo-TEM)
Peprotech®, #300-02) and 10 pg/mL of lipopolysaccharide.
For cryo-TEM, 5 μL of the EV sample was applied to
Primary human umbilical vein endothelial cells a copper grid coated with perforated lacy carbon 300
(HUVEC) from ATCC® (PCS-100-013) were cultured meshes (Ted Pella) and blotted with filter paper to obtain
with Vascular Cell Basal Medium (ATCC® PCS-100-030), a thin liquid film of solution. The blotted sample was
supplemented with the Endothelial Cell Growth Kit-VEGF immediately plunged into liquid ethane at its freezing
(ATCC® PCS-100-041) following the kit’s instruction. point (-183°C) with an automatic plunger (Lieca EM GP).
The vitrified samples were stored in liquid nitrogen until
2.3. Neonatal rat cardiomyocytes isolation analysis. Sample analysis was carried out with a FEI Tecnai
The study was performed with the approval and according 12G2 TEM at 120kV with a Gatan cryo-holder maintained
to the guidelines of the Institutional Animal Care and Use at -180°C. Images were recorded digitally using the Digital
Committee of Ben-Gurion University of the Negev, Beer Micrograph 3.6 software (Gatan, Munich, Germany).
Sheva Israel (IL-72-09-2020A). Neonatal cardiac cells Crystal size of purified EVs was measured using ImageJ
were isolated from the ventricles of 1–2-day-old neonatal 1.53q software (U.S. National Institutes of Health, Bethesda,
Sprague-Dawley rats using 6–7 cycles (20 min each) of Maryland, http://imagej.nih.gov/ij/).
enzymatic digestion with collagenase type II (95 U/mL;
Worthington, Lakewood, NJ) and pancreatin (0.6 mg/mL; 2.4.4. Western blotting for exosome markers
Sigma). The cell pellets were resuspended in M-199 culture THP-1-derived MΦs and isolated EVs were lysed in RIPA
medium, supplemented with 100 U/mL penicillin and Buffer (Thermo Fisher Scientific) supplemented with
100 mg/mL streptomycin and 5% (v/v) FBS. To enrich the protease and phosphatase inhibitor cocktails (Roche),
CM population, the cell suspension was pre-plated twice followed by centrifugation at 14,000 ×g for 15 min at 4°C.
for 1 h each on an uncoated plate to separate the non-CM. Protein quantities of the lysates were quantified using the
After pre-plating, the non-attached cells (mostly CM) were Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
collected. The isolated cells consisted of over 60% CMs For Western blotting, fractionation of lysates was carried in
(Figure S1 in Supplementary File). sodium dodecyl sulfate polyacrylamide gel electrophoresis
on NuPAGE 4%–12% Bis-Tris Protein Gels (Thermo Fisher
2.4. EV isolation and characterization Scientific). Then, fractionated samples were transferred
2.4.1. EV isolation onto nitrocellulose membranes for immunoblotting. The
EVs were isolated from culture media using differential membranes were blocked in Tris-buffered saline containing
centrifugation and ultrafiltration methodologies. THP-1 5% powdered bovine serum albumin (BSA) for 1 h at room
derived MΦs were washed with phosphate-buffered saline temperature, and then incubated overnight at 4°C with
(PBS), and the medium was replaced with fresh, serum-free anti-TSG101 rabbit polyclonal antibody (Abcam) and anti-
RPMI 1640 medium. To increase EV yield, the medium Calnexin rabbit polyclonal antibody (Abcam), which were
was supplemented with 100 mM ethanol. After incubation diluted 1:1000. After incubation with an appropriate anti-
for 48 h, culture media was collected on ice, centrifuged HRP conjugate (Thermo Fisher Scientific), bands were
at 3,500 ×g for 10 min to remove cell debris. Supernatant visualized with a chemiluminescence reagent (Biological
was collected and centrifuged at 10,000 ×g for 30 min Industries) and detected using the ChemiDoc system (Bio
for apoptotic body elimination. Supernatant was filtered Rad, CA).

Volume 9 Issue 2 (2023) 318 https://doi.org/10.18063/ijb.v9i2.670