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

2.12. Printability analysis Pharmingen) according to the manufacturer’s protocol.
The printability analysis examined the effectiveness of the The cells were immunostained with relevant antibodies in
extruded strands in the test grids to form square holes FACS buffer (2% FBS in PBS); 1 h with primary antibodies
between strands, as previously described . Circularity followed by 30 min secondary antibody. Data acquisition
[42]
(C) of an enclosed area is based on the shape perimeter and analysis were performed using a FACS-Canto machine
and area, where a perfect circle has a circularity of 1. For a (BD Biosciences) utilizing Cellquest Pro software (BD
square shape, circularity is equal to π/4, and the printability Biosciences) and FlowJo X 10.0.7. For quantification
is given by Equation I: of cardiac protein expression levels, mean fluorescent
L 2 intensity was calculated based on the intensity distribution
Pr = (I) plot of each protein marker. Gating of CM population
16 A was determined according to the intensity of sarcomeric
where L is the perimeter, and A is the area of a shape. A α-actinin (Clone EA-53, 1:300, Sigma), setting the
printability of 1 is equal to a perfect square and indicates threshold based on secondary antibody control and pre-
optimal printing conditions of a bioink. Confocal images of plated cardiac fibroblasts culture population expression
three-layer high test grids, printed as described above, were levels.
evaluated by measuring the perimeter and area of several
holes in each sample, and printability was calculated, with 2.15. Cell metabolic activity and DNA content
4 constructs and at least 25 holes measured. Cell metabolic activity was measured by PrestoBlue
reagent (Invitrogen). The 3D-bioprinted constructs were
2.13. Cryogenic scanning electron transferred into new wells and incubated with 600 µL
microscopy (Cryo-SEM) of PrestoBlue reagent mixture (1:9 in DMEM culture
Samples of a cellular, 3D-bioprinted CPs, were fabricated medium supplemented with 20% v/v FBS) for 3 h at 37°C.
as described above. Dissected samples were placed and Aliquots (300 µL) were placed in a 96-well plate and the
sandwiched between two aluminum disks (3 mm in fluorescence was measured at excitation wavelength of
diameter, each 25-μm thick) and cryo-immobilized in a 560 nm and emission wavelength of 590 nm using a plate
high-pressure freezing device (EM ICE; Leica). reader (model ELX 808, BIO TEK Instruments, Winooski,
The frozen samples were then mounted on a holder VT). Samples of the reagent mixture incubated under
under liquid nitrogen in a specialized loading station (EM the same conditions without cells were used as a blank.
VCM; Leica) and transferred under cryogenic conditions Cell DNA content was quantified using bisbenzimidazole
(EM VCT500; Leica) to a sample preparation freeze fracture Hoechst 33258 fluorescent dye (Sigma). The constructs
device (EM ACE900; Leica). In that device, the samples were dissolved in 500 µL sodium citrate (4% w/v in
were fractured by the rapid stroke of a cryogenically cooled PBS) to release encapsulated cells, and then centrifuged
knife, exposing the inner part of the sandwiched disks. for 10 min at 6000 rpm and 4°C. The cell pellet was
After fracturing, the samples were etched at -100°C for suspended in 100 µL lysis buffer (0.02% v/v SDS) in
10 min to sublime ice from the sample surface, and were sodium citrate (0.015 M-saline, pH 7) and incubated for
coated with 3 nm carbon. 1 h at 37°C. Then, 100 µL of Hoechst 33258 assay solution
(2 mg/mL) was added, followed by a 10-min incubation.
Samples were imaged in a Gemini SEM (Zeiss) by a Aliquots (180 µL) were placed in a 96-well plate and the
secondary electron in-lens detector, while maintaining fluorescence was measured at excitation wavelength of
an operating temperature of -120°C (Figure S3 in 485 nm and emission wavelength of 530 nm using a plate
Supplementary File). The measurements were done at the reader. Samples of the reagent mixture incubated under
Ilse Katz Institute for Nanoscale Science and Technology the same conditions without cells were used as a blank.
at Ben-Gurion University of the Negev, Beer Sheva, Israel. Specific metabolic activity was calculated as cell metabolic
activity intensity normalized to DNA content quantified
2.14. Flow cytometry
On days 1 and 5 post-printing, the cardiac patches were by Hoechst.
dissociated with citrate buffer (4% w/v in PBS, pH 7.4) to 2.16. Immunocytochemistry and confocal imaging
release encapsulated cells, and were washed twice in FACS For immunofluorescence of cell monolayers, cells were
buffer (2% FBS in PBS). For cellular viability analysis, cells fixed in 4% v/v warm methanol-free formaldehyde in
were stained using the Live/Dead viability/cytotoxicity kit PBS for 20 min, washed twice in PBS, permeabilized with
(Invitrogen) according to the manufacturer’s protocol.
0.1% Triton-X, and blocked with 1% BSA (Millipore,
For protein analysis, suspended cells were fixed Bedford, MA) for 1 h at room temperature. The samples
and permeabilized with the Cytofix/Cytoperm kit (BD were incubated overnight with the primary antibodies,

Volume 9 Issue 2 (2023) 321 https://doi.org/10.18063/ijb.v9i2.670