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International Journal of Bioprinting GradGelMA 3D-bioprinted vascular skin

with gold-palladium. The morphology of samples was 2.7. Cell viability and proliferation assays
observed using scanning electron microscopy (SU-8010, The HFF cells (1.0 × 10 cells/mL) were added to 5%, 10%,
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Hitachi, Japan), with a working distance of 9 mm and an 15%, and 20% concentrations of GelMA. Cell viability
accelerating voltage of 15 kV, and analyzed using ImageJ was measured by red or green fluorescent dye uptake with
®
(ImageJ2 2.14.0). The rheological properties of GelMA LIVE/DEAD Viability/Cytotoxicity Kit. The samples
hydrogel samples were measured using an MCR302 were cultured in a humidified 5% carbon dioxide incubator
rotational rheometer (Anton Paar, Austria). At a fixed at 37°C for 1, 3, and 7 days. The fluorescence images of
frequency of 1 Hz, the storage modulus (Gʹ) and loss the cell-laden structures were collected using confocal
modulus (G˝) were measured as the temperature decreased laser scanning microscopy (ZEISS LSM780, Oberkochen,
from 40 to 4°C at a rate of 5°C/min. To assess the effect Germany). Cell proliferation was determined using cell
of temperature on viscosity, measurements were taken at counting kit-8 assays (Medchemexpress, United States).
a constant shear rate of 50/s within the same temperature The cells encapsulated in the GelMA were cultured in a
range. At 25°C, the relationship between the shear rate and humidified 5% carbon dioxide incubator at 37°C for 1, 2,
viscosity was observed within the 0–500/s range. 3, 4, 5, 6, and 7 days. The samples were incubated at 37°C
for 2 h, and cell viability was quantified by measuring the
2.5. Cell culture ultraviolet absorbance of the solution at 450 nm using a
Human umbilical vein endothelial cells (HUVECs), human microplate reader (RT-6100, Rayto, China).
foreskin fibroblasts (HFF), and human immortalized
epidermal cells (HaCaT) (SUNNCELL, China) were 2.8. F-actin fluorescent labeling
cultured in Dulbecco’s modified Eagle Medium/Nutrient For the fluorescent staining of F-actin, the samples
Mixture F-12 supplemented with 15% fetal bovine serum were washed twice in PBS and then fixed with 4%
and 1% penicillin/streptomycin at 37°C and 5% carbon paraformaldehyde for 10 min at room temperature. After a
dioxide. HUVECs and HFF were routinely passaged 3× 10-min rinse with PBS, the samples were permeabilized
onto a tissue culture flask and were discarded after 8 and with 5% Triton-X 100 for 3 min, washed thrice for 10 min
12 passages, respectively, to ensure the representation of in PBS, and stained with TRITC phalloidin for 30 min at
key characteristics. Culture media was changed every 37°C in the dark. It was then washed thrice for 5 min in
2 days, and cells were sub-cultured upon reaching PBS. The samples were then counterstained with 100 nM
80–90% confluency. 4ʹ,6-diamidino-2-phenylindole (DAPI; Slarbio, China) for
15 min at room temperature in the dark and then washed
2.6. Printability and biocompatibility twice for 5 min in PBS. The images of the samples were
According to the gel point test results of the GelMA observed using confocal laser scanning microscopy (Leica,
hydrogel, printing tests were carried out for GelMA with Germany) and analyzed using ImageJ software.
concentrations of 5%, 10%, 15%, and 20% (w/v) within
the temperature ranges of 13–17, 15–19, 17–21, and 20– 2.9. Western blot
24°C, respectively. The printing speed was 6 mm/s, the Western blot was performed using a previously
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pressure was 0.1–1.2 bar, the needle type was 30 G, and described method. The HFF cells (1 × 10 cells/mL)
a two-layer grid structure was printed. A smaller needle encapsulated in the GelMA were cultured at 37°C and
diameter typically improves printing resolution but may 5% carbon dioxide for 7 days. The samples were lysed
compromise cell viability due to higher shear stress. in radioimmunoprecipitation assay buffer (Solarbio,
Therefore, we selected a 30 G needle to balance resolution China) with phenylmethanesulfonyl fluoride (Servicebio,
and cell viability. China) on ice, followed by centrifugation for 5 min
(12,000 rpm, 4°C). The protein was quantified using the
For cell-loaded printing, 10% (w/v) GelMA loaded bicinchoninic acid protein assay kit (HK Bio, China). The
with HFF cells (1 × 10 /mL) were used to print the grid and supernatants (10 μg protein) were subjected to SDS-PAGE
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film structures. During printing, the nozzle temperature (Servicebio, China) electrophoresis and transferred onto
was controlled at 18°C, and the low-temperature platform polyvinylidene difluoride membranes (Servicebio, China).
temperature was controlled at 8°C; the needle type selected The non-specific sites were blocked with 5% fat-free milk
was 30 G. After printing, the samples were irradiated with in tris-buffered saline with Tween-20 buffer (Servicebio,
405 nm blue light for 60 s, followed by the addition of China) on a shaker at room temperature for 2 h. The
culture medium and incubation. Both samples were dyed membranes were then incubated with primary antibodies
with LIVE/DEAD® Viability/Cytotoxicity Kit (Thermo for collagen I (1:1000) and GAPDH (1:1000) (Servicebio,
Fisher, United States) within an hour after printing and on China) overnight at 4°C. Subsequently, the membranes
the 7th day after printing. were incubated with goat anti-Rabbit immunoglobulin

Volume 11 Issue 4 (2025) 332 doi: 10.36922/IJB025090069