Page 438 - IJB-10-5

International Journal of Bioprinting Bioprinting for large-sized tissue delivery

a final working solution containing 2 μmol/L calcein-AM
and 4.5 μmol/L PI. Cell-laden GP samples without light Fluorescence intensity ratio = live cells (III)
exposure were used as the control group. All the samples live cells +deadcells
were incubated in the live/dead staining working solution × 100%
for 15 min in the dark to stain the live (green) and dead
(red) cells. The samples were observed and recorded using Three independent samples were analyzed for
a laser scanning confocal microscope (LSCM; Z2; Nikon, each group.
Japan). Quantitative analysis was performed using the
count/size tool in Image J. Cell viability was calculated 2.4.6. Immunofluorescence staining
using the following equation: Immunofluorescence staining was performed based on
previously described protocols, with slight modification.
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Briefly, planar cultured cells were fixed for 30 min using 4%
number of living cells paraformaldehyde (P1110; Solarbio, China), permeabilized
Cell viability = × 100% (II) by 0.3% Triton-X100 (T8200; Solarbio, China) for 20 min
totalcellnumber
on ice, and blocked with 10% bovine serum albumin
(BSA) (containing 0.1% Triton-X100) (9048-46-8; Yeasen
Three independent samples were analyzed for each Biotech Company, China) for 1 h at room temperature. The
group unless otherwise stated. specimens were then incubated with primary antibodies
2.4.3. Uniaxial compression test (diluted with PBS buffer, containing 0.3% Triton-X and
To determine the elastic modulus of GP hydrogel, we 1% BSA) at 4°C overnight, followed by incubation with
performed the uniaxial compression tests using the the corresponding secondary antibodies for 2 h in the dark
dynamic mechanical testing device. GP samples (10G2.5P at room temperature. Cell nuclei were stained with DAPI
with 10 min exposure) were loaded on the pressure head (D9542; Sigma-Aldrich, USA) for 30 min in the dark. PBS
and compressed with a 25% deformation at a loading was used to wash specimens between every step.
speed of 0.2 mm/s. The real-time data were recorded For the bioprinted architectures, samples were fixed for 4
during the compression process to plot the stress–strain h with 2.5% glutaraldehyde (G5882; Sigma-Aldrich, USA),
curve. The slope of the curve represents the compressive permeabilized for 20 min on ice, and blocked for 2 h at room
elastic modulus. The smoothed curve was generated from temperature. The specimens were washed three times with
the treated data using the signal processing function in PBS and incubated with primary antibodies at 4°C overnight.
OriginPro 2018C (OriginLab Corporation, USA). Three The residual primary antibodies were washed out, and the
independent samples were tested. specimens were incubated with secondary antibodies at 4°C

2.4.4. Cell-laden bioprinting and culture of overnight in the dark. The cell nucleus was then stained with
the architectures DAPI for 30 min in the dark. The reagent and concentration
M14A cells were used to evaluate the cell-bioprinting of solutions used were similar to that for the 2D samples
suitability and biocompatibility of GP hydrogel. M14A unless stated otherwise. Detailed information on the used
cells were collected using 0.25% Trypsin-EDTA. Cells were antibodies is listed in Table S1, Supporting Information.
counted and gently mixed to obtain a final cell-laden GP A laser-scanning confocal microscope was used to capture
precursor consisting of 10% GelMA, 2.5% PEGDA, 4× EY, immunofluorescence images. Images were captured using a
and 9 × 10 cells/mL. 3D architectures were bioprinted, 3D configuration with 100 μm total height for the printed
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photocrosslinked using white light for 10 min and washed specimens, and the maximum density projection images
with PBS to remove the residual photoinitiator. The cell- are presented accordingly.
laden architectures were cultured in an incubator with the
culture medium changed daily. The microscopic images of 2.4.7. Chemical staining
cell-laden architectures were captured on days 1, 5, 9, and For planar cultured specimens, M14A and HepaRG cells
16. Semi-quantitative analysis was performed as described were cultured in 35 mm culture dishes for 14 days, with the
in Section 2.3.3. culture medium changed daily. Bioprinted architectures on
day 16 were used for chemical staining. To assess glycogen
2.4.5. Injection of cell-laden architectures storage functions, a glycogen periodic acid-Schiff (PAS) kit
After 16 days of culture, the cell-laden architectures were (G1360; Solarbio, China) was used for glycogen staining,
injected using dispensing needles with an inner diameter following the manufacturer’s instructions. To assess
of 1.5 mm. Cell viability before and after injection was hepatocyte functions, indocyanine green (ICG) powder
assessed by live/dead staining. Semi-quantitative analysis (S46424; Shanghai Yuanye Biotechnology Company,
of cell viability was performed using Image J as follows: China) was completely dissolved in dimethyl sulfoxide

Volume 10 Issue 5 (2024) 430 doi: 10.36922/ijb.3898