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Liu, et al.
regular intervals of 15, 30, 60, 90, and 120 min using After treatment with 5% w/v blotting grade blocker, the
a 50 μL culture medium from the basal side. The membranes were incubated with corresponding primary
following equation determined the apparent permeability antibodies: Na /K -ATPase and GAPDH (diluted
+
+
coefficients P (cm/s): at 1:1000 v/v in Tris Buffered Saline with Tween
®
app
dQ 1 20 (TBST), Beyotime, China). The corresponding
P app = dt × AC (2.3) signals were then captured using horseradish peroxidase
0
dilution v/v, Beyotime, China) on a chemiluminescence
Where, A is the surface area of the culture substrates (conjugated secondary anti-rabbit IgG antibodies, 1:5000
(cm ), C is the concentration of a target molecule in the imaging system. Quantification of bands was performed
2
0
apical chamber (μg/cm ), and dQ/dt is the linear slope of using ImageJ software, and data were analyzed and
3
the molecular flux (μg/s). compared. Western blot was performed in triplicates in
three independent experiments.
2.11. Immunofluorescence, confocal microscopy
analysis, and 3D visualization 2.13. Transepithelial potential (TEP) and
After 7 days of culture, the samples were fixed with 4% transepithelial electrical resistance (TEER)
paraformaldehyde treated with 0.1% Triton X-100 in PBS TEP and TEER across the ARPE-19 cells were measured
for 30 min. After blocking for 1 hour with 5% normal goat using a Millicell-ERS-2 voltmeter (Millipore, USA)
serum in PBS with 0.1% Tween-20 (PBST), cells were at 1, 2, and 3 weeks. The TEER value was calculated
treated with the following primary antibodies: Zonula according to the following equation:
occludens (ZO)-1 (1:200 dilution, Cell Signaling, USA) TEER (Ω∙cm ) = (R – R ) × A (2.4)
2
0
and Na /K -ATPase (1:50 dilution, Beyotime, China) Where, R is the resistance measured (Ω), R (Ω)
+
+
0
overnight at 4°C. Cells were washed and incubated is the resistance of the insert, and A is the membrane
with the following secondary antibodies: Alexa Fluor area (cm ) of the insert.
2
488-labeled goat anti-rabbit IgG (H+L) (1:200 dilution,
Cell Signaling, USA) and Alexa Fluor 555-labeled 2.14. Statistical analysis
donkey anti-rabbit IgG (H+L) (1:200 dilution, Beyotime, All experiments were performed thrice, and values were
China) for 1 h at 37°C. Cell nuclei were counterstained expressed as the mean ± standard deviation, unless
using Hoechst 33342 (10 μg/mL in complete culture otherwise stated. Statistical analysis was performed
medium, Beyotime, China) for 10 min, followed by by multiple unpaired t-tests with False Discovery Rate
washing with PBS thrice. Cell-scaffold complexes were (FDR) approach (desired FDR = 1%) using GraphPad
visualized using a confocal laser scanning microscope Prism (version 9.1.1, GraphPad Software, USA).
(CLSM, LSM880 with Airyscan, Carl Zeiss, Germany).
A 3D reconstructed z-stack confocal image was obtained 3. Results and discussion
and processed using ZEN software (Carl Zeiss).
For a better 3D visualization of cell nuclei and 3.1. EHDJ printing of ultrathin scaffolds with
scaffold, spatial normalization is performed on the CLSM small pore size
images to ensure the same resolution of each axis. Then, 3D printing inks suitable for EHDJ printing are usually
the scaffolds were manually rendered with white color made of insulating polymeric materials, which led to
(Figure 6D), and the visualization was generated using the charge accumulation in the printed fibers, cause
Mayavi2 with maximum intensity projection . Coulombic repulsion, and may result in the drift of fiber
[23]
deposition [18,24] . Fabricating scaffolds with a small pore
2.12. Western blot analysis of proteins
size (≤50 μm) demand the fiber line spacing being closer
ARPE-19 cells cultured on scaffolds for total protein to the pore size. According to Coulomb’s law, as the
extraction were washed with cold PBS before being printed fibers are close to the jet (Figure 2A), Coulombic
lysed with RIPA lysis buffer at 4˚C for 30 minutes. forces will significantly decrease the accuracy of the
Cell lysates were centrifuged, and the supernatant was fiber deposition and alignment. To reduce such effects,
collected. The protein concentrations were determined by we developed a manufacturing workflow to minimize
the BCA protein assay kit (Beyotime, China). The same the effects of Coulombic forces and allow printing of
amount of proteins in each sample was mixed with a scaffolds with small pore sizes and well-aligned fibers.
loading buffer and heated at 100°C for 5 min to denature It was suggested that the mechanical drawing
proteins. The protein samples were dissolved in sodium force dominated by the stage speed is the determining
dodecyl sulfate-polyacrylamide gel electrophoresis factor on resolution, positioning, and alignment of the
(SDS-PAGE) and transferred to PVDF membranes. EHDJ-printed fibers . Since the key parameters in the
[16]
5 International Journal of Bioprinting (2022)–Volume 8, Issue 3