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International Journal of Bioprinting Kidney hydrogel print for renal cancer model

followed by DAPI staining. All samples were imaged (n = limited influence on GelMA shear-thinning (Figure 1H),
6 images at different spots per group) immediately using but its addition increased the gelation temperature to
confocal microscopy to avoid fluorescence quenching, and 25°C (Figure 1I). The appearance of the GelMA hydrogel
ImageJ was used to determine the fluorescence intensity. at different dECM concentrations is shown in Figure 1F.
It is evident that the hydrogel samples became much less
2.23. Evaluation of the resistance to sunitinib transparent after more dECM powder was added. The
We further evaluated the resistance of ACHN cells in all mechanical properties of all hydrogel compositions were
groups to exogenous sunitinib, an anti-kinase cancer evaluated using compressive tests. In this study, the Young’s
treatment drug. To do so, GelMA samples from all groups modulus for 10% GelMA, 10% GelMA-1% Kidney, 10%
were cultured for 5 days, and 2D cells, which were used as GelMA-2% Kidney, and 10% GelMA-3% Kidney samples
control, were seeded with a concentration of 50,000 cells/ was 19.77 ± 1.05 kPa, 20.46 ± 3.32 kPa, 39.51± 4.71 kPa,
well after 4 days of hydrogel culture. All groups (n = 5) were and 58.03 ± 8.65 kPa, respectively. These results indicate
then cultured with 10 μM sunitinib using dimethylsulfoxide that the Young’s modulus had a positive correlation with
(DMSO) as the vehicle. Cell viability was determined with increasing dECM concentration (Figure 1J). The stability
CCK-8 assays after 72 h of drug exposure, and viability of all hydrogel compositions was then analyzed with a
was calculated by evaluating sample absorbance relative to swelling test (Figure 1K and L), where the 10% GelMA, 10%
vehicle absorbance for all groups. GelMA-1% Kidney, and 10% GelMA-2% Kidney samples
had a similar swelling rate, which was however significantly
2.24. Statistical analysis lower than the rate of 10% GelMA-3% Kidney sample.
Unless otherwise mentioned, all results were analyzed
using GraphPad version 8.02. All experiments were 3.2. Printability of GelMA with dECM powders
performed in triplicate. Data are expressed as mean To further analyze the influence of dECM on GelMA
± standard deviation. Student’s t-tests and analysis of properties, we then assessed the printability of the 10%
variance (ANOVA) were performed to evaluate the GelMA, 10% GelMA-1% Kidney, 10% GelMA-2% Kidney,
statistical significance of differences in group means for all and 10% GelMA-3% Kidney samples. All compositions
data. A P value of <0.05 indicates significant difference (*P were printed with designated patterns to determine the
<0.05, ** P <0.01, ***P <0.001). impact of dECM powders on pore closure and filament
fusion (Figure 2A). The PAI for each was then quantified
3. Results (Figure 2B), and we found that the inclusion of an increasing
3.1 Characterization of GelMA with dECM powders amount of dECM powder significantly improved the
First, porcine kidney tissues were sliced into tiny fragments, PAI for all pore sizes, indicating a more stable structure
decellularized for 3 days, ground into powder, and imaged was formed. On the other hand, the results on material
by means of SEM before mixing with GelMA (Figure 1A). spreading index and cubic ratio (Figure 2C–F) attested that
The degree of decellularization was then assessed via DNA the dECM powders were able to better enhance GelMA
concentration measurements, Western blotting, and H&E printability.
staining. As shown in Figure 1B, the control sample without Subsequently, to further demonstrate the effect of
showing sign of decellularization had a significantly higher dECM powders on printability, all samples were utilized
DNA concentration than decellularized samples. Similar to 3D-print more intricate structures, including a 15-layer
results were also observed for GAPDH protein, as visualized thin-wall (0.4 mm) tube (diameter = 3.5 mm, and height
3
using Western blotting (Figure 1C). H&E staining images = 10 mm), a five-layer Chinese knot (10 × 10 × 1 mm ),
3
depicted in Figure 1D show that the cytoplasm and nucleus and a ten-layer cubic (7 × 7 × 4 mm ). A high-resolution
were almost completely removed, while the ECM was camera was utilized to capture the images of the top, side,
preserved in most decellularized samples. dECM powders and zoom-up views of each of these structures (Figure 3),
were further mixed with 10% GelMA precursors as shown which portrayed the positive influence of dECM inclusion
in Figure 1E, where different concentrations of dECM were on GelMA printability.
added. Here, the precursor solutions with higher dECM 3.3 Effect of kidney dECM on morphology,
concentration were obviously more viscous, a finding proliferation, and gene expression of ACHN cells
consistent with rheological evaluation. Moreover, the Next, the effect of dECM powder on RCC cellular behavior
viscosity of GelMA solution significantly increased with was assessed in a series of tests run on ACHN cells
dECM concentration (Figure 1G).
cultured in the bioprinted nephron structure. The ACHN
Viscosity under various shear rate was also determined cellular morphology was evaluated using phalloidin
for all precursor solutions. Here, the dECM exerted a staining on days 1, 3, 5, and 7 for all hydrogel compositions

Volume 10 Issue 2 (2024) 284 doi: 10.36922/ijb.1413