Page 372 - IJB-10-6

International Journal of Bioprinting 3D-printed NAFLD model

without ELA was introduced into the NAFLD model 3D structure, promoting cell growth and migration. The
from days 4 to 7. Lipid accumulation and liver function results (Figure 1D) indicated that cell viability increased
were assessed on day 7. To assess the reversible effects, with increasing concentrations of fibrinogen and laminin,
the lipogenic medium was added to the culture medium both on days 1 and 7 of cultivation. However, cell viability
from days 4 to 6, followed by the culture medium with or did not further improve when fibrinogen and laminin
without ELA introduced for 24 h. Lipid accumulation and concentrations exceeded 1 mg/mL and 30 μg/mL,
liver function were evaluated on day 7. respectively. Considering the experimental costs, 1 mg/
mL fibrinogen and 30 μg/mL laminin were selected as the
2.13. Statistical analysis optimal concentrations. The optimal bioink formulation
The data are presented as the mean ± standard deviation of for liver tissue printing was determined to be gelatin (5%),
at least three samples per test. The p-values were calculated sodium alginate (1%), fibrinogen (1 mg/mL), and laminin
via two-tailed Student’s t-test for comparisons between (30 μg/mL). To evaluate the effects of the optimized bioink
two groups, or one-way analysis of variance (ANOVA) on liver cell function, we measured the gene expression
for multiple group comparisons. Significance levels are levels of liver-specific proteins (CYP1A2, CYP3A4, and
indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001; and ALB). The results (Figure 1E) demonstrate that after 7 days
N.S.: no significant difference. of cultivation, liver tissues grown with bioink enriched
with fibrinogen and laminin presented significantly higher
3. Results and discussion expression levels of liver-specific proteins than those
3.1. Optimization of the bioink formulation cultured with bioink containing only Gelatin Methacryloyl
To prepare bioink for liver tissue printing with good (GelMA) and alginate. This result is consistent with the
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printability and biocompatibility, 12 different ratios of literature, indicating that the addition of fibrinogen and
gelatin and sodium alginate were tested, and the optimal laminin can effectively enhance liver cell function. This
concentrations of fibrinogen and laminin were explored. formulation ensures good printability and significantly
The experimental results demonstrated that when the improves cell viability, providing a solid foundation for
concentrations of gelatin and sodium alginate were too future liver tissue engineering applications.
low, the bioink remained in droplet form at 5°C, making 3.2. Evaluation of 3D-printed liver tissues
extrusion printing impossible. Conversely, when the To evaluate the growth status of 3D-printed liver tissue,
concentrations were too high, the bioink formed a hard gel the viability of hepatocytes was first analyzed. The results
at 5°C, which was difficult to extrude. Only at appropriate (Figure 2A and B) revealed that on days 1, 3, 5, and 7 of
ratios could the bioink be extruded into suitable filaments culture, high cell viability was maintained (i.e., >90%), with
for printing (Figures 1A and S1D, Supporting Information). most areas exhibiting green fluorescence and no significant
It was statistically determined (Figure 1B) that the printable necrosis. To further validate the advantages of 3D-printed
concentration range for gelatin at 5°C is 3–5%. When the liver tissue, a comparison was made between the 3D-printed
gelatin concentration reached 7%, the bioink became liver tissue model (bioprinting) and the hepatocyte gel-
too hard for extrusion printing. For sodium alginate, the spheroid model constructed via suspension droplet
printable concentration range was 0.5–2%. Below 0.5%, technology (microsphere). The results (Figures 2C, D, and
crosslinking with CaCl was difficult, and above 2%, the S1Ε, Supporting Information) indicated that after 1 day
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bioink was too viscous for printing. Therefore, the suitable of culture, no significant difference in cell diameter was
gelatin/alginate ratios for extrusion printing were 3/0.5, observed between the bioprinting and microsphere groups,
3/1, 3/1.5, 3/2, 5/0.5, and 5%/1%, respectively. Further with an average diameter of approximately 12 μm. However,
analysis of cell viability under these six ratios revealed by day 7 of culture, hepatocytes in the bioprinting group
(Figure 1C) that the highest cell viability was achieved had proliferated to form organoid-like cell clusters, which
with 5% gelatin/0.5% alginate and 5% gelatin/1% alginate. were significantly larger (50.2 ± 6.4 μm) than those in the
The bioink with 3% gelatin resulted in lower overall cell microsphere group (25 ± 1.2 μm). This was primarily due
viability, possibly due to its lower hardness, leading to to the large scale (millimeter scale) of the gel spheroids in
structural collapse and slow degradation during the the microsphere group, where mass transfer limitations
cultivation process, which negatively affected cell viability. led to insufficient diffusion of nutrients and oxygen to the
Sodium alginate at 0.5% also tended to degrade during the center, resulting in nutrient and oxygen deficiency in the
later stages of cultivation. Based on these results, 5% gelatin central cells, thus hindering cell proliferation and growth
and 1% alginate were selected for further experiments. (Figure S1F, Supporting Information). In contrast, a grid-
Fibrinogen and laminin were added to the gelatin/alginate like structure was provided by the bioprinting group, which
bioink to support cell adhesion and fixation within the mimicked the vascular channels in the liver, offering a similar

Volume 10 Issue 6 (2024) 364 doi: 10.36922/ijb.4312