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Bioprinting of a Hepatic Tissue Model
(triplicates at each sampling time point) were used for no longer significantly wider than that in the SW model
data analysis. from day 6 to day 8, while that in the edge region of
the 3DP model was still found to be significantly wider.
2.7. Evaluation of drug-induced hepatotoxic Notably, a significant difference in spheroid diameter
response between the central and edge regions of the 3DP model

In this study, acetaminophen (APAP) (A800441, Macklin, was observed on day 8, with 46.06 ± 6.38 μm and 70.25
Shanghai, China) was used as a model drug for in vitro ± 11.65 μm in the central and edge region, respectively.
Figure 2C shows the live/dead fluorescent images of the
hepatotoxicity testing. Drug-induced hepatotoxicity in the SW and 3DP models on day 8. The average diameter of
2D, SW, and 3DP models was evaluated after 8 days in the spheroids located at different distances from the edge
culture. APAP stock solution was prepared by dissolving of the hydrogel structure was analyzed. We observed that
APAP powder in dimethyl sulfoxide, which made up 1% most large spheroids were distributed toward the edge
of the culture medium volume. Consecutive dilutions, of the hydrogel structure in the SW and 3DP models. In
ranging from 0 to 80 mM, were selected for drug testing. the 3DP model, the spheroid diameter decreased rapidly
Cell viability was measured after 24 or 48 h of treatment as the distance to the edge of the hydrogel structure
using the CCK8 assay kit. Dose–response curves were increased. However, this phenomenon was not seen in
obtained using the four-parameter variable slope-fitting the SW model. These results suggest that the 3DP model
method (GraphPad Prism 7.0). Internal controls were is permissive for hiPSC-Heps to form larger spheroids,
prepared with the same volume of culture medium without despite a concentrated distribution at the edge of the
APAP. The half maximal inhibitory concentrations (IC50) hydrogel fibers.
were determined from respective dose-response curves.
Samples of each group were prepared in triplicate. 3.2. Cell viability and proliferation in the SW
2.8. Statistical analysis and the 3DP hepatic tissue model
As mentioned above, cells in the two 3D-cultured models
Statistical analyses were performed using GraphPad (SW and 3DP model) both formed spheroids after 8 days
Prism 7.0. All data are presented as the mean ± standard of culture. It is essential to compare the cell viability and
deviation (SD). Two-tailed Student’s t-test or one-way proliferation to evaluate the cell growth during spheroid
analysis of variance was used for two-group and multiple- formation. As shown in Figure 3A, fluorescence images
group comparisons, respectively. Statistical significance of living and dead cells on days 0, 2, 4, 6, and 8 were
is defined as *P < 0.05, **P < 0.01, and ***P < 0.001. captured using the live/dead double staining method.

3. Results The cell viability rates in the SW and 3DP models were
calculated and are shown in Figure 3B. On day 0, cell
3.1. Spheroid formation in the SW and the 3DP viability in the 3DP model after the printing process was
hepatic tissue model only 70.94% ± 3.44%, while hiPSC-Heps in the SW
model maintained high viability close to 100%. However,
In the 2D model, hiPSC-Heps attached to the bottom of after 2 days of culture, cell viability in the 3DP model
culture plates and grew in a monolayer with no spheroid significantly increased to 97.52% ± 0.80%, and a massive
formed. In the SW and 3DP models, hiPSC-Heps were cell death occurred in the SW model with a sharp decrease
embedded in an alginate-gelatin hydrogel and surrounded in cell viability to 68.02% ± 5.09%. After 8 days of
by 3DP microenvironments. Cells proliferated and culture, cell viability in the 3DP model was significantly
gradually formed spheroids during the culture period higher than that in the SW model. In both models, cell
(Figure 2A). Figure 2B shows the average diameter viability decreased moving from the peripheral to the
of hiPSC-Heps spheroids in the SW and 3DP model. central area of the hydrogel structure. According to the
Interestingly, we observed that the spheroid growth was results in Figure 2C, it could be speculated that spheroid
different between the central and edge regions of the 3DP diameter may be a good proxy for cell viability.
model. Considering that the diffusion limit in avascular Cell densities were calculated using the CCK8 assay
tissue is approximately 200 μm , we defined the to determine cell proliferation in the two models. As shown
[28]
region within 200 μm of the hydrogel edges as the edge in Figure 3C, cell densities in the two models exhibited
region and that more than 200 μm from the edges as the a slight decrease on day 2, followed by a continuous
central region. The average diameter of the hiPSC-Heps increase until day 8. On day 8, the cell density of the 3DP
spheroids in both the central and edge regions of the 3DP model reached at (9.79 ± 1.29) × 10 cells/cm and the SW
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model was significantly wider than that in the SW model model reached a density of (6.67 ± 0.30) × 10 cells/cm ,
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from day 2 to day 4. However, the average diameter of resulting in an approximately five- and three-fold increase
the spheroids in the central region of the 3DP model was from the initial seeding density (2 × 10 cells/cm ),
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180 International Journal of Bioprinting (2022)–Volume 8, Issue 3