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He, et al.
tissue model. Alginate, a natural polysaccharide with good 3DP models showed significantly higher expression of
biocompatibility, can be rapidly crosslinked by divalent albumin than did those in the 2D model according to
ions, such as calcium ions (Ca ). Despite their excellent the immunostaining results. Compared to monolayer
2+
formability, the lack of cell-binding domains leads to cultures, the 3D spheroids in the hydrogel structures
poor cell adhesion in pure alginate hydrogels . Thus, provide tight cell junctions for attachment-dependent
[40]
peptide-containing gelatin is often added to enhance the hepatocytes, which promotes hepatocyte polarization
cellular affinity of the bioink. By optimizing the bioink and function maintenance . In addition, the SW
[45]
concentrations and printing parameters in our preliminary and 3DP models showed a higher mRNA expression
experiment, we successfully created a 12 × 12 × 1.2 mm of liver function-related biomarkers than did the 2D
grid hydrogel structure. Considering the high oxygen model. Notably, high SDs in the mRNA expression
consumption of hepatocytes , the distance between were observed in the SW model. On the one hand, the
[41]
adjacent hydrogel fibers was set to 2 mm to ensure results were analyzed using only 3 data points, which
sufficient oxygen supply to the medium. could lead to large random errors. On the other hand,
In our study, we compared the cell growth, liver- it could be also due to the rupture of the hydrogel
specific function, and drug-induced hepatotoxicity of structure in some samples, leading to unwanted changes
constructed 2D, SW, and 3DP models. In contrast to in culture conditions. However, the secretion levels of
the 2D monolayer culture, cells are 3D-cultured in both AAT, albumin, and BUN were significantly higher in the
the SW and 3DP models. Since the same material was 3DP model than those in either of the other two models,
used in the SW and 3DP models, topological structure which clearly demonstrate the stronger liver functions of
is considered the major difference in microenvironment the 3DP model.
between the SW and 3DP models. In hydrogel structures, Finally, we conducted drug screening using
oxygen and nutrient supply to cells relies on diffusion APAP to evaluate drug-induced hepatotoxicity in the
through the culture medium. Cells in the peripheral constructed hepatic tissue models. APAP overdose
region consume oxygen and nutrients and consequently is the leading cause of acute liver failure in many
hinder their penetration . In the SW models, the cells countries. APAP induces liver impairment by causing
[42]
in the inner region of a consolidated hydrogel layer mitochondrial damage and subsequent hepatocyte
experienced impaired medium supply. Compared to necrosis [46] . PHHs, HepaRG, and HepG2 cell lines are
the consolidated hydrogel layer in the SW model, the commonly used in APAP-induced hepatotoxic assays [47] .
interconnected microchannels of the 3DP grid structure As an alternative hepatic cell source, the response
allow greater inflow of culture medium, and thus may have behavior of hiPSC-Heps to APAP in the hepatic tissue
facilitated mass transfer, supporting greater cell growth. models requires investigation. After 24 h of drug
In relevant studies of 3D bioprinting of cervical [43] and treatment, stronger drug resistance was observed in
cholangiocarcinoma tumor models, comparing 3DP the 3DP model than in the SW model, possibly due
[44]
models to SW models, it was found that cell spheroids to the larger and more viable spheroids formed in the
in 3DP models were larger than those in SW models, 3DP model. However, cell viability in the 3DP model
indicating better cell growth in 3DP models. Similarly, significantly decreased after 48 h of drug treatment.
in our study, hiPSC-Heps maintained active proliferation A pronounced upregulation of the drug metabolism-
and formed larger spheroids in the 3DP model than those related enzyme CYP1A2 increased drug sensitivity,
in the SW model. Nevertheless, diffusional gradients of which is consistent with the findings of recent studies
oxygen and nutrients considerably limited the volume of using other types of hepatocytes [8,48] . In addition, the
spheroids formed in the inner region of the hydrogel fibers. hiPSC-Heps spheroids in our 3DP model displayed
We observed that a certain number of cell deaths occurred drug resistance comparable to that of hiPSC-derived
in the 3DP model immediately after the printing process, hepatocyte spheroids generated by nanopillar plates [49]
which could be attributed to shear stress during bioink and 3D cellulosic scaffolds [50] , suggesting good efficacy
extrusion. However, hiPSC-Heps rapidly recovered their of the 3DP model in the assessment of APAP-induced
high viability and started steady proliferation in the 3DP hepatotoxicity. Overall, the 3DP model performed well
model. Conversely, in the SW model, the poor oxygen in APAP drug test and proved its application value in
and nutrient supply in the inner region of the hydrogel in vitro hepatotoxicity evaluation.
layer could not support such a high density of cell culture, Overall, 3D bioprinting of hiPSC-Heps proved
leading to substantial cell death. Therefore, 3D bioprinting to be an effective way to construct a functional hepatic
plays a key role in efficient 3D culture of hiPSC-Heps by tissue model. High cell viability, rapid cell proliferation,
ameliorating oxygen and nutrient supply conditions. and enhanced liver-specific functions were observed
Regarding biofunction in the 2D, SW, and 3DP in this 3DP model compared to other common models
models, spheroid-formed hiPSC-Heps in the SW and including SW and 2D models. This hepatic tissue model

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