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International Journal of Bioprinting 3D-printed NAFLD model

levels of these two proteins. The immunofluorescence 3.3. Establishment of a 3D-printed NAFLD model
results (Figure 2G and 2H) demonstrated that both ALB An NAFLD model was established based on the
and CYP3A4 were normally expressed in 3D-printed liver 3D-printed liver tissues. Notably, research reports on
tissues, and a uniform distribution was observed. The qRT- 3D-printed NAFLD in vitro models are relatively limited.
PCR quantitative analysis revealed that the gene expression To investigate the time required to induce NAFLD,
levels of ALB and CYP3A4 in 3D-printed liver tissues were preliminary experiments were conducted to determine
significantly greater than those in traditional 2D cultured the optimal induction period. The results (Figure S2,
hepatocytes (Figure 2I and 2J). Overall, 3D-printed liver Supporting Information) indicated that the lipid droplet
tissues were found to be superior to conventional 2D and content in 3D-printed liver tissues rapidly increased
3D hepatocyte models in both structure and function. during the first 3 days of culture in the induction medium
The 3D-printed liver tissues not only closely resembled containing 600 or 900 μM FFAs and then gradually
stabilized. Therefore, further studies were conducted 3 days
the morphology of in vivo liver tissues but also exhibited post-induction. The accumulation of intracellular lipid
significantly enhanced expression of functional proteins. droplets in the 3D-printed liver tissue model was studied
These advantages are attributed to the 3D microenvironment after 3 days of induction with NAFLD medium to evaluate
provided by 3D printing technology, which better the effect of NAFLD induction medium on lipid droplet
simulates the tissue structure and function of the in vivo accumulation. The experimental procedure is illustrated in
liver, particularly its vascular-like functionality, thereby Figure 3A. The cells were cultured in an ordinary medium
promoting cell-cell interactions and nutrient transfer for the first 4 days and then transferred to the NAFLD
and consequently enhancing hepatic function. Therefore, induction medium on the fourth day. The experimental
the significant structural and functional advantages of results (Figure 3B and C) demonstrated that the control
3D-printed liver tissues highlight their broad application group exhibited only minimal lipid droplets, whereas
potential in liver disease modeling and drug testing. significant lipid droplet accumulation was observed in

Figure 3. Construction of the non-alcoholic fatty liver disease (NAFLD) model. (A) Experimental procedure for constructing the NAFLD model. (B)
Analysis of lipid droplet accumulation in NAFLD models; ***p < 0.001. (C) Images of lipid droplet accumulation in the NAFLD model. (D) Impact of
different free fatty acid (FFA) concentrations on hepatocyte viability. Scale bar: 200 µm.

Volume 10 Issue 6 (2024) 367 doi: 10.36922/ijb.4312