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INTRODUCTION: The integration of biomaterials with living cells presents a critical bottleneck in the field of tissue engineering and regenerative medicine. Despite the rapid progress in biofabrication technologies, designing functional and biologically relevant tissue constructs that mimic native organ complexity remains challenging. Here, we assembled semi-synthetic liver-like tissues using alginate microgels and hepatocytes. We employed 3D bioprinting to construct centimeter-scale semi-synthetic tissues made of either HepG2 cells or HepaRG cells along with functionalized microgels. These microgels were loaded with a small molecule that mimicked a cytochrome P450 (CYP450) enzyme activity (Figure 1a).1 Notably, HepaRG cells, which exhibit more pronounced hepatic characteristics compared to HepG2 cells, were used to develop a more representative liver model (Figure 1b).2 Upon differentiation, HepaRG cells gave rise to both mature hepatocytes and biliary cells, offering a minimal yet biologically relevant semi-synthetic liver tissue analog.
METHODS: Alginate microgels were fabricated using an Encapsulator, optionally incorporating artificial enzymes. Cell aggregates of HepG2 cells or HepaRG cells were cultured for 48 hours before being suspended in a bio-ink mixture containing alginate, gelatin methacryloyl (GelMA), and microgels. This composite ink was then 3D-bioprinted and maintained in culture for up to 35 days. Cellular proliferation was assessed via dsDNA quantification and LIVE/DEAD staining. Expression of relevant hepatic markers was evaluated using RT-qPCR. To model liver steatosis, differentiated HepaRG cell-based tissues were treated with free fatty acids for two weeks and analyzed for lipid accumulation through fat staining techniques.
RESULTS: Cell aggregates were successfully formed and integrated into 3D-bioprinted constructs using a composite ink made of alginate/GelMA and microgels. HepG2 cells and HepaRG cells maintained viability and proliferated over a 35-day culture period. Differentiation of HepaRG cells initiated at day 14 resulted in upregulated markers of mature hepatocytes and biliary cells by day 28, confirmed by immunostaining. The steatotic tissue model, created via fatty acid incubation, exhibited pronounced lipid accumulation and elevated reactive oxygen species (ROS), as demonstrated through confocal imaging and gene expression analysis. Functional assays using tert-butyl hydroperoxide (tBuOOH) and resorufin ethyl ether revealed enhanced CYP1A2 activity in the presence of catalytic microgels.
DISCUSSION & CONCLUSIONS: Incorporating metalloporphyrin-functionalized microgels into 3D bioprinted semi-synthetic tissues successfully simulated CYP450 enzymatic activity, enhancing the metabolic capacity of co-cultured HepG2 cells over extended periods. Furthermore, employing HepaRG cells enabled the generation of differentiated liver-like tissues. The addition of free fatty acids produced a high-fidelity steatotic model, replicating key pathological features such as lipid accumulation and oxidative stress.
Altogether, these findings demonstrate that engineered semi-synthetic tissues combining functional microgels and liver cells support prolonged viability and hepatic function. This platform represents a significant step forward in developing modular, biologically integrated liver models for research and therapeutic applications.
REFERENCES:
1I. N. Westensee, L. J. Paffen, S. Pendlmayr, P. Dios Andres et al., 2024, 10.1002/adhm.202303699
2P. de Dios Andres et al. (In preparation)
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