Long-term functional maintenance of primary hepatocytes in vitro using macroporous hydrogels engineered through liquid-liquid phase separation

Preserving the functionality of hepatocytes in vitro poses a significant challenge in liver tissue engineering and bioartificial liver, as these cells rapidly lose their metabolic and functional characteristics after isolation. Inspired by the macroporous structures found in native liver tissues, here we develop synthetic hydrogel scaffolds that closely mimic the liver's structural organization through the phase separation between polyethylene glycol (PEG) and polysaccharides. Our hydrogels exhibit interconnected macroporous structures and appropriate mechanical properties, providing an optimal microenvironment conducive to hepatocyte adhesion and the formation of sizable aggregates. Compared to two-dimensional hepatocyte cultures, enhanced functionalities of hepatocytes cultured in our macroporous hydrogels were observed for 14 days, as evidenced by quantitative reverse-transcription–polymerase chain reactions (qRT-PCR), immunofluorescence, and enzyme linked immunosorbent assay (ELISA) analyses. Protein sequencing data further confirmed the establishment of cell-cell interactions among hepatocytes when cultured in our hydrogels. Notably, these hepatocytes maintained a protein expression lineage that closely resembled freshly isolated hepatocytes, particularly in the Notch and tumor necrosis factor (TNF) signaling pathways. These results suggest that the macroporous hydrogels are attractive scaffolds for liver tissue engineering.