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