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.

Engineering Reversible Hydrogels for 3D Cell Culture and Release Using Diselenide Catalyzed Fast Disulfide Formation

Hydrogels crosslinked by dynamic covalent bonds can effectively mimic the viscoelastic properties of native extracellular matrix and have been widely explored for 3D cell culture.Disulfide is one of the most common dynamic bonds in biological systems whose formation and cleavage are catalyzed by a set of dedicated enzymes.However,in vitro formation of disulfide bonds is a slow process and requires harsh catalysts.Therefore,it is difficult to use disulfide crosslinked hydrogels for cell culture.n this work,we show that disulfide bonds can be formed by thiol-diselenide(Dise)exchange under blue light llumination.This reaction is fast,reversible,and biocompatible.Moreover,residual diselenide in the hydrogel network can also accelerate thiol-disulfide exchange reactions leading to faster cell release from the hydrogels upon the addition of thiol-containing agents.We anticipate that disulfide crosslinked hydrogels catalyzed by diselenide can find broad biomedical applications,such as cell culture,celldelivery,and drug-controlled release.