Galactosylated reversible hydrogels as scaffold for HepG2 spheroid generation

Various galactosylated scaffolds have been developed for hepatocyte culture because galactose ligands help maintain cell viability, facilitate the formation of multicellular spheroids and help maintain a high level of liver-specific functions. However, it is difficult to harvest the cell spheroids generated inside the three-dimensional scaffolds for their further biological analysis and applications. Here we developed a new galactosylated hydrogel scaffold which solidifies in situ upon heating to physiological temperature, but liquefies again upon cooling back to room temperature. The new scaffold is composed of poly(N-isopropylacrylamide) (PNIPAM) microgel and poly(ethylene glycol) (PEG). Because of the thermosensitivity of PNIPAM microgel, the mixed dispersions gel upon heating and liquefy upon cooling. PEG was added to reduce the shrinkage of the gels. Part of the PNIPAM microgel was replaced with a galactosylated one to provide a series of blend gels with various galactose ligand contents. HepG2 cells, a human hepatocarcinoma cell line, were encapsulated in the in situ-formed gels. As expected, the cell viability increases with increasing content of galactose ligands. In addition, compact multicellular spheroids were obtained in gels containing galactose ligands, while loose spheroids formed in gel without galactose ligands. The cells cultured in galactose-containing gels also exhibit a higher level of liver-specific functions, in terms of both albumin secretion and urea synthesis, than those cultured in gel without these ligands. The new galactosylated scaffold not only promotes the formation of hepatocyte spheroids, but also allows for their harvest. By cooling back to room temperature to liquefy the gel, the hepatocyte spheroids can be facilely harvested from the scaffold. The reversible galactosylated scaffold developed here may be used for large scale fabrication of hepatocyte spheroids.