Manipulating hepatocellular carcinoma cell fate in orthogonally cross-linked hydrogels

De-differentiation and loss of function in hepatocytes during two-dimensional (2D) tissue culture significantly hinders the progress of liver research. An ideal three-dimensional (3D) in vitro liver parenchymal cell culture platform should restore cell-cell and cell-matrix interactions, as well as normal hepatocyte polarity. Here, we report an orthogonal thiol-ene hydrogel system for culturing liver cell lines (e.g. Huh7 and HepG2). The hydrogels were prepared by a radical-mediated orthogonal thiol-norbornene photo-click chemistry using poly(ethylene glycol)-tetra-norbornene (PEG4NB) macromer and di-thiol containing linker (e.g., dithiothreitol (DTT) or bis-cysteine matrix metalloproteinase (MMP)-sensitive peptide). This system also allows facile incorporation of bioactive peptides (e.g., fibronectin-derived RGDS) to improve cell-matrix interactions. Encapsulated Huh7 and HepG2 cells showed elevated urea secretion and CYP3A4 enzymatic activities, as well as up-regulated mRNA levels of multiple hepatocyte genes (e.g., CYP3A4, BESP, and NTCP). Importantly, this is the first 3D hydrogel system that up-regulates the expression of NCTP in encapsulated Huh7 and HepG2 cell lines without any genetic modification or the addition of growth factors and chemical additives. Furthermore, the encapsulated cells displayed hepatocyte-like polarity distinctively different from the polarity displayed in 2D culture. These characteristics not only allow the study of hepatology in 3D using inexpensive cell lines, but also permit large-scale small-molecule screening. The up-regulation of NTCP expression and restoration of hepatocyte-like polarity in our hydrogels also shed light on future study of hepatitis B virus infection in vitro.