Modular Cross-Linking of Gelatin-Based Thiol-Norbornene Hydrogels for in Vitro 3D Culture of Hepatocellular Carcinoma Cells

Gelatin-based hydrogels are increasingly used to promote cell fate processes in 3D. Here, we report the use of orthogonal thiol-norbornene photochemistry to prepare modularly cross-linked gelatin-based hydrogels for studying the influence of independent matrix properties on hepatocellular carcinoma cell fate in vitro. In addition to demonstrating the ability to independently tune the mechanical and biological properties of modular gelatin-norbornene (GElNB) hydrogels, we also determined that network cross-linking density plays a key role in the mechanisms of proteolytic gel degradation. During in vitro degradation studies, GElNB hydrogels with lower cross-linking density degraded faster and followed a surface erosion mechanism, whereas dense GElNB hydrogels degraded in a bulk degradation mechanism. Hepatocellular carcinoma cells, Huh7, were encapsulated and grown in GElNB hydrogels with modularly tuned stiffness, bioactive motifs, and heparin content. We systematically evaluated the effect of matrix properties on cell viability and functions in vitro, including CYP3A4 activity and urea secretion. We found that encapsulated Huh7 cells exhibited higher cellular metabolic activity when encapsulated in modular GElNB hydrogels composed of higher gelatin contents or gels with lower stiffness. Interestingly, altering gelatin content and matrix stiffness did not significantly affect hepatocyte-specific cellular functions. To improve cellular function, we prepared norbornene and heparin dual-functionalized gelatin through a two-step synthesis protocol. Heparin-functionalized GElNB (i.e., GElNB-Hep) hydrogels were able to sequester and slowly release hepatocyte growth factor (HGF) in vitro. Finally, the conjugation of heparin on GElNB led to suppressed Huh7 cell metabolic activity and improved CYP3A4 activity and urea secretion.