Strategies to improve neurite outgrowth from primary neurons ingelatin methacrylate hydrogels polymerized with visible lightexposure inside a microscale 3D model

Gelatin methacrylate (GelMA) hydrogels offer many advantageous properties such as excellent biocompatibility, tunable stiffness, and rapid fabrication, making them attractive materials for neural systems-on-a-chip devices used to study axonal outgrowth in three-dimensional (3D) environments. In this study, we evaluate properties of GelMA hydrogels crosslinked with visible blue light and application of these hydrogels for encapsulation of primary dorsal root ganglion (DRG) explants inside a custom microscale device. We selected low GelMA polymer concentrations of 3 % and 6 % w/v for hydrogel preparation to create soft materials and demonstrate the effect of degree of functionalization (DoF) on stiffness of GelMA in a blue light-induced crosslinking system. Additionally, a decrease in crosslinking efficiency and stiffness of GelMA was observed upon reconstitution of the polymer in DMEM cell culture media compared to PBS. To evaluate the axonal outgrowth, DRG explants were encapsulated in various GelMA formulations and cultured for 7 days. Following incubation, the explants were fixed and immunochemically labelled with anti-beta-III-tubulin antibody. We further describe an application of custom-developed semi-automated image analysis tool together with linear mixed effects modeling to evaluate axonal outgrowth across different GelMA formulations. Among the formulations tested in our study, 3 % GelMA 95 % DoF hydrogels supported the longest neurite outgrowth suggesting that matrix properties influenced neurite extension.