Dual-Stiffness Hydrogel-Based Glioblastoma In Vitro Model to Observe Cell Behavior at Interfaces

There is renewed interest in three-dimensional in vitro bioengineered models that replicate key aspects of the in vivo environment for the study of cellular behavior, with one key aspect being cell interactions with matrix interfaces. Here, we developed a dual-stiffness hydrogel-encapsulated glioblastoma (GBM) spheroid model to investigate GBM spreading along a stiffness interface. GBM is an aggressive brain cancer with a patient prognosis of 12-18 months, which is known to spread to distant brain regions by following stiffness interfaces. Our model consisted of a soft, 5% w/v, polyethylene glycol (PEG) hydrogel to mimic the native brain tissue and a stiff, 10% w/v, PEG hydrogel to replicate the stiffer GBM microenvironment. To ensure spheroids fall along the boundary, we adjusted the gelation time of the gel by varying the pH of the gel precursor solution. Encapsulated spheroids were assessed for infiltration and viability for up to 7 days. Spheroids exhibited high viability in all hydrogels. Spheroids showed a higher infiltration index in the soft hydrogel, and migration across the stiffness interface occurred only from the soft to the stiff hydrogel in the dual-stiffness gels. The developed model has a simple, robust design for studying GBM behavior in vitro, a high degree of imageability, requires no specialized equipment to prepare, and is compatible with a multiwell plate format for easy handling and analysis.