Protocol for in vitro evaluation of effects of stiffness on patient-derived glioblastoma

Glioblastoma (GBM) is the most common and lethal type of primary brain tumor. Physiologically, GBM cells experience a heterogeneous mechanical landscape. Here, we present an in vitro method to study the effects of tissue stiffness on patient-derived GBM that utilizes hyaluronic acid (HA)-based, mechanically tunable scaffolds for three-dimensional (3D) culture of patient-derived GBM spheroids. We describe steps to fabricate and characterize HA-based scaffolds, culture GBM spheroids within 3D hydrogel scaffolds, and prepare cultured cells for a variety of experimental assessments. For complete details on the use and execution of this protocol, please refer to Sohrabi et al.¹.