Reproducible 3D Glioblastoma Migration Assay with Magnetic Nanoparticle Mediated Spheroid Localization Under Hypoxic Conditions.

We describe a reproducible 3D migration assay to model the migratory and invasive potential of patient-derived glioblastoma gliomaspheres from newly diagnosed and recurrent tumors under clinically relevant hypoxic conditions. Uniform sized gliomaspheres are transferred onto a thin layer of extracellular matrix and co-cultured with magnetic iron oxide nanoparticles, which enable their centralized localization within culture wells via placement onto a magnetic plate holder. The inclusion of magnetic iron oxide nanoparticles on seeding facilitates precise, localized imaging of individual patient derived gliomaspheres via a gentle magnetic force and optimizes automated image processing pipelines by reducing positional variability. This assay supports detailed study of glioblastoma migratory behavior in a physiologically relevant microenvironment and allows direct comparison of invasive potential and migratory behavior between newly diagnosed and recurrent patient derived gliomaspheres. The method is compatible with live cell imaging and multiplexed analysis, offering a scalable platform for preclinical investigation of glioblastoma migration, invasive potential, and therapeutic response.