Biofabrication of 3D glioblastoma–neuron models with a fiber-reinforced anisotropic extracellular matrix

The progression of glioblastoma (GBM) is closely associated with the brain’s highly ordered anisotropic neural tissue. However, in vitro models have generally lacked simulation of the anisotropic neural tissue microenvironment into which GBM infiltrates, which limits the in-depth investigation of GBM–neuron interactions and the development of therapeutic strategies. To address this challenge, a bioinspired artificial anisotropic extracellular matrix (ECM) based on a gel/fiber composite was designed to guide neurons to form anisotropic neural tissue, thereby providing a biomimetic microenvironment for GBM–neuron interplay. By combining melt electrowriting (MEW) with hydrogel molding, a stable, ordered distribution of submicron fiber topographical cues within a three-dimensional hydrogel was achieved through the precise control of fiber deposition and the optimization of supporting structures, thus constructing a controllable anisotropic ECM. Mouse cortical neurons were embedded within this ECM, and fiber structural parameters were optimized using a neuronal morphology index system. The optimized ECM provided synergistic fiber-orientation cues and spacing constraints, which induced highly oriented neuronal alignment (orientation index, S =0.987). Furthermore, a neural network model was reconstructed by embedding mouse cortical tissue blocks in this ECM, in which migration, morphological coupling, and functional connections among neuronal populations were promoted, with an electrical conduction velocity of approximately 459–467 µm/ms. Finally, a GBM–neuron interaction model was established by combining mouse cortical tissue blocks with patient-derived GBM tissue blocks, in which bidirectional chemotactic migration, microtubule-associated protein 2-positive/epidermal growth factor receptor-positive (MAP2+/EGFR+) double-positive events at the fusion region, and pathological GBM–neuron circuits relevant to clinical phenomena were observed. An anisotropic brain ECM microenvironment was constructed and validated in this study, providing oriented guidance for both neural cells and cell populations and thereby offering a potentially useful model for dissecting GBM invasion mechanisms and supporting future therapeutic strategy development.