cell genotypes compared to standard cell lines). [4,5] Current in vitro models for example, rapidly lose high copy epidermal growth factor receptor (EGFR) amplification and have limited ability to monitor cells and (microtube-based) networks. [6,7] High copy EGFR amplification and microtube-based networks are two important features that are known to promote glioma progression. [8][9][10][11] To further study the role of microtubes in glioma progression and also the potential of EGFR-targeting treatments in glioma, in-vitro cell culture models that can retain these features are needed. [10] Currently, in vitro cell culture research predominantly involves the use of 2D planar surfaces. Although these 2D surfaces are cheap, easy-to-use, and reproducible, they often do not mimic the 3D spatial configuration of cells in real tissues. Indeed, cells behave differently in 3D environments [12,13] and can have differences in terms of cellular morphology, formation of cell-cell junctions, cell proliferation, gene and protein expression levels, and even in responses to treatments. [14][15][16][17] 3D tumor spheroids, which are generally employed for glioma research, can overcome these limitations by better mimicking tissue-like features. However, they are difficult to monitor especially when analyzing subcellular structures like microtubes. [6,14,16] A major obstacle in glioma research is the lack of in vitro models that can retain cellular features of glioma cells in vivo. To overcome this limitation, a 3D-engineered scaffold, fabricated by two-photon polymerization, is developed as a cell culture model system to study patient-derived glioma cells. Scanning electron microscopy, (live cell) confocal microscopy, and immunohistochemistry are employed to assess the 3D model with respect to scaffold colonization, cellular morphology, and epidermal growth factor receptor localization. Both glioma patient-derived cells and established cell lines successfully colonize the scaffolds. Compared to conventional 2D cell cultures, the 3D-engineered scaffolds more closely resemble in vivo glioma cellular features and allow better monitoring of individual cells, cellular protrusions, and intracellular trafficking. Furthermore, less random cell motility and increased stability of cellular networks is observed for cells cultured on the scaffolds. The 3D-engineered glioma scaffolds therefore represent a promising tool for studying brain cancer mechanobiology as well as for drug screening studies.