Glioblastoma (GBM) is the most common and aggressive primary brain tumour with poor prognosis and no cure. To improve the efficacy of preclinical research, there is a need for reliable in vitro models that accurately recapitulate the tumour microenvironment. GBM cells and
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Glioblastoma (GBM) is the most common and aggressive primary brain tumour with poor prognosis and no cure. To improve the efficacy of preclinical research, there is a need for reliable in vitro models that accurately recapitulate the tumour microenvironment. GBM cells and their protrusions use the brain tumour vasculature as scaffolds for migration, promoting tumour invasion. In addition, GBM cells form extensive cellular networks, which are associated to increased therapy resistance. In this study, we explored the use of two-photon polymerised scaffolds featuring elements that mimic brain tumour blood vessel geometries to study the intercellular networks of glioblastomas. We employed two distinct micro-scaffold designs to assess the effects of various geometrical design features on cell colonisation: the “Spider Web” scaffold and the “Grid-like structure”. The “Spider Web” design provides a 3D environment that consists of beams mimicking branching blood vessels. We first tested whether the presence of diagonal beams allowed for improved cell colonisation. For this, the vertical cell occupancy of cells on scaffolds with and without diagonal beams was quantified using confocal imaging. Results show that the presence of diagonal beams did not significantly improve cell occupancy of the top tier, although it led to a significant decrease in total cell count. The “Grid-like structure” was designed with pore sizes ranging from 4 μm to 75 μm. The expression of several immunofluorescent markers, as measure of cell occupancy across different pore size regions was compared. The area fraction of Hoechst, actin and tubulin expression was found to be significantly higher in the regions with larger pores compared to those with smaller pores. Despite a difference in cell density, the expression of gap junction protein CX43 was similar across pore sizes. On both scaffold designs, confocal and scanning electron microscopy revealed a variety of cellular protrusion morphologies, as well as punctate CX43 expression. This study shows that these biomimetic micro-scaffolds can be used to evaluate the effect of geometrical features on GBM cell colonisation, and hold potential for the investigation of the 3D GBM intercellular tumour network in vitro.