Microglia, the resident macrophages of the central nervous system, are key players in neuroinflammation and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. These cells represent promising cellular targets for therapeutic intervention, therefore a detailed cellular biological knowledge of microglia is of paramount importance. Current microglia in vitro cell culture models are limited in their ability to recapitulate in vivo microglia in terms of gene expression and cell morphology. There is growing evidence that mimicking the cellular environment of microglia in the central nervous system will contribute to a more physiologically accurate culture model. Three-dimensional geometries, and physical and biochemical cues similar to the brain’s extracellular matrix, are necessary to create a 3D biomimetic in vitro environment, as microglia cells can sense these environmental cues through mechanosensing. In this thesis, the effect of two-photon polymerized 2.5D and 3D microstructures on microglia morphology and phenotype was studied through quantitative and qualitative cell analysis. Also, an attempt was made to create in vitro ramified microglia that resemble in vivo microglia from a morphological point of view, by con-trolling the effective shear modulus through dimensional optimization of compliant micro-and nanopillars. It was found that culturing microglia especially on nanopillars enabled an increase of the ramified pheno-type and a more complex (i.e. more ramifications) cell morphology as compared to microglia cultured onflat fused silica substrates. Furthermore, we report that microglia cultured on 3D structures exhibit similarphenotypes observed in the 2D and 2.5D cultures, and were more resemblingin vivomicroglia in terms of3D cell morphology.