The most widely employed approach by cell biologists to performing in vitro cell culture assays is the one using 2D plastic culture ware systems, which allows reproducibility and ease of use. Moreover, this method is cost-effective. However, in most cases, these flat surfaces lead to the formation of unrealistic 2D cell monolayers, which do not reproduce the complex configuration characteristics of native tissues in terms of dimensionality, rigidity, and topography. For this reason, a new generation of interdisciplinary scientists, working across microengineering and cell biology has started to develop engineered cell microenvironments (Huang et al., 2017) by employing advanced materials and fabrication approaches (Fan et al., 2019) over the last two decades. Depending on the level of resolution of the adopted manufacturing technique, the geometrical features of these structures can reach micrometric or even sub-micrometric dimensions comparable to the ones of cellular somas or cellular filopodia, therefore fostering cell-biomaterial interactions. The developed structures are pivotal for a better investigation of fundamental mechanobiology (Lemma et al., 2019), the optimization of in vitro disease modeling, drug/treatment screening (Gao et al., 2021), and tissue engineering (Mani et al., 2022).@en

Microglia are the resident macrophages of the central nervous system and contribute to maintaining brain’s homeostasis. Current 2D “petri-dish” in vitro cell culturing platforms employed for microglia, are unrepresentative of the softness or topography of native brain tissue. This often contributes to changes in microglial morphology, exhibiting an amoeboid phenotype that considerably differs from the homeostatic ramified phenotype in healthy brain tissue. To overcome this problem, multi-scale engineered polymeric microenvironments are developed and tested for the first time with primary microglia derived from adult rhesus macaques. In particular, biomimetic 2.5D micro- and nano-pillar arrays (diameters = 0.29–1.06 µm), featuring low effective shear moduli (0.25–14.63 MPa), and 3D micro-cages (volume = 24 × 24 × 24 to 49 × 49 × 49 μm3) with and without micro- and nano-pillar decorations (pillar diameters = 0.24–1 µm) were fabricated using two-photon polymerization (2PP). Compared to microglia cultured on flat substrates, cells growing on the pillar arrays exhibit an increased expression of the ramified phenotype and a higher number of primary branches per ramified cell. The interaction between the cells and the micro-pillar-decorated cages enables a more homogenous 3D cell colonization compared to the undecorated ones. The results pave the way for the development of improved primary microglia in vitro models to study these cells in both healthy and diseased conditions.@en