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

Two-photon polymerization (2PP) has provided the field of cell biology with the opportunity to fabricate precisely designed microscaffolds for a wide range of studies, from mechanobiology to in vitro disease modelling. However, a multitude of commercial and in-house developed photosensitive materials employed in 2PP suffers from high auto-fluorescence in multiple regions of the spectrum. In the context of in vitro cell biological studies, this is a major problem since one of the main methods of characterization is fluorescence microscopy of immuno-stained cells. This undesired auto-fluorescence of microscaffolds affects the efficiency of such an analysis as it often overlaps with fluorescent signals of stained cells rendering them indistinguishable from the scaffolds. Here, we propose two effective solutions to suppress this auto-fluorescence and compare them to determine the superiority of one over the other: photo-bleaching with a powerful UV point source and auto-fluorescence quenching via Sudan Black B (SBB). The materials used in this study were all commercially available, namely IP-L, IP-Dip, IP-S, and IP-PDMS. Bleaching was shown to be 61.7–92.5% effective in reducing auto-fluorescence depending on the material. On the other hand, SBB was shown to be 33–95.4% effective. The worst result in presence of SBB (33%) was in combination with IP-PDMS since the adsorption of the material on IP-PDMS was not sufficient to fully quench the auto-fluorescence. However, auto-fluorescence reduction was significantly enhanced when activating the IP-PDMS structures with oxygen plasma for 30 s. Moreover, we performed a cell culture assay using a human neuroblastoma cell line (SH-SY5Y) to prove the effectiveness of both methods in immunofluorescence characterization. SBB presented a lower performance in the study especially in presence of 2PP-fabricated microchannels and microcages, within which the differentiated SH-SY5Y cells migrated and extended their axon-like processes, since the SBB obstructed the fluorescence of the stained cells. Therefore, we concluded that photo-bleaching is the optimal way of auto-fluorescence suppression. In summary, this study provides a systematic comparison to answer one of the most pressing issues in the field of 2PP applied to cell biology and paves the way to a more efficient immunofluorescence characterization of cells cultured within engineered in vitro microenvironments.

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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