Two-Photon Polymerization of 2.5D and 3D Microstructures Fostering a Ramified Resting Phenotype in Primary Microglia

Abstract

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.

Engineered cell culture microenvironments for mechanobiology studies of brain neural cells

“…However, the surface topography can alter the cell’s perceived stiffness of its substrate. For example, cells cultured on high aspect-ratio and bendable pillars behave similarly to those on soft ECMs ( Nikkhah et al, 2012 ; Petzold and Gentleman, 2021 ; Sharaf et al, 2022 ). The interaction between cells but also between cells and their environment in vitro can alter the cell phenotype and ECM structure ( Holle et al, 2018 ).…”

“…(A) Patterned 2.5 substrates. (I) Continuous topographies ( Fozdar et al, 2010 ), (II) Discontinuous topographies ( Limongi et al, 2013 ; Sharaf et al, 2022 ) and (III) Random topographies ( Cesca et al, 2014 ). (B) 3D architectures.…”

“…(B) 3D architectures. (I) 3D random porous architectures (a) Li et al, 2013 ; (b) Sandhurst et al, 2022 (II) 3D ordered architectures (a) Harberts et al, 2020 ; (b) Sharaf et al, 2022 ; (c) Agrawal et al, 2021 ; (d) Turunen et al, 2017 ; (e) Left (phase contrast image): Huang et al, 2021 and middle and right: Koroleva et al, 2021 . (C) .…”

“…Lastly, the microglia phenotype seems also to be modulated by discontinuous topographies. Sharaf et al demonstrated that primary primate microglia displayed an increased ramified resting phenotype when cultured on micropillars arrays ( Figure 5AII , bottom) when compared to stiff, flat surfaces ( Sharaf et al, 2022 ).…”

“…Additive manufacturing is one of the methods that can be employed to manufacture engineered cell environments. Examples of additive manufacturing techniques are two-photon polymerization ( Accardo et al, 2018a ; Lemma et al, 2019 ; Weisgrab et al, 2020 ; Akolawala et al, 2022 ; Barin et al, 2022 ; Sharaf et al, 2022 ), bioprinting ( Rider et al, 2018 ), stereolithography apparatus ( SLA, Qiu et al, 2020 ), and digital light processing ( DLP, Qiu et al, 2020 ). Other techniques used to recreate microenvironments are (optical) lithography techniques ( Pardo-Figuerez et al, 2018 ), chemical synthesis ( Marcus et al, 2017 ), electrospinning ( Honkamäki et al, 2021 ), salt leaching ( Worthington et al, 2017 ), and foaming ( D’Abaco et al, 2018 ).…”