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Two-Photon Polymerization of 2.5D and 3D Microstructures Fostering a Ramified Resting Phenotype in Primary Microglia
Ahmed Sharaf1, Brian Roos1, Raissa Timmerman2
1Department of Precision and Microsystems Engineering, Delft University of Technology, Delft, Netherlands.
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.
Insights
New biomimetic microenvironments improve in vitro microglia models. Engineered micro- and nano-pillar arrays and 3D micro-cages promote a homeostatic ramified phenotype in primary microglia, enhancing their study in brain health and disease.
Area of Science:
- Neuroscience
- Biomaterials Engineering
- Cell Biology
Background:
- Microglia, the central nervous system's resident macrophages, are crucial for brain homeostasis.
- Traditional 2D cell culture platforms inadequately mimic the brain's native microenvironment, altering microglial morphology and phenotype.
- Existing methods often result in an amoeboid microglial phenotype, unlike the ramified phenotype observed in healthy brain tissue.
Purpose of the Study:
- To develop and evaluate novel multi-scale engineered polymeric microenvironments for primary microglia culture.
- To assess the impact of biomimetic micro- and nano-pillar arrays and 3D micro-cages on microglial morphology and phenotype.
- To create more representative in vitro models for studying microglia in both healthy and diseased states.
Main Methods:
- Fabrication of 2.5D micro- and nano-pillar arrays (0.29-1.06 µm diameters) with low effective shear moduli (0.25-14.63 MPa).
- Creation of 3D micro-cages (24³ to 49³ µm³) with and without micro-/nano-pillar decorations (0.24-1 µm diameters) using two-photon polymerization (2PP).
- Culturing primary microglia from adult rhesus macaques on these engineered substrates and flat controls.
Main Results:
- Microglia cultured on pillar arrays exhibited increased expression of the ramified phenotype compared to those on flat substrates.
- Cells on pillar arrays showed a higher number of primary branches per ramified cell, indicating a more homeostatic morphology.
- Micro-pillar-decorated 3D cages facilitated more homogenous 3D cell colonization than undecorated cages.
Conclusions:
- Engineered polymeric microenvironments, particularly pillar arrays and decorated 3D cages, effectively promote a homeostatic microglial phenotype in vitro.
- These advanced microenvironments overcome limitations of traditional 2D cultures, offering a more accurate representation of native brain tissue.
- The developed platforms provide a foundation for improved in vitro microglia models essential for advancing research in neuroinflammation and neurological diseases.
