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| Home > Publications Database > 3D culture systems as a tool for recapitulating in vivo-like phenotypes in murine and human microglia |
| Journal Article | DZNE-2026-00708 |
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2026
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Please use a persistent id in citations: doi:10.70401/mc.2026.0006
Abstract: Aims: Conventional two-dimensional (2D) culture systems fail to recapitulate the structural and functional complexity of the brain parenchyma, driving microglia toward an activated, non-homeostatic state. Here, we therefore developed a simple, robust, and scalable three-dimensional (3D) culture system using a Matrigel-collagen composite matrix to promote a more in vivo-like microglial phenotype. Methods: Primary mouse microglia and human induced pluripotent stem cell-derived microglia (iMGLs) from multiple donor lines were cultured either in conventional 2D conditions or embedded in a Matrigel-collagen 3D matrix. Phenotypes were characterized using confocal microscopy and morphometric analysis, live-cell imaging, transcriptomic profiling, electrophysiology, cytokine secretion assays upon inflammatory stimulation, and flow cytometry. Results: In 3D culture, mouse microglia developed a markedly more ramified and structurally complex morphology, supported by filamentous actin-rich terminal processes. Live-cell imaging demonstrated enhanced dynamics of cell processes and a more sessile cell body, consistent with physiological microglia surveillance behavior. Transcriptomic profiling further revealed that microglia cultured in 3D downregulated genes associated with activation and proliferation while upregulating homeostatic markers, yet retained a robust cytokine response to inflammatory stimulation. Electrophysiological profiling similarly indicated a shift toward a more resting, in vivo-like state. Human iMGLs showed comparable morphological adaptations in 3D, with protein expression and functional readouts reflecting both shared homeostatic features and cell line- and context-dependent characteristics. Conclusion: Together, our results demonstrate that a simple and accessible 3D culture system shapes microglial morphology, behavior, and molecular identity, establishing a versatile platform for investigating microglial physiology in a controlled yet physiologically more relevant environment.
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