Spatial mapping, isolation, and culture of primary human microglia and astrocytes: A systematic review and framework...

Microglia and astrocytes are essential glial cells in the CNS that play key roles in brain development, homeostasis, and disease. While immortalized cell lines are convenient, they lack the physiological relevance of primary cells, highlighting the need for reliable human-based isolation protocols. This systematic review synthesizes 21 studies describing methods for isolating and culturing primary human microglia and astrocytes from adult surgical resections, postmortem tissues, and fetal brains. Using the Human Brain Cell Atlas v1.0 single-nuclei RNA-Seq dataset and voxel-wise mapping on the MNI152 template, we provide a spatial transcriptomic context for region-specific glial isolation. We critically assess tissue dissociation techniques, enrichment strategies, and culture conditions, including substrate coatings and growth factors. Additionally, we summarize phenotypic and functional assays used for characterization including surface marker expression, phagocytosis, and cytokine profiling. Despite variability across protocols, we propose an optimized, spatially informed workflow to reproducibly isolate viable, pure glial populations from human brain tissues. This approach supports New Approach Methodologies and the 3Rs-Replacement, Reduction, and Refinement by promoting ethical, human-relevant in vitro models. Our framework advances glial cell-based systems, improving the translational relevance of neurodegenerative and neuroinflammatory disease research and aligns with multiple United Nations Sustainable Development Goals through responsible scientific practices.