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bioRxiv · 10.64898/2025.12.13.693524

A validated method for banking mixed glial cultures that yield responsive microglia

Sturno, A. M.·Hassell, J. E.·Baker, K. M.·Potter, G. J.·Bruce, K. D.
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Abstract

Microglia, the primary immune cells of the brain, orchestrate immune responses to both external and internal stimuli in health and disease. Although several cell culture methods exist to model microglia in vitro, isolating loosely adherent microglia from primary murine mixed glial cultures remains valuable for recapitulating in vivo cell states from complex transgenic lines at relatively low cost. However, these methods are constrained by limited temporal control and scalability. To address these challenges, we established a protocol for generating microglia from frozen mixed glial cultures. While freezing introduced measurable differences in microglial morphology and lipid droplet content, microglia derived from frozen cultures responded to environmental stimuli (high glucose and LPS) in the same way as those from fresh cultures. Thus, while frozen primary microglia exhibit similar functional responses, maintaining consistency in using fresh or frozen cells within a given experiment is strongly recommended. MotivationAlthough our understanding of microglias role in brain development and disease continues to grow, a standardized in vitro model of primary mouse microglia remains absent in the field. Primary cell culture systems are essential for studying microglia, enabling the empirical assessment of gene function in transgenic models and testing novel interventions before committing to time- and cost-intensive in vivo studies. However, mixed glial culture-derived primary microglia (pMG) systems are constrained by the timing of animal availability and have limited scalability. In this study, we sought to establish a protocol for freezing and subsequently reviving primary mixed glial cultures from which responsive pMG could be isolated for downstream analysis. We validated this approach by quantifying microglia responses to distinct environmental stimuli before and after a freeze-thaw cycle. Research topicsCP: Cell biology Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/693524v2_ufig1.gif" ALT="Figure 1000"> View larger version (54K): org.highwire.dtl.DTLVardef@1b0e291org.highwire.dtl.DTLVardef@1eacfb2org.highwire.dtl.DTLVardef@e9c18aorg.highwire.dtl.DTLVardef@a151a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Sturno, A. M., Hassell, J. E., Baker, K. M., Potter, G. J., Bruce, K. D.. 2025-12-16. A validated method for banking mixed glial cultures that yield responsive microglia. https://doi.org/10.64898/2025.12.13.693524

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