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

Microglia, the resident immune cells of the central nervous system, play essential roles in maintaining brain homeostasis. Primary cell culture systems are indispensable for investigating microglial biology, allowing direct evaluation of gene function in complex transgenic models prior to committing to resource-intensive in vivo studies. However, traditional mixed glial culture-derived primary microglia (pMG) systems are limited by strict temporal constraints concerning the time of harvest, and poor scalability. To overcome these barriers, here we have developed, and validated, the first protocol that enables long-term cryopreservation of mixed glial cultures and isolation of viable, responsive microglia. Microglia isolated from previously frozen mixed glial cultures showed some baseline differences compared to those obtained from fresh cultures, including modest alterations in morphology. Despite these phenotypic shifts, frozen-culture-derived microglia responded similarly to environmental stimuli compared to microglia derived from fresh cultures. When exposed to metabolic (high glucose), inflammatory (LPS, amyloid beta) stimuli, microglia from frozen cultures exhibited morphological, phagocytic, and inflammatory changes comparable to those observed in fresh-derived pMG. These findings demonstrate that freezing mixed glial cultures preserves microglial capacity to respond appropriately to environmental cues. Together, this work establishes the first validated approach for generating microglia from frozen murine mixed glial cultures, providing a scalable and temporally flexible alternative to conventional methods.