A new method for obtaining bankable and expandable adult-like microglial cells

The emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia because current in vitro methods for microglial study have many limitations, including a limited proliferative capacity, low cell yield, immature form, and too many experimental animals use. Here, we developed a new method for obtaining bankable and expandable adult-like microglial cells using the head neuroepithelial layer (NEL) of mouse E13.5. The NEL includes microglia progenitors that proliferate and ramify over time. Functional validation with a magnetic-activated cell sorting system using the NEL showed that the isolated CD11b-positive cells (NEL-MG) exhibited microglial functions, such as phagocytosis (microbeads, amyloid β, synaptosome), migration, and inflammatory changes following lipopolysaccharide (LPS) stimulation. NEL was subcultured and the NEL-MG exhibited a higher expression of microglia signature genes than the neonatal microglia, a widely used in vitro surrogate. Banking or long-term subculture of NEL did not affect NEL-MG characteristics. Transcriptome analysis revealed that NEL-MG exhibited better conservation of microglia signature genes with a closer fidelity to freshly isolated adult microglia than neonatal microglia. This new method effectively contributes to obtaining adult-like microglial cells, even when only a small number of experimental animals are available, leading to a broad application in neuroscience-associated fields.