Background:The emerging role of microglia in neurological disorders requires a novel method for obtaining massive amounts of adult microglia. We aim to develop a new method for obtaining bankable and expandable adult-like microglia in mice. Methods:The head neuroepithelial layer (NEL) that composed of microglial progenitor and neuroepithelial cells at mouse E13.5 was dissected and then cultured or banked. Microglia (MG) isolated from the cultured NEL by magnetic-activated cell sorting system were obtained and named NEL-MG. Results:The NEL included microglia progenitors that proliferate and ramify over time with neuroepithelial cells as feeder. In functional analysis, NEL-MG exhibited microglial functions, such as phagocytosis (microbeads, amyloid β, synaptosome), migration, and inflammatory response following lipopolysaccharide (LPS) stimulation. NEL was passage cultured 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 passage culture 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. NEL-MG could be re-expandable when they were plated again on neuroepithelial cells. Conclusions:This new method effectively contributes to obtaining sufficient matured form of microglia (adult-like microglia), even when only a small number of experimental animals are available, leading to a broad application in the field of neuroscience.Keywords:Banking; Microglia; Neuroepithelial layer; Subculture.