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Updated: Oct 9, 2025
Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Published on: February 16, 2018
A new method for obtaining bankable and expandable adult-like microglia in mice
Min-Jung You1, Chan Rim1, Youn-Jung Kang2
1Department of Pharmacology, Research Institute for Basic Medical Science, School of Medicine, CHA University, CHA BIO COMPLEX, 335 Pangyo, Bundang-gu, Seongnam-si, Gyeonggi-do, 13488, Republic of Korea.
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.
Insights
Researchers developed a novel method to obtain abundant, expandable adult-like microglia from mouse neuroepithelial layers. This technique provides a reliable source of microglia for neurological disorder research.
Area of Science:
- Neuroscience
- Cell Biology
- Developmental Biology
Background:
- Microglia play a critical role in neurological disorders.
- Current methods for obtaining adult microglia are insufficient for large-scale research.
- A novel method is needed to generate bankable and expandable adult-like microglia.
Purpose of the Study:
- To develop a new method for obtaining bankable and expandable adult-like microglia from mice.
- To establish a reliable in vitro model for studying microglia in neurological conditions.
Main Methods:
- Dissection and culture of the head neuroepithelial layer (NEL) from E13.5 mouse embryos.
- Isolation of microglia (NEL-MG) using magnetic-activated cell sorting.
- Functional assays, gene expression analysis, and long-term culture of NEL-MG.
Main Results:
- NEL contains proliferating microglial progenitors supported by neuroepithelial cells.
- NEL-MG demonstrated key microglial functions, including phagocytosis and inflammatory response.
- NEL-MG showed higher expression of microglia signature genes and better fidelity to adult microglia compared to neonatal microglia.
- NEL-MG maintained their characteristics after banking and long-term culture, and were re-expandable.
Conclusions:
- The developed method effectively yields sufficient adult-like microglia, even from limited animal samples.
- This technique offers a valuable tool for broad applications in neuroscience research.
- The method facilitates the study of microglia in various neurological disorders.
