High yield primary microglial cultures using granulocyte macrophage-colony stimulating factor from embryonic murine cerebral cortical tissue

• Published in: Journal of neuroimmunology 2017-06, Vol.307, p.53-62
• Main Authors: Yu, Adam C, Neil, Sarah E, Quandt, Jacqueline
• Format: Article
• Language: English
• Subjects: Allergy and Immunology; Animals; Calcium-Binding Proteins - metabolism; Cell Differentiation - drug effects; Cell Proliferation - drug effects; Cells, Cultured; Cerebral Cortex - cytology; Cytokines - metabolism; Cytokines - pharmacology; Embryo, Mammalian; Flow Cytometry; Gene Expression Regulation - drug effects; GM-CSF; Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology; Lipopolysaccharides - pharmacology; Mice; Mice, Inbred C57BL; Microfilament Proteins - metabolism; Microglia; Microglia - drug effects; Microglia - physiology; Neurology; Primary culture; Time Factors
• ISSN: 0165-5728; 1872-8421
• DOI: 10.1016/j.jneuroim.2017.03.018

Abstract Background Microglia play vital roles in neurotrophic support and modulating immune or inflammatory responses to pathogens or damage/stressors during disease. This study describes the ability to establish large numbers of microglia from embryonic tissues with the addition of granulocyte-macrophage stimulating factor (GM-CSF) and characterizes their similarities to adult microglia examined ex vivo as well as their responses to inflammatory mediators. Method Microglia were seeded from a primary embryonic mixed cortical suspension with the addition of GM-CSF. Microglial expression of CD45, CD11b, CD11c, MHC class I and II, CD40, CD80, and CD86 was analyzed by flow cytometry and compared to those isolated using different culture methods and to the BV-2 cell line. GM-CSF microglia immunoreactivity and cytokine production was examined in response to lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Results Our results demonstrate GM-CSF addition during microglial culture yields higher cell numbers with greater purity than conventionally cultured primary microglia. We found that the expression of immune markers by GM-CSF microglia more closely resemble adult microglia than other methods or an immortalized BV-2 cell line. Primary differences amongst the different groups were reflected in their levels of CD39, CD86 and MHC class I expression. GM-CSF microglia produce CCL2, tumor necrosis factor-α, IL-6 and IL-10 following exposure to LPS and alter costimulatory marker expression in response to LPS or IFN-γ. Notably, GM-CSF microglia were often more responsive than the commonly used BV-2 cell line which produced negligible IL-10. Conclusion GM-CSF cultured microglia closely model the phenotype of adult microglia examined ex vivo . GM-CSF microglia are robust in their responses to inflammatory stimuli, altering immune markers including Iba-1 and expressing an array of cytokines characteristic of both pro-inflammatory and reparative processes. Consequently, the addition of GM-CSF for the culturing of primary microglia serves as a valuable method to increase the potential for studying microglial function ex vivo.