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

Abstract

BackgroundMicroglia 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. MethodMicroglia 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). ResultsOur 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. ConclusionGM-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.