Simplified procedure for producing large yields of functional microglial cells in culture: application for studying neuroinflammatory responses

SEPULVEDA DIAZ, J.; OUIDJA O.; SOCIAS, S.B.; HAMADAT, S.; GUERREIRO, S.; RAISMAN VOZARI R.; MICHEL, P.

San Diego

Congreso; Neuroscience 2016; 2016

Society for Neuroscience (USA)

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolation of pure microglial cells from post-natal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEM medium. The effects of PEI were not reproduced with other synthetic or biological substrates and they were context-dependent as replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation onto PEI coating. Noticeably, the absence of culture medium renewal during the progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to make this process as efficient as possible. PEI-isolated microglial cells expressed the macrophage antigen-1 (MAC-1) receptor and also the Fc receptor-like S (Fcrls), a biomarker that specifically distinguishes these cellsfrom other myeloid subsets. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1-10 ng/mL) treatment by secreting proinflammatory cytokines such as TNF-alpha, IL-6, IL-1beta and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed byappropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays.