A Simplified Approach for Efficient Isolation of Functional Microglial Cells: Application for Modeling Neuroinflammatory Responses In Vitro

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

GLIA

WILEY-LISS, DIV JOHN WILEY & SONS INC

Lugar: New York; Año: 2016 vol. 64 p. 1912 - 1924

0894-1491

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields ofthese cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolationof pure microglial (MAC-11/Fcrls1/Ccr2-) cells from postnatal brain tissue through a simple culture procedure that mainlyrelies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEMmedium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions.Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating thatPEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolationstrongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated inlarge culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subculturesfor functional studies. These cultures responded to lipopolysaccharide (LPS, 1?10 ng/ml) treatment by secreting proinflammatorycytokines such as TNF-a, IL-6, IL-1b and by generating nitric oxide and reactive oxygen species. Most interestingly,this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogeniccytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its highyield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies orscreening assays.