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Artículo

A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro

Sepulveda Diaz, Julia E.; Ouidja, Mohand O.; Socias, Sergio BenjaminIcon ; Hamadat, Sabah; Guerreiro, Serge; Raisman Vozari, Rita; Michel, Patrick P.
Fecha de publicación: 11/2016
Editorial: Wiley-liss, Div John Wiley & Sons Inc
Revista: Glia
ISSN: 0894-1491
Idioma: Inglés
Tipo de recurso: Artículo publicado
Clasificación temática:
Otras Biotecnologías de la Salud

Resumen

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 (MAC-1+/Fcrls+/Ccr2-) cells from postnatal 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. 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 that PEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. 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 pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β 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 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. GLIA 2016;64:1912–1924.
Palabras clave: Cell Culture , Cytokines , Lipopolysaccharide , Nitrogen And Oxygen Reactive Species , Polyethyleneimine
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info:eu-repo/semantics/openAccess Excepto donde se diga explícitamente, este item se publica bajo la siguiente descripción: Creative Commons Attribution-NonCommercial-ShareAlike 2.5 Unported (CC BY-NC-SA 2.5)
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URI: http://hdl.handle.net/11336/50615
URL: https://onlinelibrary.wiley.com/doi/full/10.1002/glia.23032
DOI: https://doi.org/10.1002/glia.23032
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Articulos(INSIBIO)
Articulos de INST.SUP.DE INVEST.BIOLOGICAS
Citación
Sepulveda Diaz, Julia E.; Ouidja, Mohand O.; Socias, Sergio Benjamin; Hamadat, Sabah; Guerreiro, Serge; et al.; A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro; Wiley-liss, Div John Wiley & Sons Inc; Glia; 64; 11; 11-2016; 1912-1924
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