title: A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro.
abstract: 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.
authors.affiliation: Institut National De La Santé Et De La Recherche Médicale, U 1127, CNRS, Unité Mixte De Recherche (UMR) 7225, Sorbonne Universités, UPMC Univ Paris 06, UMR S 1127, Institut Du Cerveau Et De La Moelle Epinière, ICM, Paris, France.
authors.forename: Julia E
authors.initials: JE
authors.lastname: Sepulveda-Diaz
authors.affiliation: Laboratoire Croissance, Régénération, Réparation Et Régénération Tissulaires (CRRET)/EAC CNRS 7149, Université Paris Est Créteil, Université Paris Est, Créteil, France.
authors.forename: Mohand O
authors.initials: MO
authors.lastname: Ouidja
authors.affiliation: Institut National De La Santé Et De La Recherche Médicale, U 1127, CNRS, Unité Mixte De Recherche (UMR) 7225, Sorbonne Universités, UPMC Univ Paris 06, UMR S 1127, Institut Du Cerveau Et De La Moelle Epinière, ICM, Paris, France.|Facultad De Bioquímica, Química Y Farmacia (UNT), Instituto Superior De Investigaciones Biológicas, INSIBIO (CONICET-UNT) and Instituto De Química Biológica "Dr Bernabé Bloj,", Tucumán, Argentina.
authors.forename: Sergio B
authors.initials: SB
authors.lastname: Socias
authors.affiliation: Institut National De La Santé Et De La Recherche Médicale, U 1127, CNRS, Unité Mixte De Recherche (UMR) 7225, Sorbonne Universités, UPMC Univ Paris 06, UMR S 1127, Institut Du Cerveau Et De La Moelle Epinière, ICM, Paris, France.
authors.forename: Sabah
authors.initials: S
authors.lastname: Hamadat
authors.affiliation: Institut National De La Santé Et De La Recherche Médicale, U 1127, CNRS, Unité Mixte De Recherche (UMR) 7225, Sorbonne Universités, UPMC Univ Paris 06, UMR S 1127, Institut Du Cerveau Et De La Moelle Epinière, ICM, Paris, France.
authors.forename: Serge
authors.initials: S
authors.lastname: Guerreiro
authors.affiliation: Institut National De La Santé Et De La Recherche Médicale, U 1127, CNRS, Unité Mixte De Recherche (UMR) 7225, Sorbonne Universités, UPMC Univ Paris 06, UMR S 1127, Institut Du Cerveau Et De La Moelle Epinière, ICM, Paris, France.
authors.forename: Rita
authors.initials: R
authors.lastname: Raisman-Vozari
authors.affiliation: Institut National De La Santé Et De La Recherche Médicale, U 1127, CNRS, Unité Mixte De Recherche (UMR) 7225, Sorbonne Universités, UPMC Univ Paris 06, UMR S 1127, Institut Du Cerveau Et De La Moelle Epinière, ICM, Paris, France. julia.esd@gmail.Com.
authors.forename: Patrick P
authors.initials: PP
authors.lastname: Michel
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000970:Antineoplastic Agents / Q000494:pharmacology; D001921:Brain / Q000166:cytology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D007797:Laminin / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D016177:Macrophage-1 Antigen / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008840:Microfilament Proteins / Q000378:metabolism; D017628:Microglia / Q000187:drug effects / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011094:Polyethyleneimine / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: C491049:Aif1 protein, mouse; D000970:Antineoplastic Agents; D002135:Calcium-Binding Proteins; D016207:Cytokines; D005904:Glial Fibrillary Acidic Protein; D007797:Laminin; D008070:Lipopolysaccharides; D016177:Macrophage-1 Antigen; D008840:Microfilament Proteins; D009842:Oligopeptides; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; C047981:arginyl-glycyl-aspartic acid; D003907:Dexamethasone; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011094:Polyethyleneimine
keywords: cell culture; cytokines; lipopolysaccharide; nitrogen and oxygen reactive species; polyethyleneimine