title: Fast astrocyte isolation by sedimentation field flow fractionation.
abstract: Astrocytes play a key role during central nervous system (CNS) repair and glial scar formation. After CNS damage, an extensive deposition of the extracellular matrix produced by the activated astrocytes limits the extension of the lesion but impairs axon outgrowth and functional recovery. Until now, methods to obtain astrocytes need long culture period and laborious cell culture conditions and do not allow the isolation of pure astrocyte preparation. In this study, we used sedimentation field flow fractionation (SdFFF) to rapidly sort well preserved astrocyte population. Four main cell fractions, the total eluted population (TP), and fractions F1, F2, and F3, were isolated by SdFFF from rat newborn cortex. After elution, cells were cultured for one week, and analyzed by immunocytofluorescence using antibodies against specific epitopes: glial fibrillary acidic protein (GFAP), O4, β-III tubulin, and CD 68, labelling respectively astrocytes, oligodendrocytes, neurons, and microglial cells. SdFFF eluted cells were compared with the cells obtained with the classical method. Results showed that SdFFF appeared to be a rapid (one week) and effective method to sort enriched populations of viable and functional astrocytes. In particular, F1 and F3 fractions contained high percentage of GFAP expressing cells (95.6% and 98.0%, respectively). Results also showed that F1 derived cell cultures contained large astrocytes that spread in the culture dish while in fraction F3 derived cell cultures, astrocytes were small, showing a tendency to aggregate and displaying higher migratory capacities than those of fraction F1. Thanks to SdFFF, isolation of almost pure astrocyte populations was rapidly obtained. In addition, the isolation of different astrocyte subpopulations showing different behaviors offers a new perspective to better understand the glial scar formation and remodeling after CNS damage.
authors.affiliation: Département de Physiologie, and EA 6309 Maintenance myélinique et neuropathies périphériques, FR 3503, Facultés de Médecine et de Pharmacie, Université de Limoges, Limoges, F-87025, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Sarrazy
authors.forename: Nicolas
authors.initials: N
authors.lastname: Vedrenne
authors.forename: Nelly
authors.initials: N
authors.lastname: Bordeau
authors.forename: Fabrice
authors.initials: F
authors.lastname: Billet
authors.forename: Philippe
authors.initials: P
authors.lastname: Cardot
authors.forename: Alexis
authors.initials: A
authors.lastname: Desmoulière
authors.forename: Serge
authors.initials: S
authors.lastname: Battu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000737:chemistry* / Q000166:cytology; D031521:Fractionation, Field Flow / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley