Optimised isolation and characterisation of adult human astrocytes from neurotrauma patients

Abstract

BackgroundAstrocytes are the main cellular constituent in the central nervous system. Astrocyte cultures from rodent brains are most commonly used in the experimental practice. However, important differences between rodent and human astrocytes exist. The aim of this study was to develop an improved protocol for routine preparation of primary astrocyte culture from adult human brain, obtained after trauma. New MethodTissue obtained during neurotrauma operation was mechanically decomposed and centrifuged. The cell sediment was resuspended in cell culture medium, plated in T25 tissue flasks and incubated for one month at 37 °C in 5% CO2. The medium was replaced twice weekly and microglia were removed. Once confluent, the purity of cultures was assessed. The culture was characterised immunocytochemically for specific astrocytic markers (GFAP, GLAST and S100B). Cell morphology was examined through the actin cytoskeleton labelling with fluorescent phalloidin. ResultsUnder basal conditions, adult astrocytes exhibited astrocyte-specific morphology and expressed specific markers. Approximately 95% of cells were positive for the main glial markers (GFAP, GLAST, S100B). Comparison with Existing MethodWe established an easy and cost-effective method for a highly enriched primary astrocyte culture from adult human brain. ConclusionThe isolation technique provides sufficient quantities of isolated cells. The culture obtained in this study exhibits the biochemical and physiological properties of astrocytes. It may be useful for elucidating the mechanisms related to the adult brain, exploring changes between neonatal and adult astrocytes, novel therapeutic targets, cell therapy experiments, as well as investigating compounds involved in cytotoxicity and cytoprotection.