Development of a serum free astrocyte culture method that mimic resting in vivo astrocyte phenotype
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Purpose Primary astrocyte cultures have been extensively used for characterization of astrocytes functions in physiological and pathological conditions. The current primary astrocytes are mostly maintained in fetal bovine serum (FBS) containing medium. Although FBS contains growth elements that fulfills many metabolic needs of cultured astrocytes, it alters the genotypic and morphological profiles of primary astrocytes as well as induces astrocyte activation. The aim of this study was to establish a serum-free astrocyte culture medium that maintains primary astrocytes in a quiescent state with phenotypes that mimic in vivo astrocytes. Methods Primary astrocytes were isolated from the cerebral cortex of postnatal day 1 C57BL/6 mice and cultured in serum-free astrocyte basal medium containing FGF2 and EGF (ABM-FGF2-EGF). The phenotype of primary astrocytes cultured in ABM-FGF2-EGF were compared with astrocytes cultured in FBS supplemented DMEM medium (MD-10% FBS). Growth assays, immunostaining, Western blot, quantitative polymerase chain reaction, and metabolic assays were used to access the growth rates, metabolic phenotype, mRNA expression profiles and quiescent or reactive states of astrocytes. Results and Conclusions We demonstrated that the novel serum free ABM-FGF2-EGF medium supports astrocytes growth and enhanced glycolytic metabolism with higher glycogen content, lower GFAP and vimentin expression, and increased glutamate transporter mRNA levels as compared to astrocytes cultured in the MD-10% FBS medium. Our study suggests that our serum free culture method produces astrocytes with a biosynthetic phenotype and morphology similar to in vivo resting astrocytes. Additionally ABM- FGF2-EGF cultured primary astrocytes could be activated by various pathological conditions. The developed serum-free and EGF/FGF2-containing astrocyte basal medium will provide a critical tool for defining the precise function of astrocytes under physiological and pathological conditions.