TY - JOUR
T1 - A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype
AU - Prah, Jude
AU - Winters, Ali
AU - Chaudhari, Kiran
AU - Hersh, Jessica
AU - Liu, Ran
AU - Yang, Shao Hua
N1 - Funding Information:
This work was partly supported by National Institutes of Health grants 1R21NS087209-01A1 (SY) and R01NS088596 (SY) .
Publisher Copyright:
© 2019 Elsevier B.V.
PY - 2019/5/15
Y1 - 2019/5/15
N2 - Background: Primary astrocyte cultures have been used for decades to study astrocyte functions in health and disease. The current primary astrocyte cultures are mostly maintained in serum-containing medium which produces astrocytes with a reactive phenotype as compared to in vivo quiescent astrocytes. The aim of this study was to establish a serum-free astrocyte culture medium that maintains primary astrocytes in a quiescent state. New method: Serum free astrocyte base medium (ABM) supplemented with basic fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) (ABM-FGF2-EGF) or serum supplemented DMEM (MD-10%FBS) was used to culture primary astrocytes isolated from cerebral cortex of postnatal day 1 C57BL/6 mice. Results: Compared to astrocytes cultured in MD-10%FBS medium, astrocytes in ABM-FGF2-EGF had higher process bearing morphologies similar to in vivo astrocytes. Western blot, immunostaining, quantitative polymerase chain reaction and metabolic assays revealed that astrocytes maintained in ABM-FGF2-EGF had enhanced glycolytic metabolism, higher glycogen content, lower GFAP expression, increased glutamine synthase, and glutamate transporter-1 mRNA levels as compared to astrocytes cultured in MD-10% FBS medium. Comparison to existing methods: These observations suggest that astrocytes cultured in ABM-FGF2-EGF media compared to the usual FBS media promote quiescent and biosynthetic phenotype similar to in vivo astrocytes. Conclusion: This media provides a novel method for studying astrocytes functions in vitro under physiological and pathological conditions.
AB - Background: Primary astrocyte cultures have been used for decades to study astrocyte functions in health and disease. The current primary astrocyte cultures are mostly maintained in serum-containing medium which produces astrocytes with a reactive phenotype as compared to in vivo quiescent astrocytes. The aim of this study was to establish a serum-free astrocyte culture medium that maintains primary astrocytes in a quiescent state. New method: Serum free astrocyte base medium (ABM) supplemented with basic fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) (ABM-FGF2-EGF) or serum supplemented DMEM (MD-10%FBS) was used to culture primary astrocytes isolated from cerebral cortex of postnatal day 1 C57BL/6 mice. Results: Compared to astrocytes cultured in MD-10%FBS medium, astrocytes in ABM-FGF2-EGF had higher process bearing morphologies similar to in vivo astrocytes. Western blot, immunostaining, quantitative polymerase chain reaction and metabolic assays revealed that astrocytes maintained in ABM-FGF2-EGF had enhanced glycolytic metabolism, higher glycogen content, lower GFAP expression, increased glutamine synthase, and glutamate transporter-1 mRNA levels as compared to astrocytes cultured in MD-10% FBS medium. Comparison to existing methods: These observations suggest that astrocytes cultured in ABM-FGF2-EGF media compared to the usual FBS media promote quiescent and biosynthetic phenotype similar to in vivo astrocytes. Conclusion: This media provides a novel method for studying astrocytes functions in vitro under physiological and pathological conditions.
KW - Astrocyte
KW - Astrogliosis
KW - Basic fibroblast growth factor
KW - Epidermal growth factors
KW - Fetal bovine serum
KW - Primary culture
UR - http://www.scopus.com/inward/record.url?scp=85063254364&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2019.03.013
DO - 10.1016/j.jneumeth.2019.03.013
M3 - Article
C2 - 30904500
AN - SCOPUS:85063254364
SN - 0165-0270
VL - 320
SP - 50
EP - 63
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
ER -