A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype

Jude Prah, Ali Winters, Kiran Chaudhari, Jessica Hersh, Ran Liu, Shaohua Yang

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)50-63
Number of pages14
JournalJournal of Neuroscience Methods
Volume320
DOIs
StatePublished - 15 May 2019

Fingerprint

Astrocytes
Phenotype
Serum
Fibroblast Growth Factor 2
Epidermal Growth Factor
Amino Acid Transport System X-AG
Serum-Free Culture Media
Glutamine

Keywords

  • Astrocyte
  • Astrogliosis
  • Basic fibroblast growth factor
  • Epidermal growth factors
  • Fetal bovine serum
  • Primary culture

Cite this

Prah, Jude ; Winters, Ali ; Chaudhari, Kiran ; Hersh, Jessica ; Liu, Ran ; Yang, Shaohua. / A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype. In: Journal of Neuroscience Methods. 2019 ; Vol. 320. pp. 50-63.
@article{d7b4fb5a3119405ca524a9e7aeaebc60,
title = "A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype",
abstract = "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.",
keywords = "Astrocyte, Astrogliosis, Basic fibroblast growth factor, Epidermal growth factors, Fetal bovine serum, Primary culture",
author = "Jude Prah and Ali Winters and Kiran Chaudhari and Jessica Hersh and Ran Liu and Shaohua Yang",
year = "2019",
month = "5",
day = "15",
doi = "10.1016/j.jneumeth.2019.03.013",
language = "English",
volume = "320",
pages = "50--63",
journal = "Journal of Neuroscience Methods",
issn = "0165-0270",
publisher = "Elsevier",

}

A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype. / Prah, Jude; Winters, Ali; Chaudhari, Kiran; Hersh, Jessica; Liu, Ran; Yang, Shaohua.

In: Journal of Neuroscience Methods, Vol. 320, 15.05.2019, p. 50-63.

Research output: Contribution to journalArticle

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, Shaohua

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

VL - 320

SP - 50

EP - 63

JO - Journal of Neuroscience Methods

JF - Journal of Neuroscience Methods

SN - 0165-0270

ER -