TY - JOUR
T1 - Insulin and heparin-binding epidermal growth factor-like growth factor synergistically promote astrocyte survival and proliferation in serum-free medium
AU - Jia, Mei
AU - Shi, Zhongfang
AU - Yan, Xu
AU - Xu, Lixin
AU - Dong, Liping
AU - Li, Jiaxin
AU - Wang, Yujiao
AU - Yang, Shaohua
AU - Yuan, Fang
N1 - Funding Information:
This work was supported by the National Natural Science Foundation of China [No. 81271286 (to FY)], Natural Science Foundation of Beijing [No. 7152027 (to FY)] and Innovation Foundation of Beijing Neurosurgical Institutes [No. 2017-5 (to FY)]. Mei Jia performed the experiments and drafted the manuscript; Zhongfang Shi performed the Western blot; Xu Yan performed flow cytometry; Lixin Xu contributed to the cell culture; Liping Dong performed the MTT assay; Jiaxin Li and Yujiao Wang analyzed the data; Shaohua Yang contributed to the conception of the work and revised the manuscript; Fang Yuan designed the studies and wrote the manuscript. All authors revised and approved the final version of the manuscript to be published and agreed to be held accountable for all aspects of the work. At last, we thank Elsevier's WebShop for carefully editing language of the manuscript.
Publisher Copyright:
© 2018 The Authors
PY - 2018/9/1
Y1 - 2018/9/1
N2 - Background: In vitro systems allowing maintenance and experimentation on primary astrocyte cultures have been used for decades. Astrocyte cultures are most maintained in serum-containing medium which has been found to alter the morphology and gene profiles of astrocytes. New method: Here, we reported a new serum-free medium for astrocyte culture, which consisted of DMEM and NB media supplemented with insulin and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (SF-I-H medium). Meanwhile FBS-containing (FBS) medium composed of DMEM medium containing 10% FBS were used for comparison study. Cerebral cortex was harvested from postnatal day 1 Wistar rats and brain cells were isolated and seeded to poly-L-lysine coated culture dishes after 15 min differential velocity adherence. Results: Compared with FBS medium, astrocytes in SF-I-H medium were smaller and exhibited process bearing morphologies. MTT assays showed that cell density and proliferation rate were higher in SF-I-H medium than in FBS medium all the time, and flow cytometry analysis revealed that SF-I-H medium promoted cell mitosis in a manner comparable to FBS medium. Consistently, western blot analysis further revealed that insulin and HB-EGF synergistically activated the PI3K/AKT and MAPK/ERK1/2 signaling cascades as FBS. Comparison with existing method(s): Astrocytes cultured in SF-I-H medium grew faster than FBS medium. Conclusion: Taken together, our results indicated that SF-I-H medium, in which cell morphology was similar with astrocytes in brain, was more effective for astrocyte survival and proliferation than FBS medium, providing a new cell model to study astrocyte functions without the interference of serum.
AB - Background: In vitro systems allowing maintenance and experimentation on primary astrocyte cultures have been used for decades. Astrocyte cultures are most maintained in serum-containing medium which has been found to alter the morphology and gene profiles of astrocytes. New method: Here, we reported a new serum-free medium for astrocyte culture, which consisted of DMEM and NB media supplemented with insulin and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (SF-I-H medium). Meanwhile FBS-containing (FBS) medium composed of DMEM medium containing 10% FBS were used for comparison study. Cerebral cortex was harvested from postnatal day 1 Wistar rats and brain cells were isolated and seeded to poly-L-lysine coated culture dishes after 15 min differential velocity adherence. Results: Compared with FBS medium, astrocytes in SF-I-H medium were smaller and exhibited process bearing morphologies. MTT assays showed that cell density and proliferation rate were higher in SF-I-H medium than in FBS medium all the time, and flow cytometry analysis revealed that SF-I-H medium promoted cell mitosis in a manner comparable to FBS medium. Consistently, western blot analysis further revealed that insulin and HB-EGF synergistically activated the PI3K/AKT and MAPK/ERK1/2 signaling cascades as FBS. Comparison with existing method(s): Astrocytes cultured in SF-I-H medium grew faster than FBS medium. Conclusion: Taken together, our results indicated that SF-I-H medium, in which cell morphology was similar with astrocytes in brain, was more effective for astrocyte survival and proliferation than FBS medium, providing a new cell model to study astrocyte functions without the interference of serum.
KW - Astrocytes
KW - Cell proliferation
KW - Cell survival
KW - Heparin-binding epidermal growth factor-like growth factor
KW - Insulin
KW - Serum-free medium
UR - http://www.scopus.com/inward/record.url?scp=85048711932&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2018.06.002
DO - 10.1016/j.jneumeth.2018.06.002
M3 - Article
C2 - 29890195
AN - SCOPUS:85048711932
SN - 0165-0270
VL - 307
SP - 240
EP - 247
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
ER -