Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 240-247 |
| Number of pages | 8 |
| Journal | Journal of Neuroscience Methods |
| Volume | 307 |
| DOIs | |
| State | Published - 1 Sep 2018 |
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Keywords
- Astrocytes
- Cell proliferation
- Cell survival
- Heparin-binding epidermal growth factor-like growth factor
- Insulin
- Serum-free medium
Cite this
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Insulin and heparin-binding epidermal growth factor-like growth factor synergistically promote astrocyte survival and proliferation in serum-free medium. / Jia, Mei; Shi, Zhongfang; Yan, Xu; Xu, Lixin; Dong, Liping; Li, Jiaxin; Wang, Yujiao; Yang, Shaohua; Yuan, Fang.
In: Journal of Neuroscience Methods, Vol. 307, 01.09.2018, p. 240-247.Research output: Contribution to journal › Article › Research › peer-review
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
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
VL - 307
SP - 240
EP - 247
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
ER -