Department of Pathophysiology, Beijing Neurosurgical Institute, Beijing Tiantan Hospital, Capital Medical University, Beijing, China; China National Clinical Research Center for Neurological Diseases, Beijing, China; Beijing Key Laboratory of Central Nervous System Injury, Beijing, China; Center of Stroke, Beijing Institute for Brain Disorders, Beijing, China; Beijing Key Laboratory of Translational Medicine for Cerebrovascular Disease, Beijing, China.
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA.
J Neurosci Methods. 2018 Sep 1;307:240-247. doi: 10.1016/j.jneumeth.2018.06.002. Epub 2018 Jun 8.
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.
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.
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.
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.
几十年来，允许在原代星形胶质细胞培养物上进行维持和实验的体外系统一直被使用。星形胶质细胞培养物最常保存在含有血清的培养基中，而这种培养基已被发现会改变星形胶质细胞的形态和基因谱。
在这里，我们报道了一种用于星形胶质细胞培养的新的无血清培养基，它由 DMEM 和 NB 培养基组成，补充了胰岛素和肝素结合表皮生长因子样生长因子 (HB-EGF)（SF-I-H 培养基）。同时，使用含有 FBS 的培养基（由含有 10% FBS 的 DMEM 培养基组成）进行对比研究。从出生后 1 天的 Wistar 大鼠中收获大脑皮质，在 15 分钟的差速粘附后，将脑细胞分离并播种到多聚-L-赖氨酸包被的培养皿中。
与 FBS 培养基相比，SF-I-H 培养基中的星形胶质细胞较小，呈具有突起的形态。MTT 分析表明，SF-I-H 培养基中的细胞密度和增殖率始终高于 FBS 培养基，流式细胞术分析表明，SF-I-H 培养基以类似于 FBS 培养基的方式促进细胞有丝分裂。一致地，Western blot 分析进一步表明，胰岛素和 HB-EGF 协同激活 PI3K/AKT 和 MAPK/ERK1/2 信号通路，与 FBS 相同。
在 SF-I-H 培养基中培养的星形胶质细胞比在 FBS 培养基中生长得更快。
综上所述，我们的结果表明，SF-I-H 培养基更有效地促进星形胶质细胞的存活和增殖，其细胞形态与大脑中的星形胶质细胞相似，比 FBS 培养基更有效，为研究星形胶质细胞功能提供了一种新的细胞模型，而不会受到血清的干扰。
