Chinese Journal of Tissue Engineering Research ›› 2018, Vol. 22 ›› Issue (32): 5186-5190.doi: 10.3969/j.issn.2095-4344.0383

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A method of high purity primary neuron culture and high efficiency transfection

Li Xing-tong1, Tang Hong-yan3, Ma Wei1, Yang Jin-wei1, 2, Wang Xian-bin1, Dai Yun-fei1, Li Jun-yan4,
Guo Jian-hui2, Li Li-yan1   

  1. 1Institute for Neuroscience, Kunming Medical University, Kunming 650500, Yunnan Province, China; 2Second Department of General Surgery, the First People’s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China; 3Department of Neurosurgery, the First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan Province, China; 4Department of Neurosurgery, the First Hospital of Kunming, Kunming 650032, Yunnan Province, China
  • Received:2018-04-21 Online:2018-11-18 Published:2018-11-18
  • Contact: Li Li-yan, MD, Professor, Institute for Neuroscience, Kunming Medical University, Kunming 650500, Yunnan Province, China Corresponding author: Guo Jian-hui, Chief physician, Second Department of General Surgery, the First People’s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China Corresponding author: Li Jun-yan, Associate chief physician, Departement of Neurosurgery, the First Hospital of Kunming, Kunming 650032, Yunnan Province, China
  • About author:Li Xing-tong, Master, Institute for Neuroscience, Kunming Medical University, Kunming 650500, Yunnan Province, China Tang Hong-yan, Nurse-in-charge, Department of Neurosurgery, the First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan Province, China Li Xing-tong and Tang Hong-yan contributed equally to this work.
  • Supported by:

    the National Natural Science Foundation of China, No. 31560295 and 31260253; the Applied Basic Research Project of Yunnan Province, No. 2015FB098; the Health, Science and Technology Project of Yunnan Province, No. 2014NS202

Abstract:

BACKGROUND: Neuron culture and cell transfection technologies are important means to study the development and pathophysiologic mechanism of neurons in vitro, but the purity and transfection efficiency of primary cultured cortical neurons are poor.
OBJECTIVE: To establish a simple, efficient and stable method of culture and transfection method of primary neurons in vitro.
METHODS: Primary cortical neurons were harvested from neonatal 1-day rat brains under aseptic condition, which was digested with 0.25% trypsin prior to centrifugation and made into cell suspensions, followed by being seeded into Neurobasal-A medium (5×109 L-1 per pore). The morphological characteristics of neurons were observed by inverted microscope; two neuron-specific markers (MAP2 and Tuj1) were used for immunolabeling to identify the cultured cells; the transfection efficiency of neurons was tested by Lipofectamine 2000 and Block-iT Transfection Kit.
RESULTS AND CONCLUSION: The neurons cultured in vitro exhibited interconnected networks after culture for 7 days. All the cultured neurons displayed MAP2 and Tuj1 immunoreactivity. The highly effective transfection was observed under fluorescence microscope using Lipofectamine 2000 transfected neurons. In summary, the culture method of primary cerebral cortex neurons can be adopted to obtain highly purified neurons. Besides, high transfection efficiency of primary neurons can be realized by artificial liposome.

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松组织工程

Key words: Neurons, Cell Culture Techniques, Transfection, Liposomes, Tissue Engineering

CLC Number: