Spinal motor neurons from neonatal rats: purification, culture and identification

doi:10.3969/j.issn.2095-4344.2015.42.011

Abstract

Abstract:

BACKGROUND: neurons are post-mitotic cells that are difficult to survive. Isolation, purification and culture of spinal motor neurons are technical difficulties of cell culture technology.
OBJECTIVE: To establish the culture system of spinal motor neurons from neonatal rats and to identify and determine the purity of spinal cord neurons.
METHODS: Ventral spinal cord tissues from neonatal rats were made into cell suspension that was subjected to density gradient centrifugation and differential adherence followed by purification culture. Then, the cells on cover plates were identified and classified using immunocytochemical double staining method, and the content of cell components was calculated in combination with fluorescent Hoechst nuclear staining.
RESULTS AND CONCLUSION: The cells adhered well, and the neurons accounted for 85.8%, among which, motor neurons reached 71.6%, astrocytes accounted for 7.8%, cells negative for neurofilament 200 and glial fibrillary acidic protein were 6.4%. These findings indicate that the ventral spinal cord tissues from neonatal rats combined with density gradient centrifugation and differential adherence can develop high-purity spinal motor neurons.
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, Cells, Cultured, Microglia

Yang Lin, Liu Yang, Lv De-cheng. Spinal motor neurons from neonatal rats: purification, culture and identification[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(42): 6782-6786.

Figures/Tables 3

References

[1] Kuhn TB.Growing and working with spinal motorneurons. Methods cell Biol.2013;71(1):67-87.
[2] 孙毅,赵冬梅,张烨,等.胎鼠前角运动神经元的取材与培养研究[J].滨州医学院学报,2009,32(6):17-19.
[3] Elliott P,Wallis DI,Foster GA.Ionic mechanisms underlying excitatory effects of serotonin on embryonic rat motoneurons in long-term culture.Neuroscience.2013;90(4):1311-1323．
[4] 赵合庆,杨晓玲,严志强.胚胎大鼠脊髓运动神经元的体外培养与鉴定[J].中华神经科杂志,2004,37(5): 435-437.
[5] 宋学琴,李春岩,吴淑玉,等.胚胎大鼠脊髓运动神经元的体外培养及鉴定[J].基础医学与临床,2004,24(1):83-87.
[6] Son EY, Ichida JK, Wainger BJ, et al.Conversion of Mouse and Human Fibroblasts into Functional Spinal Motor Neurons. Cell Stem Cell. 2011;9(3):205-218.
[7] Haastert K, Grosskreutz J, Jaeckel M, et al.Ratembryonic motoneurons in long -term co-culture with Schwann cells-a system to investigate motoneuron diseases on a cellular level invitro.J Neurosci Method.2012;142(2):275-284.
[8] Das M, Rumsey JW, Gregory CA, et al.Embryonc motoneuron-skeletal muscle co-culture in a defined system. Neuroscience.2014;146(2): 481-488.
[9] Larsen KE, Benn SC, Ay I, et al. Aglial cell line-derived neurotroph factor (GDNF):tetanus tox in fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice.Brain Res.2006;1120(1):1-12.
[10] Andersona KN, Potterb AC, Piccenna LG, et al. Isolation and culture of motor neurons from the newborn mouse spinal cord.Brain Res Protoc.2014;12: 132-136.
[11] Taguchi T, Huchet M,Roa M,et al. A sub population of embryonic telecephalic neurons survive and develop in vitro in response to factors derived from the periphery. Dev Brain Res.2012;37:125-132.
[12] Rochefort NL,Narushima M,Grienberger C.Development of direction selectivity in mouse cortical neurons.Neuron.2001; (3):425-432.
[13] 杨浩,梁喆,鞠躬,等.体外培养脊髓神经元过程中关于提高细胞产率以及活性的技术探讨[J].解剖学杂志,1997,20(3):239-242.
[14] 杨浩,王春婷,鞠躬.不同方法纯化体外培养脊髓神经元的比较[J].中国组织化学与细胞化学杂志2000,9(3).
[15] Rakowicz WP,Staples CS,Milbrandt J. Glial cell line-derived neuro-trophic factor promotes the survival of early postnatal spinal motor neurons in the lateral and medial motor columns in slice culture.J Neurosci.2012;22:3953-3962.
[16] Carriedo SG,Yin HZ,Weiss JH.Motor neurons are selectively vulnerable to AMPA/Kainate receptor-mediated injury in vitro. J Neurosci.2006;16:4069-4079.
[17] 赵林普.某些神经营养因子对培养大鼠脊髓腹侧神经元生长分化作用[J].细胞生物学杂志, 2010(增刊),117(3).
[18] Bataille S,Potalier P,Coulon P.Influence of acety-l2 cholinesterase on embryonic spinal rat motoneurous grousth in culture:a quantitative morphometric study.Eur J Neurosci. 2008;10:560-572.
[19] 宋学琴,王丽琴,吴淑玉.生长底物对培养胚胎大鼠脊髓运动神经元的作用[J].中国免疫学杂志,2004,20(6): 405-409.
[20] 杨晓玲,赵合庆,严志强.体外培养大鼠脊髓运动神经元的存活率及形态观察[J].基础医学与临床,2007,27(3):344-345.
[21] Martin FC,Wiley CA.A serum-free,pyruvate-free medium that supports neonatal/ glial cultures.J Neurosci Res.2005; 41(2): 246.
[22] Banker GA,Cowan WM.Rat hippocampal neurons in dispersed cell culture.Brain Res.2012;126(2):397.
[23] Taylor AR, Robinson MB, Milligan CE.In vitro methods to prepare astrocyte and motoneuron cultures for the investigation of potential in vivo interactions. Nat Protoc.2007; 2(6):1499-1507.
[24] 余磊, 潘树义, 秦建强.脊髓神经细胞培养的方法.中国临床解剖学杂志,2002,20(4),267.
[25] Lin LF,Doherty DH,Lile JD.GDNF:a glia cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science. 2013;260(5111):1130.
[26] Zum AD,Baetge EE,Hammang JP.Glia cell line-derived neurotrophic factor(GDNF),a new neurotrophic factor for motoneurons.Neuro Report.2014;6(1):113.
[27] 陈波,袁琼兰.胶质细胞源性神经营养因子与突触可塑性[J].四川解剖学杂志,2006(4):38-39,44.
[28] 宋学琴,吴淑玉,王丽琴.胚胎大鼠脊髓运动神经元体外培养方法的优化.河北医科大学学报,2004,25(3),133-135.
[29] Lucius R, Mentlein R.Development of a culture system for pure rat neurons: advantages of a sandwich technique.Anat Anz.2013;177(5):447-454.
[30] 司徒镇强,吴军正.细胞培养[M].西安:世界图书出版公司,2004: 150-152.