Cryopreservation and Culture of Neural Stem Cells Isolated from Postnatal Rat
Abstract: Objective: to isolate and culture neural stem cells from postnatal rat and to observe the viability and biological property of these cells after cryopreservation. Methods: the neural stem cells isolated from hippocampus of the postnatal rat were cultured in culture solution without blood serum. After amplification, the cells were cultured in different concentration of freeze-stored liquid which contained 0、5%、10%、15%、20%DMSO and fetal calf serum, then stored in refrigerator of 80 centigrade below zero one week, two weeks and one month respectively. Through resuscitation training and testing, then we detected the recovery rate of the NSCs. Results: after cryopreservation and culture of neural stem cells isolated from postnatal rat, most of them were growth well and formed into the new cell clones, then the recovery rate of the cells were de-tected with Trypan Blue. There was significant difference in 10% DMSO and it has the highest recovery rate，which is 54.00 ± 1.73, 59.00 ± 1.16, 58.00 ± 2.08. Conclusion: the neural stem cells derived from postnatal rat were able to culture, cryopreservation and resuscitation, which did not change their biological properties.
文章引用: 张静 , 康现江 , 周辰 , 张平 , 穆淑梅 , 张晗 (2011) 新生大鼠神经干细胞的体外培养与冷冻复苏。 生物医学， 1， 25-33. doi: 10.12677/hjbm.2011.12005
 周燕琼, 刘小辉, 刘幸平等. 人肝癌细胞冷冻保存方法研究[J]. 世界华人消化杂志, 2000, 8(6): 714-715.
 X. M. Zhou, X. J. Kang, Z. Q. Shu, et al. Cryopreservation of peripheral blood stem cells using a box-in-box cooling device. Biopreservation and Boibanking, 2009, 7(2): 107-114.
 黄毓, 覃甲仁. 神经干细胞的研究进展[J]. 广西医科大学学报, 2004, 21(2): 308-310.
 A. Gritti, E. A. Parati, L. Cova, et al. Multipotential stem cells from the Adultmouse brain proliferate and self-renew in response to basic fibroblast growth factor. The Journal of Neuroscience, 1996, 16(3): 1091-1100.
 N. Uchida, D. W. Buck, D. He, et al. Direct isolation of human central nervous system stem cells. Proceeding of the National Academy Sciences of the United States of America, 2000, 97(26): 14720-14725.
 胡钧涛, 涂汉军, 张力等. 大鼠胚胎神经干细胞两种不同分离、传代方法的比较[J]. 中国神经精神疾病杂志, 2006, 32(2): 164-166.
 B. A.Reynolds, S. Weiss. Generation of neurons and astrocytes from isolated cells of adult mammalian central nervous system. Science, 1992, 255(5052): 1707-1710.
 L. J. Richards, T. J. Kilpatrick and P. F. Bartlett. De novo genera-tion of neural cells from the adult mouse brain. Proceeding of the National Academy Sciences of the United States of America, 1992, 89(18): 8591-8595.
 P. S. Eriksson, E. Perfilieva, T. Bjork-Eriksson, et al. Neuro-genesis in the adult human hippocampus. National Medecine, 1998, 4(11): 1313-1317.
 C. B. Johansson, M. Svensson, L. Wallstedt, et al. Neural stem cells in the adult human brain. Experimental Cell Research, 1999, 253(2): 733-736.
 S. Temple. The development of neural stem cells. Nature, 2001, 414(6859): 112-117.
 罗小丹, 沈岳飞. 神经干细胞定向诱导分化为多巴胺能神经元的研究进展[J]. 神经解剖学杂志, 2009, 25(4): 469-473.
 V. G. Kukekvo, E. D. Laywell, O. Suslov, et al. Multipotent stem cells with similar progerties arise from two neurogenic regions of adult human brain. Experimental Neurology, 1999, 156(2): 333-344.
 B. A. Reynolds, A. Weiss. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous sys-tem. Science, 1992, 255(5052): 1707-1710.
 A. A. Davis, S. Temple. A self-renewing multipotential stem cells in embryonic rat cerebral cortex. Nature, 1994, 372(6503): 263-266.
 J. Dietrich, M. C. Easterday. Developing concepts in neural stem cells. Trends in Neurosciences, 2002, 25(3): 129-131.
 M. Nakamura, Y. Toyama and H. Okano. Transplantation of neural stem cells for spinal cordinjury. Shinkeigaku, 2005, 45(11): 874-876.
 陈凯, 康现江, 张平等. 大鼠海马神经干细胞的分离培养与免疫荧光鉴定[J]. 医学研究教育, 2010, 27(2): 1-3.
 Z. Q. Shu, X. J. Kang, H. H. Chen, et al. Development of a reliable low-cost controlled cooling rate instruments for the cryopreserva-tion of hematopoietic stem cells. Cytotherapy, 2010, 12(2): 161- 169.
 R. Mckay. Stem cells in the central nervous system. Science, 1997, 276(5309): 66-71.
 Gaiano N, Fishell G. Transplantation as a tool to study progeni-tors within the vertebrate nervous system. Neurobiol, 1998, 36(2): 152-161.
 Y. Ogawa, K. Sawamoto, T. Miyata, et al. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogene-sis and functional recovery after spinal cord contusion injury in adult rats. Journal of Neuroscience Research, 2002, 69(6): 925- 933.
 W. W. Tornford, G. P. Duff and H. J. Mankin. Experimental freezepreservation of chondrocytes. Clinical Orthopaedics & Related Research, 1985, 7(197): 11-14.
 P. Akerud, J. M. Canals, E. Y. Snyder, et al. Neuroprotection through delivery of glial cell linederived neurotrophic factor by neural stem cells in a mouse model of Parkinson’s disease. The Journal of Neuroscience, 2001, 21(20): 8108-8118.