培养小鼠神经干细胞的前体

Biology

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Summary

本视频介绍了从胚胎小鼠的发展皮层neuroprecursors隔离所采用的方法。胚胎从子宫中取出,解剖皮层组织,消化孤立的大脑皮层的过程。

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Currle, D. S., Hu, J. S., Kolski-Andreaco, A., Monuki, E. S. Culture of Mouse Neural Stem Cell Precursors. J. Vis. Exp. (2), e152, doi:10.3791/152 (2007).

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Abstract

主神经干细胞培养为研究中枢神经系统发育的内在机制,有用的。干细胞研究将增加我们的神经系统的了解,可能使我们能够发展为目前无法治愈的大脑疾病和损伤的治疗。此外,应使用干细胞,干细胞的研究,在神经细胞分化和转分化的机制和遗传和环境信号的详细研究,直接成特定的细胞类型的细胞的专业化。本视频演示了从胚胎12.5天鼠标背侧前脑细胞用来分解的一种技术。剥离过程包括收获E12.5小鼠胚胎,从子宫精细解剖钳去除的“皮肤”,最后隔离件大脑皮层。解剖后,该组织是消化和机械分离。悬浮分离细胞,然后培养在“干细胞”的媒体,有利于神经干细胞的生长。

Protocol

  1. 隔离E12.5胚胎皮层神经前体鼠标(筹备)。
  2. 从解剖telencephalic水泡,皮肤和间质层被拆除。
  3. 囊泡在0.05%的胰蛋白酶与0.02%EDTA和0.2%,在20分钟的HBSS牛血清白蛋白在37 ° C孵育
  4. 胰蛋白酶是由1 mg / ml的大豆胰蛋白酶抑制剂等体积的HBSS(Sigma公司#T6522)停止。
  5. 组织消化是分离的火抛光的巴斯德移液器使用几轮trituration。
  6. 细胞被洗涤一次,0.2%在HBSS中牛血清白蛋白和5万细胞/层粘连蛋白,涂布盖玻片毫升镀与表皮生长因子为20毫微克/毫升,10毫微克/毫升FGF2组(研究Ð系统或Peprotech),和2微克/ ml肝素媒体( Sigma公司)。

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Discussion

我们的收获,分离和培养胚胎神经干细胞的能力,在我们的中枢神经系统发育和干细胞生物学的理解的巨大进步已成为可能。本视频演示E12.5小鼠大脑皮层和随后的分类和胚胎神经干细胞培养的夹层。许多其他的其他类似的方法已成功地采用了其他的调查。

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Materials

Name Type Company Catalog Number Comments
Straight Iris Scissors Tool Fine Science Tools 14060-11
Medium Scissors Tool Fine Science Tools 15024-10
Micro Scissors Tool Fine Science Tools 15002-08
Bent Forceps Tool Fine Science Tools 11251-35

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References

  1. Currle, D., Cheng, X., Hsu, C., Monuki, E. Direct and indirect roles of CNS dorsal midline cells in choroid plexus epithelial formation. Development. 132, (15), 3549-3559 (2005).
  2. Flanagan, L., Rebaza, L., Derzic, S., Schwartz, P., Monuki, E. Regulation of human neural precursor cells by laminin and integrins. J Neurosci Res. 83, 845-856 (2006).

Comments

3 Comments

  1. There are lots of mistakes about working in a steril conditions, the cap of the bottles should be plased upsode down.  No good lab practoce !

    Reply
    Posted by: Maryam M.
    April 20, 2009 - 2:31 PM
  2. Thanks, but no contamination in 6 years.

    Reply
    Posted by: Anonymous
    April 20, 2009 - 4:34 PM
  3. I think it would be better to say "Culture of Mouse neural Stem (precursor) cells". You can not use both stem and precursor terms together.

    Reply
    Posted by: Hassan A.
    July 13, 2010 - 9:25 PM

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