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成年鼠神经干细胞的分离和扩大使用神经干含量

Neuroscience

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Summary

这个视频协议演示,从成年小鼠脑室周围地区产生和扩大神经干细胞的神经球的含量测定方法,并提供技术见解,以确保可以实现重复性的神经球文化。

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Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay. J. Vis. Exp. (45), e2393, doi:10.3791/2393 (2010).

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Abstract

分离和扩展的假定从成年鼠大脑中的神经干细胞(NS​​Cs的)是在1992年首次描述了采用化学定义的无血清培养系统,被称为神经干法(NSA)的雷诺和Weiss。在这个实验中,多数分化的细胞类型文化的几天内死亡,但人口少的生长因子的反应前体细胞进行增殖活跃在表皮生长因子(EGF)和/碱性成纤维细胞生长因子(bFGF)的存在。这些细胞的未分化细胞,称为神经球,这反过来又可以传代培养的神经干细胞池扩大形成的殖民地。此外,细胞可以诱导分化,产生中枢神经系统,即神经元,星形胶质细胞和少突胶质细胞的三个主要类型的细胞。这个实验提供了一个宝贵的工具,提供一个一致的,可再生能源的未分化的中枢神经系统前体, 可用于在体外研究中,也为治疗目的。

本视频演示了NSA的产生和扩大,从成年小鼠脑室周围区域的神经干细胞的方法,并提供技术见解,以确保可以实现重复性的神经球文化。该过程包括收获从成年小鼠,脑室周围地区的微观解剖,组织编制和文化在NSA大脑。收获的组织是第一个使用在NSC培养基胰蛋白酶- EDTA和机械分离,实现了单细胞悬液,最后镀在NSA化学消化。经过7-10天文化,产生的初级神经球传代培养的准备,以达到未来的实验所需的细胞量。

Protocol

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Discussion

神经球的检测 2不仅获得了隔离和中枢神经系统 4-5的神经干细胞研究,但也是公认的从众多的组织干细胞 ,如乳房 6和心脏7,其他类型的隔离研究界的广泛关注和脑,乳腺癌和结肠癌肿瘤干细胞8-9表明,这种文化系统可应用于体细胞和肿瘤的整个身体的前体细胞群的鉴定。这种方法的一些优点,包括它的简单性,重复性和无限期数量从一小块组织,甚至在化学定义无血清培养细胞的少数细胞的生成。

应当强调,在培养的神经球数量并不代表干细胞的数量作为神经球,可无论是从真正的干细胞,或从更受限制的祖细胞 10派生。在这方面的检测已发展到正确枚举文化11中的神经干细胞。

使用不同的方法分解成单细胞悬液的神经球,包括机械和酶法。虽然机械方法是原始的神经干分解方法,它需要很多的经验和它的效率有所不同,取决于运营商。这种方法也可能造成重大的细胞死亡和破坏,如果一个没有经验的个人,它可以减少检测的准确性依赖于神经干离3的应用。胰蛋白酶EDTA酶解也有一些优点和缺点。酶法分解的神经球很容易,高效和可重复性,如果进行适当的时间长度和权利大小的神经球。虽然没有侧方神经球解离这两种方法的效果比较研究,我们的经验表明,一个简短的潜伏期(3-5分钟)的神经球(250微米),胰蛋白酶EDTA结果在一个高效率的解离不干扰他们的生存能力,增殖和分化能力。另一方面,长期暴露的神经球(特别是对于大型杂草丛生的神经球)胰蛋白酶EDTA可能与细胞表面受体,细胞消化和潜在的细胞死亡造成严重损害。过度胰蛋白酶EDTA也可能干扰领域的形成,并导致细胞附着到基板和区分。

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Disclosures

没有利益冲突的声明。

Acknowledgements

这项工作是从Overstreet基金会的资金支持。

Materials

Name Type Company Catalog Number Comments
NeuroCult NSC Basal Medium Medium Stem Cell Technologies 05700
NeuroCult NSC Proliferation Supplements Medium supplement Stem Cell Technologies 05701
%0.05 trypsin-EDTA Reagent GIBCO, by Life Technologies 25300-062
Soybean trypsin inhibitor Reagent Sigma-Aldrich T6522
Cell strainer Sieve BD Biosciences 352340
T25 flask Culture ware Nalge Nunc international 136196
T80 flask Culture ware Nalge Nunc international 178905
EGF Growth factor R&D Systems 2028-EG
Pen/Strep Reagent GIBCO, by Life Technologies 15140-122
*MEM Reagent GIBCO, by Life Technologies 41500-018 HEM component
*HEPES Reagent Sigma-Aldrich H4034 HEM component
*Distilled water Reagent GIBCO, by Life Technologies 15230-147
15 ml tubes Culture ware BD Biosciences 352096
50 ml tubes Culture ware BD Biosciences 352070
Fine curved forceps Surgical tools Fine Science Tools 11251‐35
Small fine forceps Surgical tools Fine Science Tools 11272‐30
Small forceps Surgical tools Fine Science Tools 11050‐10
b-FGF Growth factor R&D Systems 3139-FB
Heparin Growth factor Sigma-Aldrich H4784 Reconstituted in PBS

*To make HEM, mix 1×10L packet of MEM and160ml of 1M HEPES and bring the volume to 8.75 L using distilled water. Set the final PH to 7.4 and store it at 4°C.

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References

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  11. Louis, S. A. Enumeration of Neural Stem and Progenitor Cells in the Neural Colony Forming Cell Assay. Stem Cells. (2008).

Comments

1 Comment

  1. Please email me at azari.hassan@gmail.com if you have any questions regarding this assay.

    Reply
    Posted by: Hassan A.
    May 13, 2011 - 6:59 PM

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