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Neuroscienze

生活成像的脑皮层细胞使用二维培养系统

Published: August 09, 2017
doi:
10.3791/56063
, , , ,
1Brain Institute,Federal University of Rio Grande do Norte, 2Department of Biosystems Science and Engineering,ETH Zurich, 3The Solomon H. Snyder Department of Neuroscience,Johns Hopkins University

Summary

生动的图像是研究在实时的细胞行为的有力工具。在这里，我们描述延时视频-显微镜的脑皮层细胞，允许颁布在沿袭前进过程中主要的神经干细胞分化的神经元和神经胶质的阶段详细的审查的协议。

Abstract

大脑皮质发育过程中祖细胞经过几轮的对称和非对称细胞分裂生成新的祖细胞或分裂后神经元。后来，一些祖细胞切换到 gliogenic 的命运，将添加到的星形胶质细胞和少突胶质细胞的人口。使用延时视频显微镜的脑皮层细胞培养，有可能控制模式的细胞分裂和细胞周期参数的祖细胞的细胞和分子机制的研究。同样，命运的分裂后的细胞可以被审查，使用细胞特异性荧光记者蛋白质或后成像免疫细胞化学。更重要的是，所有这些功能，可以在单个细胞水平，允许致力于生成的特定类型的细胞的祖细胞的鉴定分析了。也可以使用病毒介导的基因转染，允许细胞自主和非细胞自主的现象的研究执行操纵基因的表达。最后，融合荧光蛋白的使用允许选定蛋白质过程划分与对比与女儿细胞命运的对称和非对称分布的研究。在这里，我们描述的延时视频显微镜方法图像几天到脑皮层小鼠细胞和细胞分裂、 细胞周期长度和新生成的细胞命运的模式进行了分析。我们还描述祖细胞，可以用于操纵基因的兴趣或简单标签与记者蛋白细胞转染的简便方法。

Introduction

神经干细胞 (NSC) 生成大脑皮质发育过程中神经元和星形细胞。在早期的-corticogenesis，神经干细胞经过几轮的对称的细胞分裂，并展开祖池。然后，神经干细胞发生不对称分裂生成神经元直接或间接通过中间体1。只有在中期-向晚-corticogenesis，祖细胞切换来生成星形胶质细胞和少突胶质细胞2，，34。然而，控制细胞增殖和分化，以及命运限制给代的独特类型的神经元或星形细胞祖细胞的贡献的完整机制仍然是激烈辩论4，，56。个人皮层神经干细胞生成神经元、 星形胶质细胞和少突胶质细胞的潜力已经广泛研究体外和体内使用大量的技术如： 生活成像在单个单元格文化7，8，9，10，11，，1213;生活影像在高密度文化文化3，，1415;生活影像在切片文化16，，1718;无性系分析使用病毒载体介导遗传标记在高密度文化14，15，19，，2021;无性系分析体内使用逆转录病毒22，23，24，25，26，27，28，29，30，31，，3233;与此同时，并用是克隆分析体内转基因动物34。

每个这些技术提出了利与弊。例如，在体内沿袭跟踪易发生混为一谈、 劈裂错误3，导致矛盾的结论，对潜在的个别皮质祖细胞。此外，在体外和体内研究基于标签祖细胞在早期的时间点和后的细胞谱系分析可能受到细胞死亡在沿袭进展35期间未检测到发生。因此，适当的系统，以分析单一神经干细胞的潜力必须允许鉴定所有细胞生成，以及细胞的命运，在世系的适当表征。原代细胞和实时成像的结合提供此设置。使用单一细胞培养和时间推移视频显微镜，寺庙等人有开关示宗族的个人大脑皮质祖细胞从神经到 gliogenesis11。后来，他们用于同一系统显示从单皮质祖12生成不同类型的神经元。然而，这一制度提出了一个重要的警告： 只有 1%的皮质祖细胞在早期 corticogenesis 孤立产生无性系 4 个或更多的后代9。后增加了 FGF2，细胞生成 4 或更多单元格的频率增加到 8-109。不过，这个数字是太小了，考虑到几乎所有皮质祖细胞增殖在这一阶段。此外，FGF2 对命运规范的潜在影响，也不能排除36。为了克服这些局限性，我们使用支持的两个心室扩散 （Pax6 表示） 的高密度细胞培养和室管膜下 （Tbr2 表示） 皮质祖细胞15。此外，这些文化的实时观察表明，NSC 血统进展的几个特征转载这些条件，如细胞分裂、 细胞周期延长、 潜力的单个细胞生成神经元和神经胶质，除其他外的模式下3，15。最近，我们有也用于此系统表明，CREB 信号影响小鼠37未成熟脑皮质神经元的细胞存活。因此，我们认为该延时视频显微镜的小鼠脑皮层细胞生长在高密度是强大和方便的工具，研究细胞和分子机制的细胞周期、 细胞分裂、 细胞存活和细胞命运规范模式。后者可以完成用转基因动物，允许特定细胞的命运，在真正的时间38，，3940鉴定或后影像学免疫细胞化学3，38，，4142使用。

在这里，我们提供一步一步的协议，准备支持的神经干细胞的增殖和对下一代的神经元和星形细胞的脑皮层细胞培养。我们还讨论了利用逆转录病毒介导基因转染来操纵基因表达的个体细胞，它可以在单个细胞水平使用时间推移视频显微镜跟踪和标识。这个协议可以用于研究脑皮层的细胞从一开始到结束了在啮齿类动物，corticogenesis 但须根据阶段14的几个调整。孤立的从其他来源的神经干细胞研究也可以使用时间推移视频显微镜的 2D 的文化，但适当的培养体系应由比较细胞行为在体外和体内38，43。

Protocol

All experiments involving live animals described in this protocol are conducted according to the National and International laws and were approved by the local University Animal Care and Use Committee (CEUA/UFRN), under the license 009/2014. The following protocol is performed in a sterile environment. Familiarity with basic cell culture is expected. 1. Dorsolateral Telencephalon Microdissection Prepare the dissection medium (100 mL): 98.5 mL Hank's Balanced Salt Solution (HBSS…

Representative Results

Primary cultures of cerebral cortex cells isolated from embryos E14 contain both progenitor and neuronal cells. During the period of imaging, progenitors undergo several rounds of cell division, increasing the number of cells (Figure 5 and Video Figure 1). Retroviral-mediated transfection of a few progenitor cells facilitates the identification of cell clones (F…

Discussion

脑皮层细胞的实时观察允许分析细胞的增殖、 细胞分裂、 细胞周期长度、 细胞分化和细胞生存3，14，，1537的模式。更重要的是，它允许单个细胞谱系，导致颁布了神经干细胞向神经元3过程的中间阶段的鉴定研究。最后，这个文化体系与生物工程工具来操纵基因表达组合是强大的?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作由 CNPq （委员会全国德日托 Científico 电子技术学校），披风 （也 de Aperfeiçoamento de Pessoal de Nível 高级） 和 FAPERN （该片 de Amparo 共同做格兰德北里）。

Materials

Hank's Balanced Salt Solution (HBSS) Invitrogen Life Technologies 14175129
HEPES Sigma-Aldrich H3375-25G
Penicillin/streptomycin Gibco 15140122
Dulbecco Modified Eagle's Medium (DMEM) Gibco 12400-024
Fetal Calf Serum (FCS) Gibco 10437028
Glucose Gibco A2494001
B27 Gibco 17504044
trypsin-EDTA (0.05%) Gibco 25300054
Paraformaldehyde Sigma 16005
Goat serum Sigma-Aldrich 69023
Triton X-100 VWR International Ltd. 306324N
Isoflurane Sigma 792632
anti-MAP2, mouse Sigma M4403
anti-GFP chicken Aves 0511FP12
DAPI Sigma D9542
Goat anti-mouse alexa 594 Invitrogen A11005
Goat anti-chicken alexa 488 Invitrogen A11039
ImageJ NIH
tTt ETH Zurich
Cell observer microscope Zeiss
Pasteur pipette
PBS
The Tracking Tool (tTt) software https://www.bsse.ethz.ch/csd/software/ttt-and-qtfy.html download link
DOWNLOAD MATERIALS LIST

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Tags

Keywords: Live ImagingPrimary Cerebral Cortex Cells2D Culture SystemNeural Stem CellsNeuronsGlial CellsNeurodevelopmentCell LineageCell DivisionCell CycleCell GrowthEmbryonic BrainDorsolateral TelencephalonCell DissociationTrypsin-EDTAProliferation MediumPoly-D-lysine
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Landeira, B. S., Araújo, J. A. d. M., Schroeder, T., Müller, U., Costa, M. R. Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System. J. Vis. Exp. (126), e56063, doi:10.3791/56063 (2017).
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