A neuron-benign microfluidic gradient generator for studying the response of mammalian neurons towards axon guidance factors.
A neuron-benign microfluidic gradient generator for studying the response of mammalian neurons towards axon guidance factors.
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DOI：
10.1039/c0ib00038h
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发表时间：
2010-11
期刊：
影响因子：
--
通讯作者：
Folch A
中科院分区：
文献类型：
--
作者：
Bhattacharjee N;Li N;Keenan TM;Folch A
对细胞培养系统中分离或组合的生化信号的研究对于解开支配神经发育和修复的机制至关重要。在体外诱导轴突引导的最广泛使用的实验范式使用脉动吸管、转基因细胞或加载的凝胶作为梯度的来源，产生重复性差的随时间变化的梯度，这不适合于研究生长缓慢的哺乳动物细胞。虽然微流控装置的设计可以产生稳定、复杂的可扩散分子梯度，但微通道中流动诱导的剪切力使其不可能维持足够长的时间进行哺乳动物神经元培养。在这篇文章中，我们描述了一种基于开放式小室的“神经元良性”梯度发生器装置中小鼠皮质神经元的轴突反应，该装置可以在至少6小时内建立高度稳定的可扩散分子的梯度，而剪应力可以忽略不计，并允许神经元生长至少2周。除了梯度开启的时间段外，梯度中的细胞处于与控制表面上的细胞相同的条件下，这确保了一组一致的微环境变量。该设备在细胞培养表面实现的梯度稳定性和一致性，加上我们用于获取、后处理和定量分析大量图像的软件平台，使我们即使从小数据集中也能提取有价值的信息。我们报道了体外培养的原始哺乳动物神经元(取自E14胚胎小鼠皮质)对可扩散梯度的netrin的直接反应。我们从我们的研究中推断，在梯度应用过程中，绝大多数(~73%)延伸轴突的神经元向Netrin源生长，我们的数据分析也表明Netrin对同样数量的神经元起到了生长因子的作用。
Investigation of biochemical cues in isolation or in combinations in cell culture systems is crucial for unraveling the mechanisms that govern neural development and repair. The most widely used experimental paradigms that elicit axon guidance in vitro utilize as the source of the gradient a pulsatile pipette, transfected cells, or a loaded gel, producing time-varying gradients of poor reproducibility which are not well suited for studying slow-growing mammalian cells. Although microfluidic device design have allowed for generating stable, complex gradients of diffusible molecules, the flow-induced shear forces in a microchannel has made it impossible to maintain viable mammalian neuronal cultures for sufficiently long times. In this paper, we describe axonal responses of mouse cortical neurons in a “neuron-benign” gradient-generator device based on an open chamber that can establish highly stable gradients of diffusible molecules for at least 6 hours with negligible shear stress, and also allows the neurons to thrive for at least 2 weeks. Except for the period when the gradient is on, the cells in the gradient are under the same conditions as the cells on the control surfaces, which ensure a consistent set of micro-environmental variables. The gradient stability and uniformity over the cell culture surface achieved by the device, together with our software platform for acquiring, post-processing and quantitatively analyzing the large number of images allowed us to extract valuable information even from small datasets. We report a directed response of primary mammalian neurons (from E14 embryonic mice cortex) to a diffusible gradient of netrin in vitro. We infer from our studies that a large majority (~73%) of the neurons that extend axons during the gradient application grow towards the netrin source, and our data analysis also indicates that netrin acts as a growth factor for this same population of neurons.
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影响因子：
16.2
作者：
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通讯作者：
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