A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues
A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues
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DOI：
10.1039/b713945d
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发表时间：
2008-01-01
期刊：
影响因子：
6.1
通讯作者：
Levchenko, Andre
中科院分区：
文献类型：
--
作者：
Wang, C. Joanne;Li, Xiong;Levchenko, Andre
神经元生长锥包含复杂的分子机制，精确地调节它们的迁移，以响应不同外部线索的复杂组合梯度。这项规定的细节在很大程度上仍然是未知的，部分原因是目前可用的实验技术的限制。微流体装置已被证明能够产生复杂、稳定和精确控制的化学梯度，但它们在研究生长锥迁移中的使用部分地由于剪切应力的影响而受到限制。在这里，我们描述了一种基于微流体的转向检测芯片，旨在克服这个问题。除了产生精确梯度的可溶性引导线索，芯片还可以制造复杂的复合梯度的扩散和表面结合的引导线索，模拟条件的生长锥实际上在体内计数。应用此检测爪蟾胚胎脊髓神经元，我们表明，表面结合层粘连蛋白梯度的存在下，可以微调的极性生长锥的反应（排斥或吸引）梯度的脑源性神经营养因子（BDNF），与指导结果依赖于平均BDNF浓度。在这种测定固有的灵活性，我们的神经系统发育和再生的理解的细化具有显着的潜力，并可以扩展到阐明其他细胞的过程，涉及趋化性的剪切敏感细胞。
Neuronal growth cones contain sophisticated molecular machinery precisely regulating their migration in response to complex combinatorial gradients of diverse external cues. The details of this regulation are still largely unknown, in part due to limitations of the currently available experimental techniques. Microfluidic devices have been shown to be capable of generating complex, stable and precisely controlled chemical gradients, but their use in studying growth cone migration has been limited in part due to the effects of shear stress. Here we describe a microfluidics-based turning-assay chip designed to overcome this issue. In addition to generating precise gradients of soluble guidance cues, the chip can also fabricate complex composite gradients of diffusible and surface-bound guidance cues that mimic the conditions the growth cones realistically counter in vivo. Applying this assay to Xenopus embryonic spinal neurons, we demonstrate that the presence of a surface-bound laminin gradient can finely tune the polarity of growth cone responses (repulsion or attraction) to gradients of brain-derived neurotrophic factor (BDNF), with the guidance outcome dependent on the mean BDNF concentration. The flexibility inherent in this assay holds significant potential for refinement of our understanding of nervous system development and regeneration, and can be extended to elucidate other cellular processes involving chemotaxis of shear sensitive cells.