Development of a Microfluidic System for Neuronal Differentiation Control

利用微机械加工技术，研制了由细胞培养腔、微通道、微阀和纳米孔阵列组成的微流控系统。在微通道中构造的微阀没有机械可移动部件，通过在亲水和疏水通道表面上使用不同的液体表面张力来气动驱动。利用聚焦离子束刻蚀技术，直接在腔体下方制备了用于释放神经生长因子(NGF)的纳米孔阵列。每个纳米孔的直径为500纳米，比轴突终末的直径还小。由于这些组件制造得非常接近，微阀开关允许精确控制NGF通过纳米孔释放到细胞培养室内。使用荧光溶液的实验表明，通过打开/关闭微阀，可以成功地控制通过纳米孔的化学释放。特别是，通过保持微阀打开直到浓度达到所需的值，然后以调整的占空因数重复开关微阀，即在一个开关周期中阀门打开的持续时间，可以保持细胞培养室内的化学浓度几乎恒定。此外，我们还在所研制的微流控装置上培养了PC12细胞(肾上腺嗜铬细胞瘤)，并成功地通过控制NGF的释放来引导神经元分化。结果表明，瓣膜转换的频率和占空比对细胞分化和轴突伸长有较大影响。这种神经元分化的动态控制技术有望应用于再生医学或损伤神经细胞的仿生医学。自2007年起，该研究项目继续作为科学研究资助项目(B)“神经干细胞诱导分化药物释放控制微装置的研制”继续进行。

A microfluidic system consisting of a cell-culture chamber, a microchannel, a microvalve, and a nano-hole array was fabricated using micromachining technology. The microvalve constructed in the microchannel has no mechanically movable parts and is pneumatically driven using different liquid surface tensions on hydrophilic and hydrophobic channel surfaces. The nano-hole array for release of nerve growth factor (NGF)which enhances neuronal differentiation was fabricated directly under the chamber using FIB (Focused Ion Beam)etching. Each nano-hole measures 500 nm in diameter, and is smaller than the diameter of an axonal terminal. Because these components were fabricated very close to one another, the microvalve switching allowed precise control of NGF release through the nano-holes to the cell-culture chamber. Experiments using a fluorescent solution showed that chemical release through the nano-holes was successfully controlled by opening/closing the microvalve. Especially, chemical concentration on the cell-culture chamber could be kept almost constant by keeping the microvalve open until the concentration reaches a required value and then switching the microvalve repeatedly at an adjusted duty factor that is the duration of valve being opened in one switching cycle. Also, we cultured PC12 cells (adrenal pheochromocytoma) on the fabricated microfluidic device, and succeeded in guiding neuronal differentiation by controlling NGF release. It was found that the cell differentiation and the axon elongation were affected greatly by the frequency and the duty factor of the valve switching. This dynamic control technique of neuronal differentiation is expected to be applied to regenerative medicine or bionic medicine for injured nerve cells. From 2007, this research project was continued to the Grant-in-Aid for Scientific Research (B) "Development of a drug-release controlling microdevice for differentiation induction of neural stem cells".