Wong Kevin, Ayuso-Sacido Angel, Ahyow Patrick, Darling Andrew, Boockvar John A, Wu Mingming
Biomedical Engineering Department, Cornell University, USA.
J Vis Exp. 2008 Feb 11(12):674. doi: 10.3791/674.
While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.
尽管微流控技术在大分子检测方面正达到一个新的成熟水平，但基于细胞的检测仍处于起步阶段。这主要是因为使用传统的微加工技术和材料难以创建与细胞兼容且稳定的微环境。我们通过引入一种新型微加工材料——琼脂糖凝胶作为微流控装置的基础材料来解决这个问题。琼脂糖凝胶具有高度的可塑性，并且能渗透细胞存活所需的气体和营养物质，因此是基于细胞检测的理想材料。我们之前已经表明基于琼脂糖凝胶的装置在研究细菌和中性粒细胞迁移方面取得了成功。在本报告中，在约1毫米厚的琼脂糖凝胶膜中构建了三条平行的微流控通道。使用蠕动泵在两侧通道中维持含有培养基/缓冲液的恒定流动。细胞置于中间通道进行观察。由于两侧通道中的营养物质和化学物质不断从侧面扩散到中间通道，通过沿两侧通道的流动可以轻松控制中间通道的化学环境。使用该装置，我们证明在各种化学条件下可以轻松地通过光学方式监测神经干细胞的运动，实验结果表明表皮生长因子受体（EGFR）的过表达增强了神经干细胞的运动性。神经干细胞的运动性是评估细胞侵袭性的重要生物标志物，因此也是致瘤因素。解读神经干细胞运动性的潜在机制将有助于深入了解神经发育障碍和脑癌干细胞侵袭。
