Chung Henry H, Chan Charles K, Khire Tejas S, Marsh Graham A, Clark Alfred, Waugh Richard E, McGrath James L
Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA.
Lab Chip. 2014 Jul 21;14(14):2456-68. doi: 10.1039/c4lc00326h.
Microfluidic systems are powerful tools for cell biology studies because they enable the precise addition and removal of solutes in small volumes. However, the fluid forces inherent in the use of microfluidics for cell cultures are sometimes undesirable. An important example is chemotaxis systems where fluid flow creates well-defined and steady chemotactic gradients but also pushes cells downstream. Here we demonstrate a chemotaxis system in which two chambers are separated by a molecularly thin (15 nm), transparent, and nanoporous silicon membrane. One chamber is a microfluidic channel that carries a flow-generated gradient while the other chamber is a shear-free environment for cell observation. The molecularly thin membranes provide effectively no resistance to molecular diffusion between the two chambers, making them ideal elements for creating flow-free chambers in microfluidic systems. Analytical and computational flow models that account for membrane and chamber geometry, predict shear reduction of more than five orders of magnitude. This prediction is confirmed by observing the pure diffusion of nanoparticles in the cell-hosting chamber despite high input flow (Q = 10 μL min(-1); vavg ~ 45 mm min(-1)) in the flow chamber only 15 nm away. Using total internal reflection fluorescence (TIRF) microscopy, we show that a flow-generated molecular gradient will pass through the membrane into the quiescent cell chamber. Finally we demonstrate that our device allows us to expose migrating neutrophils to a chemotactic gradient or fluorescent label without any influence from flow.
微流控系统是细胞生物学研究的强大工具，因为它们能够在小体积内精确添加和去除溶质。然而，在细胞培养中使用微流控技术时固有的流体力有时并不理想。一个重要的例子是趋化系统，其中流体流动会产生明确且稳定的趋化梯度，但也会将细胞向下游推动。在此，我们展示了一种趋化系统，其中两个腔室由分子级薄（15纳米）、透明且纳米多孔的硅膜隔开。一个腔室是微流控通道，携带由流动产生的梯度，而另一个腔室是用于细胞观察的无剪切环境。分子级薄的膜对两个腔室之间的分子扩散几乎不提供阻力，使其成为在微流控系统中创建无流腔室的理想元件。考虑到膜和腔室几何形状的分析和计算流动模型预测，剪切力降低超过五个数量级。通过观察纳米颗粒在仅15纳米外的流动腔室中高输入流量（Q = 10 μL min⁻¹；vavg ~ 45 mm min⁻¹）情况下在细胞容纳腔室中的纯扩散，证实了这一预测。使用全内反射荧光（TIRF）显微镜，我们表明由流动产生的分子梯度将穿过膜进入静态细胞腔室。最后，我们证明我们的装置使我们能够使迁移的中性粒细胞暴露于趋化梯度或荧光标记，而不受流动的任何影响。
