Ferry M S, Razinkov I A, Hasty J
Department of Bioengineering, University of California, San Diego, California, USA.
Methods Enzymol. 2011;497:295-372. doi: 10.1016/B978-0-12-385075-1.00014-7.
With the expanding interest in cellular responses to dynamic environments, microfluidic devices have become important experimental platforms for biological research. Microfluidic "microchemostat" devices enable precise environmental control while capturing high quality, single-cell gene expression data. For studies of population heterogeneity and gene expression noise, these abilities are crucial. Here, we describe the necessary steps for experimental microfluidics using devices created in our lab as examples. First, we discuss the rational design of microchemostats and the tools available to predict their performance. We carefully analyze the critical parts of an example device, focusing on the most important part of any microchemostat: the cell trap. Next, we present a method for generating on-chip dynamic environments using an integrated fluidic junction coupled to linear actuators. Our system relies on the simple modulation of hydrostatic pressure to alter the mixing ratio between two source reservoirs and we detail the software and hardware behind it. To expand the throughput of microchemostat experiments, we describe how to build larger, parallel versions of simpler devices. To analyze the large amounts of data, we discuss methods for automated cell tracking, focusing on the special problems presented by Saccharomyces cerevisiae cells. The manufacturing of microchemostats is described in complete detail: from the photolithographic processing of the wafer to the final bonding of the PDMS chip to glass coverslip. Finally, the procedures for conducting Escherichia coli and S. cerevisiae microchemostat experiments are addressed.
随着对细胞对动态环境反应的兴趣不断增加，微流控设备已成为生物学研究的重要实验平台。微流控“微恒化器”设备能够在捕获高质量单细胞基因表达数据的同时实现精确的环境控制。对于群体异质性和基因表达噪声的研究而言，这些能力至关重要。在这里，我们以我们实验室制造的设备为例，描述实验性微流控的必要步骤。首先，我们讨论微恒化器的合理设计以及可用于预测其性能的工具。我们仔细分析了一个示例设备的关键部分，重点关注任何微恒化器最重要的部分：细胞阱。接下来，我们提出了一种使用与线性致动器耦合的集成流体连接来生成片上动态环境的方法。我们的系统依靠静水压力的简单调节来改变两个源储液器之间的混合比例，并详细介绍了其背后的软件和硬件。为了提高微恒化器实验的通量，我们描述了如何构建更简单设备的更大的并行版本。为了分析大量数据，我们讨论了自动细胞跟踪的方法，重点关注酿酒酵母细胞所呈现的特殊问题。详细描述了微恒化器的制造过程：从晶圆的光刻处理到PDMS芯片与玻璃盖玻片的最终键合。最后，阐述了进行大肠杆菌和酿酒酵母微恒化器实验的步骤。
