Department of Biomedical Engineering, University of Wisconsin-Madison, Wisconsin Institutes for Medical Research, 1111 Highland Avenue, Madison, Wisconsin 53705, United States.
Anal Chem. 2013 Feb 5;85(3):1562-70. doi: 10.1021/ac3027228. Epub 2013 Jan 17.
Microfluidics is emerging as a promising platform for cell culture, enabling increased microenvironment control and potential for integrated analysis compared to conventional macroculture systems such as well plates and Petri dishes. To advance the use of microfluidic devices for cell culture, it is necessary to better understand how miniaturization affects cell behavior. In particular, microfluidic devices have significantly higher surface-area-to-volume ratios than conventional platforms, resulting in lower volumes of media per cell, which can lead to cell stress. We investigated cell stress under a variety of culture conditions using three cell lines: parental HEK (human embryonic kidney) cells and transfected HEK cells that stably express wild-type (WT) and mutant (G601S) human ether-a-go-go related gene (hERG) potassium channel protein. These three cell lines provide a unique model system through which to study cell-type-specific responses in microculture because mutant hERG is known to be sensitive to environmental conditions, making its expression a particularly sensitive readout through which to compare macro- and microculture. While expression of WT-hERG was similar in microchannel and well culture, the expression of mutant G601S-hERG was reduced in microchannels. Expression of the endoplasmic reticulum (ER) stress marker immunoglobulin binding protein (BiP) was upregulated in all three cell lines in microculture. Using BiP expression, glucose consumption, and lactate accumulation as readouts we developed methods for reducing ER stress including properly increasing the frequency of media replacement, reducing cell seeding density, and adjusting the serum concentration and buffering capacity of culture medium. Indeed, increasing the buffering capacity of culture medium or frequency of media replacement partially restored the expression of the G601S-hERG in microculture. This work illuminates how biochemical properties of cells differ in macro- and microculture and suggests strategies that can be used to modify cell culture protocols for future studies involving miniaturized culture platforms.
微流控技术作为一种有前途的细胞培养平台正在兴起，与传统的宏观培养系统（如培养板和培养皿）相比，它能够更好地控制微环境并进行集成分析。为了推进微流控器件在细胞培养中的应用，有必要更好地了解微缩化如何影响细胞行为。特别是，微流控器件的表面积与体积比显著高于传统平台，导致每个细胞的培养基体积较小，从而导致细胞应激。我们使用三种细胞系（亲本 HEK（人胚肾）细胞和稳定表达野生型（WT）和突变型（G601S）人醚-a-去甲肾上腺素相关基因（hERG）钾通道蛋白的转染 HEK 细胞）在各种培养条件下研究细胞应激。这三种细胞系通过研究微培养中的细胞类型特异性反应提供了一个独特的模型系统，因为突变型 hERG 已知对环境条件敏感，使其表达成为比较宏观和微观培养的一个特别敏感的读数。虽然 WT-hERG 的表达在微通道和培养孔中相似，但突变 G601S-hERG 的表达在微通道中降低。内质网（ER）应激标志物免疫球蛋白结合蛋白（BiP）在所有三种细胞系中的微培养中均上调。通过 BiP 表达、葡萄糖消耗和乳酸积累作为读数，我们开发了减少 ER 应激的方法，包括适当增加培养基更换频率、降低细胞接种密度以及调整培养基的血清浓度和缓冲能力。事实上，增加培养基的缓冲能力或增加培养基更换频率部分恢复了 G601S-hERG 在微培养中的表达。这项工作阐明了细胞在宏观和微观培养中的生化特性如何不同，并提出了一些策略，这些策略可以用于修改未来涉及微型化培养平台的研究的细胞培养方案。
