Effect of Microculture on Cell Metabolism and Biochemistry: Do Cells Get Stressed in Microchannels?
Effect of Microculture on Cell Metabolism and Biochemistry: Do Cells Get Stressed in Microchannels?
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DOI：
10.1021/ac3027228
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发表时间：
2013-02-05
影响因子：
7.4
通讯作者：
Beebe, David J.
中科院分区：
文献类型：
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作者：
Su, Xiaojing;Theberge, Ashleigh B.;Beebe, David J.
微流体正在成为一个有前途的细胞培养平台，与传统的孔板和培养皿等大型培养系统相比，它能够增加微环境控制和集成分析的潜力。为了推进微流体装置在细胞培养中的应用，有必要更好地了解微型化如何影响细胞行为。特别是，微流体装置比传统平台具有更高的表面积体积比，导致每个细胞的介质体积更小，这可能导致细胞应力。我们使用三种细胞系：亲代HEK（人胚胎肾）细胞和转染的稳定表达野生型（WT）和突变型（G601S）人乙醚-go-go相关基因（hERG）钾通道蛋白的HEK细胞，研究了在各种培养条件下的细胞应激。这三种细胞系提供了一种独特的模型系统，通过它可以研究微培养中的细胞类型特异性反应，因为已知突变体hERG对环境条件敏感，使其表达成为比较宏观和微培养的特别敏感的读数。WT-hERG在微通道和孔培养中的表达相似，而突变体G601S-hERG在微通道中的表达降低。内质网（ER）应激标志物免疫球蛋白结合蛋白（BiP）在三种细胞系的微培养中表达上调。利用BiP表达、葡萄糖消耗和乳酸积累作为读数，我们开发了降低内质网应激的方法，包括适当增加培养基更换频率、降低细胞播种密度、调整血清浓度和培养基缓冲能力。确实，增加培养基的缓冲容量或更换培养基的频率可以部分恢复微培养中G601S-hERG的表达。这项工作阐明了细胞的生化特性在宏观和微培养中是如何不同的，并提出了可用于修改细胞培养方案的策略，以用于未来涉及小型化培养平台的研究。
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-typespecific 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.