Effective Quenching Processes for Physiologically Valid Metabolite Profiling of Suspension Cultured Mammalian Cells
Effective Quenching Processes for Physiologically Valid Metabolite Profiling of Suspension Cultured Mammalian Cells
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DOI：
10.1021/ac8016899
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发表时间：
2009-01-01
影响因子：
7.4
通讯作者：
Dickson, Alan J.
中科院分区：
文献类型：
--
作者：
Sellick, Christopher A.;Hansen, Rasmus;Dickson, Alan J.
全球代谢物分析方法，加上复杂的数据分析和建模程序（代谢组学），可以动态读出细胞蛋白质如何与细胞和环境条件相互作用，以确定细胞状态。Ibis类型的方法对于理解和随后操纵细胞功能的调节具有深远的潜力。作为我们研究的一部分，以确定可用于最大限度地从培养的哺乳动物细胞中生产具有商业价值的重组蛋白的调控事件，我们优化了淬灭过程，以保留重组中国仓鼠卵巢（CHO）细胞样品中的生理相关细胞内代谢产物谱。在一系列候选淬灭程序的比较中，我们已经表明，在-40 ℃下在补充有0.85%碳酸氢铵（AMBIC）的60%甲醇中淬灭产生的代谢物谱代表了基于关键不稳定细胞代谢物检查的生理状态。这代表了悬浮培养哺乳动物细胞的任何代谢组学研究的关键特征，并为后续数据分析和建模程序的有效性提供了信心。
Global metabolite analysis approaches, coupled with sophisticated data analysis and modeling procedures (metabolomics), permit a dynamic read-out of how cellular proteins interact with cellular and environmental conditions to determine cell status. Ibis type of approach has profound potential for understanding, and subsequently manipulating, the regulation of cell function. As part of our study to define the regulatory events that may be used to maximize production of commercially valuable recombinant proteins from cultured mammalian cells, we have optimized the quenching process to allow retention of physiologically relevant intracellular metabolite profiles in samples from recombinant Chinese hamster ovary (CHO) cells. In a comparison of a series of candidate quenching procedures, we have shown that quenching in 60% methanol supplemented with 0.85% ammonium bicarbonate (AMBIC) at -40 degrees C generates a profile of metabolites that is representative of a physiological status based upon examination of key labile cellular metabolites. This represents a key feature for any metabolomic study with suspension cultured mammalian cells and provides confidence in the validity of subsequent data analysis and modeling procedures.