Hernández Bort Juan A, Shanmukam Vinoth, Pabst Martin, Windwarder Markus, Neumann Laura, Alchalabi Ali, Krebiehl Guido, Koellensperger Gunda, Hann Stephan, Sonntag Denise, Altmann Friedrich, Heel Christine, Borth Nicole
ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria.
ACIB GmbH, Austrian Centre of Industrial Biotechnology, Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
J Biotechnol. 2014 Jul 20;182-183(100):97-103. doi: 10.1016/j.jbiotec.2014.04.014. Epub 2014 Apr 29.
In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration. In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10-15s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching.
为了保持细胞内代谢物水平,必须迅速执行淬灭方案,以避免不稳定代谢物在化学或生物学作用下发生降解。对于在复杂培养基中培养的哺乳动物细胞培养物,淬灭前的洗涤步骤是必要的,以避免细胞沉淀被细胞外代谢物污染,这可能会扭曲代谢物的真实细胞内浓度。这通常通过一个或多个离心/洗涤步骤来实现,这些步骤会延迟淬灭时间(即使是剧烈的离心也需要几分钟才能处理完毕直至细胞被淬灭),或者通过过滤来实现。在本文中,我们描述并评估了一种基于快速过滤的两步优化方案,该方案使用真空泵进行淬灭,并随后从CHO(中国仓鼠卵巢)悬浮细胞中提取细胞内代谢物,该方案使用的是市售组件。该方法能够在采样后10 - 15秒内将洗涤后的细胞转移到液氮中,并回收整个提取溶液体积。它还有一个优点,即能去除最终样品中的残留滤丝,从而防止在后续质谱分析过程中对分离柱造成损坏。相对于目前文献中使用的其他方法,细胞内腺苷核苷酸的能量电荷从冷PBS淬灭时的0.90或冷甲醇/AMBIC淬灭时的0.82提高到了0.94。