Bordag Natalie, Janakiraman Vijay, Nachtigall Jonny, González Maldonado Sandra, Bethan Bianca, Laine Jean-Philippe, Fux Elie
Metanomics GmbH, Berlin, Germany.
Biogen Idec Inc., Raleigh-Durham, North Carolina, United States of America.
PLoS One. 2016 Jul 20;11(7):e0159389. doi: 10.1371/journal.pone.0159389. eCollection 2016.
The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench) followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli) as well as mammalian cells chinese hamster ovary (CHO) and mouse myeloma cells (NS0).The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.
代谢组可实时检测生物体对环境变化、病理生理刺激或基因修饰的适应性多参数反应，从而使生物加工中细胞培养的优化合理化。在生物加工中，测量生理细胞内代谢物水平对于成功应用至关重要。然而，目前仍缺少一种适用于所有细胞类型、几乎无需验证且能同时提供高回收率、高代谢物覆盖率并充分去除细胞外污染物的采样方法。在此，对淬灭、离心和快速过滤进行了比较，发现快速过滤与稳定洗涤液相结合是最有前景的采样方法。对不同影响因素，如过滤器类型、真空压力、洗涤液等进行了全面测试。改进后的快速过滤方法（MxP®快速淬灭法）结合常规脂质/极性提取，可为不同细胞类型，如细菌（大肠杆菌）以及哺乳动物细胞中国仓鼠卵巢细胞（CHO）和小鼠骨髓瘤细胞（NS0）提供广泛的代谢物覆盖率和回收率，很好地反映生理细胞内代谢物水平。所提出的MxP®快速淬灭法可在不到30秒的时间内完成采样，即通过洗涤和淬灭将细胞与培养基分离，其设计坚固耐用，适用于所有细胞类型。洗涤液含有碳源或13C标记的碳源，以避免采样过程中的营养应激。该方法还与自动化兼容，可以进一步减少采样时间和代谢物谱数据的变异性。
