Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.
Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.
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DOI：
10.1371/journal.pone.0159389
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Fux E
中科院分区：
文献类型：
--
作者：
Bordag N;Janakiraman V;Nachtigall J;González Maldonado S;Bethan B;Laine JP;Fux E
代谢组学提供了生物体对环境变化、病理生理刺激或遗传修饰的适应性多参数响应的真实的时间检测，从而使生物加工中细胞培养物的优化合理化。在生物加工中，测量生理细胞内代谢物水平对于成功应用是必要的。然而，仍然缺少适用于所有细胞类型的采样方法，几乎没有验证工作，同时提供高回收率，高代谢物覆盖率和充分去除细胞外污染物。在这里，淬灭，离心和快速过滤进行了比较，快速过滤与稳定洗涤液相结合，被确定为最有前途的采样方法。对不同的影响因素如过滤器类型、真空压力、洗涤液等进行了综合试验。改进的快速过滤法（MxP® FastQuench），然后进行常规脂质/极性提取，可提供广泛的代谢物覆盖率和回收率，充分反映不同细胞类型（如细菌）的生理细胞内代谢物水平（大肠杆菌）以及哺乳动物细胞中国仓鼠卵巢（CHO）和小鼠骨髓瘤细胞（NS 0）。即，通过洗涤和淬灭从培养基中分离细胞，在小于30秒内完成，并且被稳健地设计为适用于所有细胞类型。洗涤溶液分别含有碳源和13 C标记的碳源，以避免取样期间的营养应激。该方法还与自动化兼容，这将进一步减少采样时间和代谢物分析数据的可变性。
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.