NMR-based metabolomics of mammalian cell and tissue cultures
NMR-based metabolomics of mammalian cell and tissue cultures
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DOI：
10.1007/s10858-011-9490-8
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发表时间：
2011-04-01
影响因子：
2.7
通讯作者：
Reily, Michael D.
中科院分区：
文献类型：
--
作者：
Aranibar, Nelly;Borys, Michael;Reily, Michael D.
核磁共振波谱被用来评估生物医学研究中感兴趣的两个系统中的生长介质和细胞代谢体。其中第一个是中国仓鼠卵巢细胞系，该细胞系被改造为表达一种重组蛋白。在这里，核磁共振波谱和量子力学总线形分析被用来定量30种代谢物，如氨基酸、Krebs循环中间体、活性糖、辅因子等。生物反应器的规模和在培养液中添加抗凋亡剂对细胞外和细胞内代谢组的影响表明能量利用的代谢途径发生了变化。这些结果揭示了可以操纵的培养参数，以优化生长和蛋白质生产。其次，在药物代谢和毒理学研究中常用的模型--体外肝脏切片中，在灌流介质上进行代谢组学分析。在这项研究中，研究表明，48种标准介质成分中的两种，胆碱和组氨酸的消耗速度比许多其他营养物质都要快。在起始培养液中添加额外的胆碱和组氨酸可以改善肝脏切片的长期存活率，这是通过在孵育开始后94小时内较高的组织乳酸脱氢酶(LDH)、谷胱甘肽和三磷酸腺苷(ATP)水平以及较低的LDH水平来衡量的。在这两个模型中，随着时间的推移，介质成分和细胞代谢物被测量，并与当前接受的终点测量相关联。
NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.