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首页> 外文会议>Cell culture engineering conference >DYNAMICS OF INTRACELLULAR METABOLITE POOLS IN MDCK SUSPENSION CELLS DURING GROWTH AND INFLUENZA VIRUS INFECTION
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DYNAMICS OF INTRACELLULAR METABOLITE POOLS IN MDCK SUSPENSION CELLS DURING GROWTH AND INFLUENZA VIRUS INFECTION

机译：生长和流感病毒感染期间MDCK悬浮细胞中的细胞内代谢池动力学

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Influenza virus infections are responsible for millions of flu cases with hundred thousands of deaths worldwide [1]. Additionally, pandemic outbreaks of aggressive influenza virus strains are very dangerous both for livestock and human population. Seasonal vaccination campaigns are in place to reduce infections, especially among young, old or immunodeficient individuals, generating a huge demand of 500 million (2015) vaccine doses every year [2]. Besides egg-based vaccine manufacturing, production platforms based on animal cell culture increasingly contribute to an overall growing market. Thus, the use of suspension MDCK cells (MDCKsus) cultivated in chemically defined medium emerges as a modern vaccine manufacturing platform. In order to improve overall productivity and reduce costs, process analysis, process optimization, and process intensification strategies are necessary. In particular, a better understanding of the effect of virus replication on cell growth, cell morphology and cell metabolism is crucial for developing production processes. In this study, the effect of a synchronous influenza A virus infection on cell growth and central carbon metabolism was investigated. Additionally, intracellular virus replication dynamics of influenza were analyzed and correlated to metabolic pool dynamics. For analysis of intracellular metabolites, an established HPLC-MS method was used to identify and quantify extracted metabolites [3]. A mathematical model, established for adherent MDCK cells, was modified to describe cell growth, consumption and production of main extracellular metabolites [4] as well as dynamics of intracellular metabolite pools of glycolysis and TCA. Our results showed fast infection (< 2 h) of the whole MDCKsus population under the used infection conditions. Intracellular infection was very similar to the already reported dynamics in adherent MDCK cells [5]. Virus particles were released six hours post infection (hpi) for 30 h, with an overall yield of 10,000 virus particles per cell. Despite massively impaired cell growth at 6 hpi, the concentrations of extracellular metabolites did not differ significantly from mock-infected cells used as a control. The majority of intracellular TCA metabolites also followed a similar dynamics. For glycolysis, however, metabolite pools of lower glycolysis decreased rapidly after infection, whereas glucose-6-P and fructose-6-P pools where maintained at a similar level as controls. Overall it seems that influenza infected MDCK cells show primarily an alteration in the glycolysis pathway, channeling metabolites not to the lower part of glycolysis but to the pentose phosphate pathway. Energy metabolism (ATP pools and energy charge) and TCA pools seemed not be affected by virus infection. Quantitative data for mock-infected cells are described by the mathematical model. Work is in progress to explain the dynamics observed in infected cells.
机译：流感病毒感染是导致全球数百万流感病例和数十万人死亡的原因[1]。另外，侵袭性流感病毒株的大流行暴发对牲畜和人口都非常危险。开展季节性疫苗接种运动以减少感染，尤其是在年轻人，老年人或免疫缺陷患者中，每年产生5亿（2015）剂疫苗的巨大需求[2]。除了以鸡蛋为基础的疫苗生产外，基于动物细胞培养的生产平台也为整个不断增长的市场做出了贡献。因此，在化学上确定的培养基中培养的悬浮MDCK细胞（MDCKsus）的使用成为一种现代疫苗生产平台。为了提高整体生产率并降低成本，必须进行过程分析，过程优化和过程强化策略。特别是，更好地了解病毒复制对细胞生长，细胞形态和细胞代谢的影响对于发展生产过程至关重要。在这项研究中，研究了同步性甲型流感病毒感染对细胞生长和中央碳代谢的影响。另外，分析了流感的细胞内病毒复制动态，并将其与代谢池动态相关。为了分析细胞内代谢物，已建立的HPLC-MS方法用于鉴定和定量提取的代谢物[3]。修改了建立用于粘附MDCK细胞的数学模型，以描述细胞生长，主要细胞外代谢物的消耗和产生[4]以及糖酵解和TCA的细胞内代谢物池的动力学。我们的结果表明，在使用的感染条件下，整个MDCKsus群体均被快速感染（<2小时）。细胞内感染与贴壁MDCK细胞中已报道的动力学非常相似[5]。感染后六个小时（hpi）释放病毒颗粒30小时，每个细胞的总产量为10,000个病毒颗粒。尽管在6 hpi时细胞生长受到严重损害，但细胞外代谢物的浓度与用作对照的模拟感染细胞没有显着差异。大多数细胞内TCA代谢物也遵循类似的动力学。但是，对于糖酵解，较低糖酵解的代谢产物库在感染后迅速减少，而葡萄糖6-P和果糖6-P库维持在与对照相似的水平。总的来说，流感感染的MDCK细胞似乎主要表现出糖酵解途径的改变，其代谢产物不是通过糖酵解的下部而是通过戊糖磷酸途径。能量代谢（ATP池和能量电荷）和TCA池似乎不受病毒感染的影响。数学模型描述了模拟感染细胞的定量数据。目前正在进行解释感染细胞中动态的工作。

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