Abstract

Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth.

Key points

• Less and more efficient glucose utilization for suspension cell growth

• Concerted alteration of metabolic enzyme activity and protein expression

• Protein candidates to interfere glycolytic activity in MDCK cells

中文翻译：

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跟踪MDCK细胞悬浮生长适应性的变化：细胞生长与代谢物，酶和蛋白质的水平相关

摘要

动物细胞对悬浮培养中的生长的适应性特别涉及病毒疫苗的生产，在病毒疫苗的生产中，必须考虑到病毒与宿主细胞相互作用的非常特殊的方面，以实现高的细胞比产量和总体生产效率。到目前为止，对适应所需的代谢，酶和蛋白质组水平改变的复杂性了解甚少。在本研究中，我们首次结合了几种复杂的分析方法，旨在追踪不同水平的细胞变化并揭示相互联系和相关性。因此，在1-L生物反应器中培养了更适合悬浮生长的Madin-Darby犬肾（MDCK）悬浮细胞系。细胞浓度和细胞体积，细胞外代谢物浓度，测定细胞内酶活性。实验数据集用作隔离生长模型的输入，该模型已用于描述亲代粘附细胞系的生长动力学。另外，通过液相色谱法和串联质谱法使用无标记蛋白质定量方法分析细胞蛋白质组，以揭示悬浮液和粘附细胞系改变的细胞过程。四个调节机制被确定为对粘附的MDCK细胞适应悬浮生长的响应。这些调节机制与蛋白质caveolin，cadherin-1和pirin相关。结合细胞，代谢产物，酶，

关键点

•减少和更有效地利用葡萄糖来悬浮细胞生长

•代谢酶活性和蛋白质表达的协同变化

•候选蛋白质干扰MDCK细胞中的糖酵解活性