New approaches for population-based kinetic study and modeling of cell culture under high cell density

传统上认为，在生物反应器中培养的哺乳动物细胞表现出均一的和不同的动力学行为。然而，真正的细胞培养是由多个组成不同的亚群的混合物组成的，这些亚群通过培养介质相互作用。出现这种亚群的一个主要原因是单个细胞在细胞周期内的进展。在不同的细胞周期阶段，哺乳动物细胞培养的行为的潜在代谢和调节变化在文献中还没有系统的研究。实验结果往往是不完整和矛盾的，导致了激烈的争论，从细胞周期中完全没有事件的新陈代谢和调节的假设，到极其复杂和没有机制解释的细胞周期依赖的调节级联。造成这些差异的一个主要原因是不完全有效的或非生理的同步方法以及统计分析和适当的模型描述。在过去的几年中，我们团队已经建立了必要的过程控制、细胞培养技术和建模框架，以便在广泛未受干扰的情况下，对细胞培养中与细胞周期相关的代谢过程和调节进行系统的动力学和验证的实验检验，即过程相关的生理条件。首先，分析已经表明细胞质量比底物周转率随细胞周期而变化。在拟议的项目中，将利用基于群体的分析和统计评估方法，系统地阐明在工艺条件下，特别是在高细胞密度的条件下，细胞培养中相关的细胞周期特定代谢过程。重点介绍了胁迫条件下丙酮酸代谢的调控动力学以及高密度细胞对生长动力学和中枢代谢的影响。此外，我们还打算探索利用这种基于种群的动力学关系在不同栽培条件下对过程稳定性进行优化控制的可能性。

tIt is conventionally assumed that mammalian cell cultures cultivated in bioreactors exhibit a homogeneous and indifferent kinetic behavior. However, real cell cultures consist of a mixture of multiple subpopulations with variable composition that interact with each other via the culture medium. A major cause for the occurrence of such subpopulations is the progress of individual cells within the cell cycle. Potential metabolic and regulatory variations of the behavior of mammalian cell cultures during the different cell cycle phases have not been systematically examined in literature. Experimental results are often incomplete and contradictory, leading to intense disputes with a spectrum of opinions ranging from the assumption of completely eventless metabolism and regulation in the cell cycles towards extremely complex and not mechanistically explained cell-cycle dependent regulation cascades. A main reason for these discrepancies lies in incompletely validated or non-physiological synchronization methods and insufficient statistic analysis and proper model description.During the last few years, our group has established the necessary framework regarding process control, cell culture techniques and modeling in order to conduct systematic kinetic and well validated experimental examinations of cell-cycle related metabolic processes and regulations in cell cultures under widely undisturbed, that is process relevant physiological conditions. First analyses already indicated cell cycle dependent variations of cell mass specific substrate turnover rates. In the proposed project, a systematic elucidation of relevant cell cycle specific metabolic processes in cell cultures under process conditions, especially at high cell density, shall be performed utilizing population based analysis and statistic evaluation methods. The focus is especially laid on the regulation kinetics of the pyruvate metabolism under stress conditions and the influence of high cell density on growth kinetics and central metabolism. Furthermore, we intend also to explore the possibility of using such population-based kinetic relationships for optimal control of process stability under variable cultivation conditions.