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培养物表现出均匀和无关的动力学行为。然而，真实的细胞培养物由具有可变组成的多个亚群的混合物组成，这些亚群通过培养基彼此相互作用。出现这种亚群的主要原因是细胞周期内单个细胞的进展。哺乳动物细胞培养物在不同细胞周期阶段的行为的潜在代谢和调节变化尚未在文献中进行系统研究。实验结果往往是不完整的和矛盾的，导致激烈的争议与一系列的意见，从假设完全无事件的代谢和调节的细胞周期对极其复杂的，而不是机械解释的细胞周期依赖性调节级联。造成这些差异的主要原因在于不完全验证或非生理同步方法和不充分的统计分析和适当的模型描述。在过去的几年中，我们小组已经建立了必要的框架，关于过程控制，细胞培养技术和建模，以进行系统的动力学和充分验证的细胞实验检查，在广泛不受干扰的条件下，细胞培养物中与周期相关的代谢过程和调节，即与过程相关的生理条件。第一次分析已经表明细胞周期依赖的细胞团特异性底物周转率的变化。在拟定项目中，应利用基于群体的分析和统计评价方法，系统阐明工艺条件下（尤其是高细胞密度下）细胞培养物中相关细胞周期特异性代谢过程。重点是特别奠定了丙酮酸代谢的调节动力学在应力条件下和高细胞密度对生长动力学和中央代谢的影响。此外，我们还打算探索使用这种基于群体的动力学关系的最佳控制的过程中的稳定性在可变的培养条件下的可能性。

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.