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Quantification of the dynamics of population heterogeneities in CHO cultures with stably integrated fluorescent markers
Analytical and Bioanalytical Chemistry ( IF 4.3 ) Pub Date : 2020-03-04 , DOI: 10.1007/s00216-020-02401-5
Johannes Möller 1 , Marcel Rosenberg 1 , Kristoffer Riecken 2 , Ralf Pörtner 1 , An-Ping Zeng 1 , Uwe Jandt 1
Affiliation  

Abstract

Cell population heterogeneities and their changes in mammalian cell culture processes are still not well characterized. In this study, the formation and dynamics of cell population heterogeneities were investigated with flow cytometry and stably integrated fluorescent markers based on the lentiviral gene ontology (LeGO) vector system. To achieve this, antibody-producing CHO cells were transduced with different LeGO vectors to stably express single or multiple fluorescent proteins. This enables the tracking of the transduced populations and is discussed in two case studies from the field of bioprocess engineering: In case study I, cells were co-transduced to express red, green, and blue fluorescent proteins and the development of sub-populations and expression heterogeneities were investigated in high passage cultivations (total 130 days). The formation of a fast-growing and more productive population was observed with a simultaneous increase in cell density and product titer. In case study II, different preculture growth phases and their influence on the population dynamics were investigated in mixed batch cultures with flow cytometry (offline and automated). Four cell line derivatives, each expressing a different fluorescent protein, were generated and cultivated for different time intervals, corresponding to different growth phases. Mixed cultures were inoculated from them, and changes in the composition of the cell populations were observed during the first 48 h of cultivation with reduced process productivity. In summary, we showed how the dynamics of population heterogeneities can be characterized. This represents a novel approach to investigate the dynamics of cell population heterogeneities under near-physiological conditions with changing productivity in mammalian cell culture processes.



中文翻译:

使用稳定整合的荧光标记定量CHO培养中种群异质性的动力学

摘要

细胞群体异质性及其在哺乳动物细胞培养过程中的变化仍未得到很好的表征。在这项研究中,基于慢病毒基因本体(LeGO)载体系统,使用流式细胞仪和稳定整合的荧光标记物研究了细胞群体异质性的形成和动力学。为此,用不同的LeGO载体转导产生抗体的CHO细胞以稳定表达单个或多个荧光蛋白。这使得能够跟踪转导的种群,并在生物过程工程领域的两个案例研究中进行了讨论:在案例研究I中,细胞被共转导以表达红色,绿色和蓝色荧光蛋白,以及亚群和在高传代培养中(总共130天)研究了表达异质性。观察到快速增长和高产种群的形成,同时细胞密度和产物滴度增加。在案例研究II中,使用流式细胞仪(离线和自动)研究了混合分批培养中不同的预培养生长期及其对种群动态的影响。产生四种细胞系衍生物,每种表达不同的荧光蛋白,并在不同的时间间隔(对应于不同的生长阶段)下进行培养。从其中接种混合培养物,并在培养的前48小时内观察到细胞群组成的变化,降低了生产效率。总之,我们展示了如何表征种群异质性的动态。

更新日期:2020-03-04
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