Hamburg University of Technology, Bioprocess and Biosystems Engineering, Denickestr. 15, 21073, Hamburg, Germany.
Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre (UMC) Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany.
Anal Bioanal Chem. 2020 Apr;412(9):2065-2080. doi: 10.1007/s00216-020-02401-5. Epub 2020 Mar 4.
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.
哺乳动物细胞培养过程中的细胞群体异质性及其变化仍未得到很好的描述。在这项研究中，我们使用流式细胞术和基于慢病毒基因本体（LeGO）载体系统的稳定整合荧光标记物来研究细胞群体异质性的形成和动态变化。为了实现这一目标，使用不同的 LeGO 载体转导抗体产生 CHO 细胞，以稳定表达单个或多个荧光蛋白。这使得可以追踪转导群体，并在生物工艺工程领域的两个案例研究中进行了讨论：在案例研究 I 中，将细胞共转导以表达红色、绿色和蓝色荧光蛋白，并在高传代培养中（共 130 天）研究亚群和表达异质性的发展。观察到一个快速生长和更具生产力的群体的形成，同时细胞密度和产物滴度增加。在案例研究 II 中，使用流式细胞术（离线和自动）在混合分批培养中研究了不同预培养生长阶段及其对群体动态的影响。生成了四个表达不同荧光蛋白的细胞系衍生物，并在不同的时间间隔进行培养，对应于不同的生长阶段。从它们中接种混合培养物，并在培养的头 48 小时内观察到细胞群体组成的变化，导致过程生产力降低。总之，我们展示了如何描述群体异质性的动态变化。这代表了一种在哺乳动物细胞培养过程中研究接近生理条件下的细胞群体异质性动态变化及其生产力变化的新方法。
