Abstract：
Cysteine is considered an essential amino acid in the cultivation of Chinese hamster ovary (CHO) cells. An optimized cysteine supply during fed-batch cultivation supports the protein production capacity of recombinant CHO cell lines. However, we observed that CHO production cell lines seeded at low cell densities in chemically defined media enriched with cysteine greater than 2.5 mm resulted in markedly reduced cell growth during passaging, hampering seed train performance and scale-up. To investigate the underlying mechanism, seeding cell densities and initial cysteine concentrations ranging from low to high cysteine concentrations were varied followed by an analysis of cell culture performance. Additionally, cell cycle analysis, intracellular quantification of reactive oxygen species (ROS) as well as transcriptomic analyses by next-generation sequencing were carried out. Our results demonstrate that CHO cells seeded at low cell densities at high initial cysteine concentrations encountered increased oxidative stress leading to a p21-mediated cell cycle arrest in the G1/S phase. The resulting oxidative stress caused redox imbalance in the endoplasmic reticulum and activation of the unfolded protein response as well as the major antioxidant nuclear factor-like 2 response pathways. Potential signature genes related to oxidative stress and the inhibition of the pentose phosphate pathway were identified in the study. Finally, the study presents that seeding cells at a higher concentration counteract oxidative stress in cysteine-enriched cell culture media.
摘要：
半胱氨酸被认为是中国仓鼠卵巢（CHO）细胞培养中的必需氨基酸。补料分批培养过程中优化的半胱氨酸供应支持重组CHO细胞系的蛋白质生产能力。然而,我们观察到CHO生产细胞系在低细胞密度下接种在富含半胱氨酸大于2.5mm的化学成分确定的培养基中导致传代期间细胞生长显著降低,阻碍种子训练性能和规模扩大。为了研究潜在的机制，改变接种细胞密度和从低到高半胱氨酸浓度的初始半胱氨酸浓度，然后分析细胞培养性能。此外,细胞周期分析,进行了活性氧（ROS）的细胞内定量以及通过下一代测序进行的转录组学分析。我们的结果表明，在高初始半胱氨酸浓度下以低细胞密度接种的CHO细胞遇到了增加的氧化应激，导致p21介导的细胞周期停滞在G1/S期。由此产生的氧化应激导致内质网中的氧化还原失衡和未折叠蛋白反应的激活以及主要的抗氧化核因子样2反应途径。研究中鉴定了与氧化应激和戊糖磷酸途径抑制相关的潜在特征基因。最后,该研究表明，在富含半胱氨酸的细胞培养基中，以更高的浓度接种细胞可以抵消氧化应激。
