Stress-Induced Premature Senescence in High Five Cell Cultures: A Principal Factor in Cell-Density Effects

The Baculovirus Expression Vector System (BEVS) is highly valued in vaccine development, protein engineering, and drug metabolism research due to its biosafety, operational convenience, rapid scalability, and capacity for self-assembling virus-like particles. However, increasing cell density at the time of inoculation severely compromises the production capacity of BEVS, resulting in the “cell density effect”. This study aimed to explore the mechanisms of the cell density effect through time-series analysis of transcriptomes and proteomes, with the goal of overcoming or alleviating the decline in productivity caused by increased cell density. The dynamic analysis of the omics of High Five cells under different CCI (cell density at infection) conditions showed that the impact of the “cell density effect” increased over time, particularly affecting genetic information processing, error repair, protein expression regulation, and material energy metabolism. Omics analysis of the growth stage of High Five cells showed that after 36 h of culture (cell density of about 1×106 cells/mL), the expression of ribosome-related proteins decreased, resulting in a rapid decrease in protein synthesis capacity, which was a key indicator of cell aging. Senescence verification experiments showed that cells began to show obvious early aging characteristics after 36 h, resulting in a decrease in the host cell’s ability to resist stress. Overexpression and siRNA inhibition studies showed that the ndufa12 gene was a potential regulatory target for restricting the “cell density effect”. Our results suggested that stress-induced premature senescence in High Five cell cultures, resulting in reduced energy metabolism and protein synthesis capabilities, was a critical factor contributing to cell density effects, and ultimately affecting virus production. In conclusion, this study provided new insights into managing virus production limitations due to cell density effects and offered innovative strategies to mitigate the adverse effects of cellular aging in biomanufacturing technologies. To explore the intrinsic mechanisms of the cell density effect, we performed a time-series analysis of transcriptomic and proteomics for the CCI1 and CCI3, including four time points during the cell culture stage (12 h, 36 h, 60 h, 84 h) and five time points during the virus infection stage (0 hpi, 6 hpi, 12 hpi, 24 hpi, 72 hpi).

杆状病毒表达载体系统（Baculovirus Expression Vector System, BEVS）凭借其生物安全性、操作便捷性、快速可扩展性以及自组装病毒样颗粒的能力，在疫苗研发、蛋白质工程与药物代谢研究领域备受青睐。然而，接种时细胞密度的升高会严重削弱BEVS的生产能力，进而引发“细胞密度效应”。本研究旨在通过转录组与蛋白质组的时序分析，探究细胞密度效应的内在机制，以期克服或缓解因细胞密度升高引发的生产力下降问题。
对不同感染前细胞密度（cell density at infection, CCI）条件下的High Five细胞进行组学动态分析后发现，“细胞密度效应”的影响随时间推移逐渐增强，尤其会对遗传信息加工、错误修复、蛋白质表达调控以及物质能量代谢产生显著影响。对High Five细胞生长阶段的组学分析显示，培养36小时后（细胞密度约为1×10^6 个细胞/毫升），核糖体相关蛋白的表达量出现下调，导致蛋白质合成能力快速下降，这是细胞衰老的关键指征。衰老验证实验表明，细胞在培养36小时后便开始显现明显的早期衰老特征，致使宿主细胞的抗胁迫能力降低。
过表达与小干扰RNA（small interfering RNA, siRNA）抑制实验证实，ndufa12基因是限制“细胞密度效应”的潜在调控靶点。本研究结果表明，High Five细胞培养过程中由胁迫诱导的过早衰老，会引发能量代谢与蛋白质合成能力下降，这是导致细胞密度效应、最终影响病毒生产的关键因素。综上，本研究为解决因细胞密度效应导致的病毒生产限制问题提供了全新视角，并为缓解生物制造技术中细胞衰老的不利影响提供了创新性策略。为进一步探究细胞密度效应的内在机制，本研究对CCI1组与CCI3组开展了转录组学与蛋白质组学的时序分析，涵盖细胞培养阶段的4个时间点（12 h、36 h、60 h、84 h）以及病毒感染阶段的5个时间点（0 hpi、6 hpi、12 hpi、24 hpi、72 hpi）。