Genome-scale metabolic analysis reveals enhanced metabolism and antioxidative stress response in perfusion cell culture under constant hyperosmotic stimulation

Hyperosmotic stimulation is a prevalent strategy to enhance cell culture productivity in fed-batch cultures. Maintaining stable hyperosmotic conditions during perfusion cultures presents a promising strategy, but research on its application in perfusion processes remains limited. In this study, we investigated cellular responses under constant hyperosmolality in Chinese hamster ovary (CHO) cell perfusion cultures using Raman spectroscopy to maintain a constant hyperosmotic environment. Integrated genome-scale metabolic model (GEM) with real-time monitoring of the oxygen uptake rate (OUR) was employed to systematically analyze the metabolic alterations induced by constant hyperosmotic stimulation. Our findings showed that the CHO cells exhibited time-dependent metabolic responses, with rapid changes in nutrient uptake, glycolysis, and TCA cycle activity, while lactate metabolism responded more slowly. The specific productivity (q_mAb) displayed the slowest changes, stabilizing only after 6–9 days upon the simulation, resulting in a maximum increase up to 168.5 %. Notably, we found shifts in the intracellular redox environment, and the cells enhanced their antioxidative capacity at low dissolved oxygen (DO) levels under hyperosmotic conditions. Even when DO dropped to 10 %, the cells subjected to hyperosmotic stimulation maintained relatively low levels of reactive oxygen species (ROS) while preserving high q_mAb. Overall, this study provides new insights into cellular responses under constant hyperosmotic condition and provides insights for the application of hyperosmotic strategies in perfusion processes.