Hyperosmotic stimulation is a common strategy to boost cell culture productivity for batch and fed-batch cell cultures used in biomanufacturing. Continuous exposure to osmotic pressure, however, risks cell death in that environment. That risk appears to be mediated in perfusion cell cultures, though. In fact, recent research suggests continuous osmotic pressure is a viable strategy to boost cell line productivity in a continuous manufacturing environment.

Specifically, “maintain[ing] a constant hyperosmotic pressure during Chinese hamster ovary (CHO) cell perfusion culture led to a [maximum] 168.5% increase in specific productivity,” according to recent work reported by senior author Qian Ye, PhD, postdoctoral fellow, East China University of Science and Technology, and researchers from Shanghai Collaborative Innovation Center for Biomanufacturing Technology, Shanghai Austar Pharmaceutical Technology Equipment, and Shanghai Bioengine Sci-Tech Co. This implies these conditions may be used in continuous manufacturing environments with minimal risks to cell cultures.

Hypoxia reduced

Real-time monitoring of recombinant CHO cells in dense perfusion cell cultures shows that hyperosmotic conditions may actually reduce hypoxia. Ye and team reported “time-dependent metabolic responses, (and) rapid changes in nutrient uptake, glycolysis, and tricarboxylic acid cycle activity.” In contrast, specific productivity changed slowly, requiring six to nine days to stabilize after stimulation.

The benefits of hyperosmotic conditions remained even in low dissolved oxygen environments. “Cells enhanced their antioxidative capacity,” the researchers noted. “Even when dissolved oxygen dropped to 10%, the cells subjected to hyperosmotic stimulation maintained relatively low levels of reactive oxygen species while preserving high specific productivity,” the team reported.

The ability to thrive under such low oxygen levels makes the cells more robust, thus helping them continue to neutralize reactive oxygen species—harmful byproducts of cellular metabolism. Therefore, they appear to maintain high productivity under stress and thereby increase their therapeutic protein output.

“To optimize cell productivity under hyperosmotic stimulation while avoiding cell death, it is crucial to identify the appropriate osmotic pressure, tailored to the specific cell line,” the team cautions. For the CHO-DG44 cell line the team chose, optimal conditions appear to be an osmotic pressure of 380–410 mOsm/kg. Using that range, they increased specific cell productivity from 25.15 pg/cell/day to 41.81 pg/cell/day—an improvement of 66%.

In fed-batch cultures, exposing cells to continuous osmotic pressure often leads to the death of the cells, but in perfusion cultures, it leads to enhanced productivity. The difference in outcomes between these two types of cell cultures seems to be directly related to the nature of the cultures themselves. Perfusion cultures, the scientists point out, “benefit from the ongoing renewal” of the culture medium, which reduces the risks associated with osmotic pressure buildup.