(157r) Analyzing the Role of Chinese Hamster Ovary Extracellular Vesicles (CHO-EVs) in Extracellular Communication, Cellular State, and Protein Expression of CHO Cultures

Chinese hamster ovary (CHO) cells are the primary host cell line for producing protein therapeutics, resulting in the need to better understand and improve CHO cell culture conditions to increase protein production and quality. CHO cells dynamically produce and uptake extracellular vesicles (EVs) in culture to exchange small RNAs, protein material, and small DNA fragments. This exchange of protein and genetic material dynamically regulates the cellular state and protein expression of the target cell. Here, CHO cell derived EVs (CHO-EVs) harvested from various shake flask cultures are characterized in terms of size distribution, morphology, zeta potential, and RNA content. CHO-EVs are separated by differential ultracentrifugation into the heavier EV fraction and in the lighter EV fraction and the size distribution is measured by nanoparticle tracking analysis (NTA). RNA fragment analysis shows CHO-EVs are enriched in small RNAs compared to the parent cells, indicating the CHO-EVs are selectively sorting material into the CHO-EVs to regulate protein expression in target cells. Using RNA sequencing (RNA seq), the RNA profiles of the CHO-EVs were compared to the RNA profile of the CHO cells to determine which RNAs are highly enriched in CHO-EVs. To visualize the dynamic production, protein and genetic material exchange, and uptake of CHO-EVs in shake flask cultures, non-specific protein dyes (carboxyfluorescein succinimidyl ester (CFSE) and CellTracker Deep Red) were used in conjunction with correlative confocal microscopy and scanning electron microscopy (SEM). Correlating confocal images with SEM images showed the localization of exchanged CHO-EVs with the high-resolution imaging of CHO cell morphology. Additionally, flow cytometry and confocal microscopy were used to determine the proportion of CFSE and Deep Red cells in culture and the exchange of fluorescently stained CHO-EVs in culture. The characterization of CHO-EVs produced in shake flask cultures elucidates the native cell communication and material exchange mechanisms via EVs. Future steps in expanding the characterization and application of CHO-EVs include loading specific genetic cargo, such as anti-apoptotic small interfering RNAs (siRNAs), to deliver in CHO cultures to prolong CHO culture health and lifespan.