High-yield production of extracellular vesicle subpopulations with constant quality using batch-refeed cultures

The conventional manufacturing of extracellular vesicles (EVs) is characterized by low yields and batch-to-batch variability, hampering fundamental research on EVs and their practical applications. Perfusion operations have huge potential to address these limitations and increase the productivity and quality of EVs.

Here, researchers from ETH Zurich simulate perfusion cultures with batch-refeed systems and compare their productivity with that achieved using batch cultures. They show that a shift from batch to batch-refeed system can increase the space-time yields of a target EV subpopulation characterized by CD81 and CD63 biomarkers by threefold. Moreover, the researchers demonstrate that the method facilitates the consistent production of the target EVs from cells maintained under constant conditions for 13 days. These results indicate that the use of perfusion cultures is a promising strategy to increase the manufacturing yield of EVs and control the production of specific EV subpopulations with constant quality attributes, thereby improving reproducibility.

Schematic of the experimental setup

For the production of EVs, perfusion cultures were simulated with batch-refeed cultures. A perfusion culture involves a bioreactor with a cell retention device. Under normal operations, the medium is constantly harvested, fresh medium is fed, and cells are bled to keep the cell culture conditions in the bioreactors stable. In contrast to batch cultures, high cell densities can be obtained in perfusion bioreactors, which can be maintained for several days. In a batch-refeed culture, cell bleed and medium exchange are not performed continuously but at regular intervals of time. In this study, cells were partially removed from the culture every 24 h, pelleted, resuspended in a fresh medium, and further incubated in shake flasks. This daily medium replacement allowed maintenance of a high density of viable cells over several days in batch-refeed operations. For the characterization of EVs, the samples were analyzed using nanoparticle tracking analysis, microfluidic diffusion sizing, and bead-based flow cytometry. The quantification of EVs in the harvested medium was performed using bead-based flow cytometry. The surface marker CD81 was used to capture EVs on the beads, and CD63 was used to stain the EVs. With this approach, we measured the production of CD81+/CD63+ EVs in the culture. Owing to the stable culture conditions and constant harvest of the product, the EV concentration in perfusion bioreactors remains constant over time. In contrast, in batch systems, EVs reach a maximum concentration in the culture and are eventually taken up by cells.