Scale-out production of extracellular vesicles derived from natural killer cells via mechanical stimulation in a seesaw-motion bioreactor for cancer therapy

Abstract

Extracellular vesicles (EVs) derived from immune cells have shown great anti-cancer therapeutic potential. However, inefficiency in EV generation has considerably impeded the development of EV-based basic research and clinical translation. Here, we developed a seesaw-motion bioreactor (SMB) system by leveraging mechanical stimuli such as shear stress and turbulence for generating EVs with high quality and quantity from natural killer (NK) cells. Compared to EV production in traditional static culture (229 ± 74 particles per cell per day), SMB produced NK-92MI-derived EVs at a higher rate of 438 ± 50 particles per cell per day and yielded a total number of 2 × 1011 EVs over two weeks via continuous dynamic fluidic culture. In addition, the EVs generated from NK-92MI cells in SMB shared a similar morphology, size distribution, and protein profile to EVs generated from traditional static culture. Most importantly, the NK-92MI-derived EVs in SMB were functionally active in killing melanoma and liver cancer cells in both 2D and 3D culture conditions in vitro, as well as in suppressing melanoma growth in vivo. We believe that SMB is an attractive approach to producing EVs with high quality and quantity; it can additionally enhance EV production from NK92-MI cells and promote both the basic and translational research of EVs.

Modularized dynamic cell culture platform for efficient production of extracellular vesicles and sequential analysis

“…31 As is well-known, EV production increases as cells are exposed to mechanical stimuli such as shear stress during cell culture. 14,15 Therefore, the production rate of EVs collected from the modular perfusion plate was compared with that of EVs produced from the static culture (Fig. 8).…”

“…To address this problem, researchers have investigated in vitro cell culture systems capable of producing EVs with high physiological relevance and yield. 11–15 Among several alternatives, perfusion cell culture is one of the methods that can reflect the physiological activities of EVs derived from cells with high production efficiency. 14,15 As the term ‘perfusion’ implies, inspired by the motion of body fluid flowing in the human body, the perfusion cell culture system continuously applies the fluid flow of culture media to cells during cultivation.…”

“…11–15 Among several alternatives, perfusion cell culture is one of the methods that can reflect the physiological activities of EVs derived from cells with high production efficiency. 14,15 As the term ‘perfusion’ implies, inspired by the motion of body fluid flowing in the human body, the perfusion cell culture system continuously applies the fluid flow of culture media to cells during cultivation. During the process, wall shear stress occurs due to the frictional force between the moving fluid and surface of adherent cells.…”

“…Thus, external stimuli applied to cells affect the changes in cell characteristics, similar to those in vivo , thereby resulting in the secretion of EVs that reflect physiological activity with high yield. 14–18 Indeed, there exist two major methods in perfusion: circulating and non-circulating. The circulating method of perfusion is advantageous in concentrating EVs during cell culture as it circulates the culture medium in the chamber during the culture period.…”

Advances of multi-omics applications in hepatic precancerous lesions and hepatocellular carcinoma: The role of extracellular vesicles

“…These results indicate that DEX has a great application prospect as a cell-free tumor vaccine or a natural antitumor adjuvant, opening up a new avenue for HCC immunotherapy. Moreover, EVs derived from other immune cells such as natural killer cells (NK) have also shown specific tumor-killing effects on HCC cells ( Kim et al, 2022 ); Wu J. et al (2022) have developed a motion bioreactor that can produce NK EVs on a large scale, which provides technical support for promoting the clinical translation of EVs. The cytotoxic effect of immune cell-derived EVs on tumors greatly enriches the role of EVs in cell-free immunotherapy.…”