High yield and scalable EV production from suspension cells triggered by turbulence in a bioreactor

Abstract

Background & Aim Extracellular vesicles (EVs) are membrane-delimited particles between 30nm and 500nm, released by all cells, which are biological actors of far-reaching intercellular communication. As EVs can recapitulate the properties of their mother cells, they offer a promising alternative to cell therapies, mitigating the risks of cell injection while presenting storage advantages. However, poor yield and scalability issues of EV manufacturing are major bottlenecks hindering the translation of EV-based therapies to clinics. In a groundbreaking strategy, our lab developed a high yield and scalable production method for EVs, increasing the classical agitation speed of an impeller to create a turbulent flow in a bioreactor. Adherent cells cultured on microcarriers were subjected to shear stress which triggered an active and massive EV shedding. These turbulence EVs from mesenchymal stem cells demonstrated the same regenerative potency as classical EVs produced by starvation in an in vivo model of chronic heart failure and featured anti-apoptotic and immunomodulatory properties in vitro. However, some cells, mostly derived from blood, are anchorage independent, our aim was thus to decipher if this method could also increase the EV release of non-adherent suspension cells. Methods, Results & Conclusion In a proof of concept, we have shown that THP-1, a human monocytic cell line, can produce 20 000 EVs/cell or more in three hours given that the size of the smallest vortices in the flow – calculated via the Kolmogorov equation – is below 30µm. This represents a tenfold increase compared to cells in starvation which produce up to 2000 EV/cell in 72h. By monitoring cell viability and cell lysis during production for three cell lines, we have shown that cells such as THP-1, HeLa and Raji react differently to shear stress and that a trade-off can be made between cellular damage and high EV yield. Isolated EVs were analyzed by flow cytometry and Exoview, exhibiting EV markers such as CD63 and CD81, as well as markers reflecting the nature of their mother cells. Our scalable and high yield approach extends EV manufacturing perspectives to suspension cell-derived EVs, in particular from immune cells derived from blood. Extracellular vesicles (EVs) are membrane-delimited particles between 30nm and 500nm, released by all cells, which are biological actors of far-reaching intercellular communication. As EVs can recapitulate the properties of their mother cells, they offer a promising alternative to cell therapies, mitigating the risks of cell injection while presenting storage advantages. However, poor yield and scalability issues of EV manufacturing are major bottlenecks hindering the translation of EV-based therapies to clinics. In a groundbreaking strategy, our lab developed a high yield and scalable production method for EVs, increasing the classical agitation speed of an impeller to create a turbulent flow in a bioreactor. Adherent cells cultured on microcarriers were subjected to shear stress which triggered an active and massive EV shedding. These turbulence EVs from mesenchymal stem cells demonstrated the same regenerative potency as classical EVs produced by starvation in an in vivo model of chronic heart failure and featured anti-apoptotic and immunomodulatory properties in vitro. However, some cells, mostly derived from blood, are anchorage independent, our aim was thus to decipher if this method could also increase the EV release of non-adherent suspension cells. In a proof of concept, we have shown that THP-1, a human monocytic cell line, can produce 20 000 EVs/cell or more in three hours given that the size of the smallest vortices in the flow – calculated via the Kolmogorov equation – is below 30µm. This represents a tenfold increase compared to cells in starvation which produce up to 2000 EV/cell in 72h. By monitoring cell viability and cell lysis during production for three cell lines, we have shown that cells such as THP-1, HeLa and Raji react differently to shear stress and that a trade-off can be made between cellular damage and high EV yield. Isolated EVs were analyzed by flow cytometry and Exoview, exhibiting EV markers such as CD63 and CD81, as well as markers reflecting the nature of their mother cells. Our scalable and high yield approach extends EV manufacturing perspectives to suspension cell-derived EVs, in particular from immune cells derived from blood.