Design and operation of a fully controlled platform for the production and purification of well-defined mesenchymal stromal cell (MSC)-derived extracellular vesicles

Abstract

Background & Aim Currently, the production of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC) is performed in laborious and time-consuming static culture systems using serum-containing media, and their purification is through ultracentrifugation-based methods. The whole process lacks on resolution, selectivity and capacity. Additionally, several differences were observed, in terms of the cargo of EVs, between EVs isolated from culture supernatants of MSC expanded under different culture conditions, stressing the importance of controlling all culture process parameters. A robust and in-depth process to manufacture MSC-EVs may provide a new therapeutic paradigm for cell-free MSC-based therapies. Methods, Results & Conclusion The main goal of this work is to develop a fully controlled manufacturing process for the production and purification of an effective, safe and fully characterized MSC-derived EVs-based product. We firstly established a spinner flask culture system combining microcarriers and xeno free culture medium for the expansion of human MSC. The established protocol was then implemented in a stirred tank bioreactor (1.3L working volume) yielding 3 × 108 MSC and approximately 1014 EVs after 8-10 days. In order to establish a scalable downstream process platform, we explore chromatography technique since it allows higher selectivity, reproducibility and cost-effectiveness. Nonetheless, the purification of these large biomolecules using chromatography remains a challenge due to low binding capacity and inability of working at higher flowrates using traditional packed-bed resins. In this work, we evaluated the performance of new chromatographic alternatives - gigaporous resins versus traditional agarose resins and the new-in-market CIMmultus™ EV monolith kit in the purification of MSC-EVs, by comparing the resolution and resin capacity by evaluating the recovery yields, and impurities removal. The quality of the final product obtained was characterized by different methods according to the minimal experimental requirements for characterizing EVs proposed by ISEV, including nanoparticle tracking analysis, western blot, Fourier-Transform InfraRed spectroscopy and lipidomics. The manufacturing platform to be established herein is expected to pave a new way for the development of stem cell-based engineering, eliminating time- and labour-consuming procedures aiming at the scalable production of EVs secreted from well-defined MSC populations in order to boost their medical uses.