Production cell analysis and compound-based boosting of small extracellular vesicle secretion using a generic and scalable production platform

Extracellular vesicles (EVs) are a novel format of advanced therapeutical medicinal products (ATMPs). They can act regenerative or immune-modulatory as cell therapy substitutes or as a platform for designer exosomes. The biotechnological production of therapeutic EVs is still very much uncharted territory so standardized host cells, production setups, and isolation methods are not yet implemented. In this work, we present a tangential flow filtration (TFF) and fast-performance liquid chromatography (FPLC)-based size exclusion chromatography (SEC) purification setup that is compatible for industry applications. Moreover, we evaluated a series of potential host cell lines regarding their EV productivity, characteristics, and biological functionality. It was found that telomerase-immortalized Wharton's jelly mesenchymal stromal cells (WJ-MSC/TERT273) secrete high amounts of EVs per cell with regenerative capabilities. On the other hand, Cevec's amniocyte producer cells (R) (CAP (R)) and human embryonic kidney (HEK293) suspension cells are suitable platforms for designer EVs with high yields. Finally, we aimed to boost the EV secretion of HEK293 cells via chemical adjuvants and verified four compounds that heighten cellular EV secretion in a presumably cAMP-dependent manner. A combination of fenoterol, iodoacetamide, and dinitrophenol increased the EV yield in HEK293 cells threefold and cellular secretion rate fivefold.

Automated summary

This study presents a purification setup using tangential flow filtration (TFF) and fast-performance liquid chromatography (FPLC)-based size exclusion chromatography (SEC) suitable for industrial production of therapeutic extracellular vesicles (EVs). Potential host cell lines were evaluated, with telomerase-immortalized Wharton's jelly mesenchymal stromal cells (WJ-MSC/TERT273) found to secrete high amounts of regenerative EVs, and Cevec's amniocyte producer cells (R) (CAP (R)) and human embryonic kidney (HEK293) suspension cells suitable for designer EVs with high yields. Chemical adjuvants were used to enhance EV secretion in HEK293 cells, with the combination of fenoterol, iodoacetamide, and dinitrophenol increasing EV yield threefold and cellular secretion rate fivefold.