Extracellular vesicles produced by a large-scale protocol are therapeutically effective in preclinical model of Parkinson's disease.

Abstract

BACKGROUND: Therapeutic applications require large amounts of extracellular vesicles (EVs) that cannot be obtained by standard laboratory protocols. Since culturing parameters and isolation methods can significantly affect the molecular composition and therapeutic efficacy of EVs, the development of a new scale-up protocol should be followed by the molecular fingerprinting and validation of therapeutic potential in vivo. METHODS: We developed a new scale-up protocol based on microcarrier culture (3D) of immortalized human dental pulp stem cells in a spinning bioreactor and subsequent isolation of EVs by 2-step tangential flow filtration (TFF) and size exclusion chromatography (SEC). RESULTS: A new scale-up protocol increased EV yields by 463-fold. When compared with ultracentrifugation (UC), isolation using TFF/SEC substantially reduced the complexity of proteomic cargo, whereas culture conditions (2D vs. 3D) affected miRNA, but not mRNA and proteomic content of the EVs. We next compared the therapeutic efficacy of both EV products in 6-hydroxydopamine rat model of Parkinson's disease (PD). The same amounts of EVs derived from standard 2D cultures by UC and a new large-scale protocol were intranasally administered to PD rats, where they similarly improved gait and cognitive functions, preserved nigrostriatal tyrosine hydroxylase density and suppressed neuroinflammation. Notably, both EV preparations were enriched in proteins and miRNAs associated with anti-oxidative and anti-inflammatory responses. CONCLUSION: Our protocol allows large-scale production of EVs that are therapeutically effective in the pre-clinical model of PD.