Bioreactor expansion of human neural progenitor cells for exosome scalable production and miRNA-engineering.

Upscaling protocols to produce exosomes from human neural precursor cells (NPCs) are crucial for enabling broader therapeutic applications for neurodegenerative diseases with associated inflammation. Exosomes are small extracellular vesicles measuring between 30 and 150 nm in diameter that are emerging as promising delivery systems in cell-free therapies. An analysis of the US-NIH clinical trials database identifies 246 studies focused on exosome diverse applications, underscoring the growing importance of both naïve and engineered exosomes, specifically those enriched with miRNAs. NPC transplantation has faced several challenges, including immunogenicity and limitations associated with the ineffectiveness of single-dose administration. However, NPC exosomes are emerging as more promising therapeutic tools due to the possibility of multiple applications and their unique properties such as low immunogenicity, biocompatibility, ability to penetrate biological barriers and neuroregenerative properties. To tackle the challenge of producing large quantities of high-quality exosomes, our research used advanced three-dimensional cultivation techniques in vertical-wheel (PBS) and stirred-tank (DASbox) bioreactors. Bioreactor-upscaled ReNcell® VM human NPCs enhanced exosomal yield while maintaining essential stem NPC characteristics. DASbox bioreactor produced smaller, more uniformly sized neurospheres than the PBS system. DASbox-generated exosomes demonstrated superior transfection efficiency with pre-miR-124-3p, here used as a promising neuroprotective strategy, and improved microglia uptake than those from PBS or adherent cultures. Moreover, DASbox-derived exosomes were shown to be internalized by neurons and glial cells and to differently regulate inflammatory mediators upon stress conditions, while exerting better modulatory activity when transfected with pre-miR-124-3p. These results highlight the potential of exosomes from bioreactor-upscaled human NPCs as innovative therapeutic agents for targeting neuron-glia dyshomeostasis and dysfunctional miRNAs in neurodegenerative diseases, meeting the growing demand for their therapeutic application and supporting the development of more effective strategies.