Optimizing bioreactor expansion of human neural progenitor cells for exosome scalable production and engineering

Upscaling protocols to produce exosomes from human neural precursor cells (NPCs) is crucial for enabling broader therapeutic applications to neurodegenerative diseases with associated inflammation. Exosomes are small extracellular vesicles measuring between 30-150 nm that emerge as promising delivery systems in cell-free therapies. An analysis of the US-NIH clinical database identifies 246 studies focused on their diverse applications, underscoring the growing importance of both naïve and engineered exosomes, specifically those enriched with miRNAs. NPC transplantation has faced challenges that include immunogenicity and single administration limitation. However, their exosomes are emerging as promising therapeutic tools due to their unique properties like 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 originated smaller, more uniformly sized neurospheres than the PBS system. DASbox-generated exosomes demonstrated superior transfection efficiency with pre-miR-124-3p, here used as promising neuroprotective application, and better microglia uptake than those from PBS or adherent cultures. Moreover, DASbox-derived exosomes showed 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 complain with more effective strategies.