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

Optimizing bioreactor expansion of human neural progenitor cells for exosome scalable production and engineering | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimizing bioreactor expansion of human neural progenitor cells for exosome scalable production and engineering Miriam Corraliza-Gomez, Marta Ianni, Diogo E.S. Nogueira, William O.S. Salvador, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7284870/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Journal of Biological Engineering → Version 1 posted 13 You are reading this latest preprint version Abstract 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. Upscaled-exosome production Neural precursor cells Cell-free therapies Vertical-wheel bioreactors Stirred-tank bioreactors miRNA-124-engineered exosomes Paracrine signaling Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Journal of Biological Engineering → Version 1 posted Editorial decision: Revision requested 11 Sep, 2025 Reviews received at journal 11 Sep, 2025 Reviews received at journal 29 Aug, 2025 Reviews received at journal 22 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers agreed at journal 15 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers agreed at journal 13 Aug, 2025 Reviewers agreed at journal 12 Aug, 2025 Reviewers invited by journal 12 Aug, 2025 Editor assigned by journal 12 Aug, 2025 Submission checks completed at journal 12 Aug, 2025 First submitted to journal 03 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7284870","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501877645,"identity":"81b25158-762f-41f4-a789-d2c2985ed596","order_by":0,"name":"Miriam Corraliza-Gomez","email":"","orcid":"","institution":"Universidade de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"Miriam","middleName":"","lastName":"Corraliza-Gomez","suffix":""},{"id":501877646,"identity":"18cc32a9-f643-4bb7-8666-44c6b7568570","order_by":1,"name":"Marta Ianni","email":"","orcid":"","institution":"Universidade de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Ianni","suffix":""},{"id":501877647,"identity":"37d1ce19-8597-4147-a438-59d4890ba056","order_by":2,"name":"Diogo E.S. 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