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Neural stem cell-derived extracellular vesicles purified by monolith chromatography retain stimulatory effect on in vitro scratch assay

Colao, Ivano Luigi; Corteling, Randolph L; Bracewell, Daniel G; Wall, Ivan B; (2024) Neural stem cell-derived extracellular vesicles purified by monolith chromatography retain stimulatory effect on in vitro scratch assay. Cytotherapy 10.1016/j.jcyt.2024.11.007.

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Abstract

Background: Extracellular vesicles (EVs) have gained traction as potential cell-free therapeutic candidates. Development of purification methods that are scalable and robust is a major focus of EV research. Yet, there is still little in the literature which evaluates purification methods against potency of the EV product. Aims: In the present study, we examine two monolith chromatography methods with a focus on assessing the ability of purified EVs to retain stimulatory effects on firbroblasts to connect scalable purification methods with product outputs. Methods: We characterised EVs recovered from CTX0E03 (CTX) neural stem cell-conditioned medium in terms of biomarker distribution, functional capacity, and purity. We evaluated the ability of EVs to promote wound closure in an in vitro scratch assay prior to, and following two monolith chromatography steps (anion exchange, and hydrophobic interaction) to determine whether these options may better serve EV bioprocessing. Results: EVs from CTX cells were successful in initiating wound repair on a fibroblast scratch assay over 72 hours with a single 20μg dose. EV preparations presented the markers CD9, CD81, CD63, but also contained culture albumin and DNA as process impurities. EVs recovered by tangential flow filtration could be successfully purified further by both monolith chromatography steps. Post-monolith EV stimulation was conserved. Conclusion: The results indicate that monolith chromatography is a viable purification method for EVs derived from cell culture that doesn't detract from the product's ability to stimulate fibroblasts, suggesting that product functionality is conserved. Further work is needed in developing suitable downstream processes and analytics to achieve clinically relevant purities for injectable biologics. Key Words

Type: Article
Title: Neural stem cell-derived extracellular vesicles purified by monolith chromatography retain stimulatory effect on in vitro scratch assay
DOI: 10.1016/j.jcyt.2024.11.007
Publisher version: http://dx.doi.org/10.1016/j.jcyt.2024.11.007
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: Extracellular vesicles, Neural Stem Cells, Quaternary Amine, Hydrophobic Interaction, Large Scale, TFF, Scratch Assay
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10200835
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