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bioRxiv · 10.64898/2026.10.21.760728

Increased EV yield and bioactivity of matrix-bound nanovesicles compared to conditioned culture medium-derived extracellular vesicles

White, A.·Zhu, C.·Sundaram, V.·Levin, H.·Dewey, M. J.
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Abstract

Aim: Extracellular vesicles (EVs) are nanoparticles secreted by cells for cell-cell communication functions, such as wound repair, but low yields often limit their use. Several isolation methods have been explored to increase EV yield from conditioned culture medium (liquid-EVs); however, few studies compare isolation techniques and yield between liquid-EVs and matrix-bound nanovesicles (MBVs), a novel EV subtype embedded within the extracellular matrix. Methods: We isolate liquid-EVs and MBVs from mesenchymal stem cells (MSC) using size exclusion chromatography (SEC) and ultracentrifugation (UC) across various timepoints (14, 21, 24, 30 days). EVs are characterized by their yield, size, morphology, protein concentration, surface marker expression (CD9, CD81, ALIX), zeta potential, and influence on fibroblast wound closure. Results: We find that isolation method influences yield, where both SEC isolated liquid-EVs and MBVs yield higher particle counts and protein compared to UC. Interestingly, we find that regardless of isolation method, MBVs have a significantly higher yield than liquid-EVs from the same culture (15.6-fold higher using UC and 7.49-fold higher using SEC). Culturing for 24 days yielded the greatest number of MBV particles and protein. We performed a fibroblast scratch assay to assess bioactivity and found that liquid-EVs decreased wound closure compared to a medium control, whereas MBVs did not. Conclusion: Our work suggests SEC maximizes particle and protein yields for MSC-EVs. By achieving much higher yields of MBVs compared to liquid-EVs, and their promise as a wound repair strategy, we offer an alternative EV choice when EV yields are an issue towards translation.

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White, A., Zhu, C., Sundaram, V., Levin, H., Dewey, M. J.. 2026-10-23. Increased EV yield and bioactivity of matrix-bound nanovesicles compared to conditioned culture medium-derived extracellular vesicles. https://doi.org/10.64898/2026.10.21.760728

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