Richmond, K., Moore, M., Zhao, Y., Smyth, Lesley, Ayoub, S. and Nowocin, A. (2024) THE EFFECT OF DIFFERENT ISOLATION METHODS ON THE IMMUNOMODULATORY POTENCY OF EXTRACELLULAR VESICLES ISOLATED FROM AN IMMORTALISED MESENCHYMAL STROMAL CELL LINE. Cytotherapy, 26 (6). S86. ISSN 14653249

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Abstract

Background & Aim
Introduction: Mesenchymal stromal cells (MSCs) can influence immune cell behaviour through their secretome, comprising of extracellular vesicles (MSC-EVs) and other factors. Conclusions on the role and mechanism of action underlying MSC-EVs immunomodulation are hindered by their heterogeneity, which is linked to cell source as well as the EV bioprocessing. This study's aim was to develop an immortalised MSC line to allow the assessment of EV isolation methods on the EV phenotype and immune modulatory function. This will aid identifying critical quality attributes for enabling greater reproducibility when researching EV therapeutic products.

Methods, Results & Conclusion: Methods
To facilitate standardisation an immortalized MSC (ImmMSC) line, exhibiting a similar MSC phenotype and immunomodulatory potency to primary human bone marrow derived MSCs, was produced. To isolate EVs, supernatants collected from ImmMSCs underwent either Size-exclusion chromatography (SEC), differential ultracentrifugation (DUC) or ExoQuick-TC™ precipitation. Supernatants not subjected to these isolation methods, were used to assess the released secretome. EV yield and expression of specific EV markers obtained from the different isolation methods were assessed with nanoparticle tracking analysis and R-plex MSD assay, respectively. The immunomodulatory potency of the secretome and respective individual components, MSC-EV and MSC secreted proteins, were evaluated by measuring T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay.

Results
Different EV isolation methods resulted in varying yields of tetraspanin expressing nanoparticles. Of the isolation methods tested, EV-enriched fractions acquired using ExoQuick-TC™ or SEC showed significantly higher expression of EV markers than those isolated by DUC. Interestingly, we observed that although MSC-EVs inhibited T cell proliferation, it was the protein-enriched EV-free SEC fractions that suppressed T cell proliferation more effectively.

Conclusions
SEC or ExoQuick-TC™ were more efficient in isolating EVs than DUC. Secretome fractions lacking EVs supressed T cell proliferation better than the EVs, suggesting secretome proteins to be the more potent mediators of MSC immunomodulation.

Item Type:	Article
Identifier:	10.1016/j.jcyt.2024.03.161
Additional Information:	** Article version: AM ** Embargo end date: 22-05-2025 ** From Elsevier via Jisc Publications Router ** History: epub 22-05-2024; issued 30-06-2024. ** Licence for AM version of this article starting on 22-05-2025: http://creativecommons.org/licenses/by-nc-nd/4.0/
Subjects:	Medicine and health > Microbiology
SWORD Depositor:	Jisc Router
Depositing User:	Jisc Router
Date Deposited:	18 Sep 2024 05:30
Last Modified:	18 Sep 2024 05:30
URI:	https://repository.uwl.ac.uk/id/eprint/11950

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