Nguyen Vivian V T, Welsh Joshua A, Tertel Tobias, Choo Andre, van de Wakker Simonides I, Defourny Kyra A Y, Giebel Bernd, Vader Pieter, Padmanabhan Jayanthi, Lim Sai Kiang, Nolte-'t Hoen Esther N M, Verhaar Marianne C, Bostancioglu R Beklem, Zickler Antje M, Hong Jia Mei, Jones Jennifer C, El Andaloussi Samir, van Balkom Bas W M, Görgens André
Department of Nephrology and Hypertension, UMC Utrecht, Utrecht, The Netherlands.
Translational Nanobiology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
J Extracell Vesicles. 2024 Jun;13(6):e12463. doi: 10.1002/jev2.12463.
Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs - being small and non-living - are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.
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