Role of Collection Media on the Biological Activity of Extracellular Vesicles From hTERT-Immortalised Mesenchymal Stromal Cells.

Extracellular Vesicles (EVs) from Mesenchymal Stromal Cells (MSCs) are promising cell-free therapeutics due to their ability to modulate immune responses, promote tissue repair and replicate many benefits of their parental cells. However, developing standardised, clinically translatable MSC-EV preparations remains difficult because of biological and technical variability. Factors such as cell source, donor differences, passage number and culture conditions affect EV yield and function, while enrichment methods, purity and media composition further complicate interpretation of EV-specific effects. Although prior studies have explored isolation methods and serum contaminants, the impact of different collection media on EV bioactivity is poorly understood. Here, we used hTERT-immortalised MSCs, which preserve the therapeutic properties of primary MSCs, to minimise cellular variability. EVs were collected in four different xeno-free media, enriched via Tangential Flow Filtration (TFF) and assessed in terms of their anti-inflammatory, anti-fibrotic, gap closure and proliferative potential compared with unconditioned, TFF processed collection media. Our results demonstrate that collection media significantly affect MSC-EV biological and functional properties, highlighting the need to carefully select media for standardised EV production. This study advances standardisation in EV research and supports the development of consistent, clinically relevant MSC-EV products.