Impact of flow cytometry-based sorting on microRNA signature of extracellular vesicles derived from mesenchymal stromal cells: a proof-of-concept study

Aim: This proof-of-concept study aimed to evaluate the impact of fluorescence-based sorting on the microRNA (miRNA) molecular profile of extracellular vesicles (EVs) derived from adipose-derived mesenchymal stromal cells (ASCs) (ASC-EVs), with a focus on osteoarthritis (OA) as a model disease. Methods: ASCs from five human donors were characterized by flow cytometry and cultured to collect conditioned media. EV isolation was performed by fluorescence-based sorting using a high-sensitivity cell sorter calibrated for particles ≥ 100 nm. In both ASC-EVs and sorted ASC-EVs (sASC-EVs), EV and MSC markers were analysed by flow cytometry, while size and morphology were assessed via nanoparticle tracking analysis and electron microscopy. Small RNA sequencing was used to profile miRNAs, followed by differential expression and functional enrichment analyses. Results: EVs displayed comparable size distributions and surface marker profiles before and after sorting. In contrast, small RNA sequencing revealed a marked reduction in the number of detectable miRNAs in sASC-EVs relative to EVs isolated by standard ultracentrifugation (285 vs. 749). Among these, 271 miRNAs were shared between groups, exhibiting a strong correlation in relative abundance and functional enrichment in angiogenesis, inflammation modulation and gene silencing pathways. Nevertheless, differential expression analysis identified 32 upregulated and 6 downregulated miRNAs in sASC-EVs, with 14 miRNAs detected exclusively after sorting. Despite these transcriptional differences, the overall balance between protective and detrimental OA-related miRNAs remained positive and was preserved across both ASC-EVs and sASC-EVs. Conclusion: While sorting reduces EV-associated miRNA diversity, it retains core functional signals. The difference in recovered miRNAs may be due to lower EV recovery, exclusion of miRNA-bound non-vesicular particles or loss of small EVs undetectable by fluorescence-based sorting techniques. Overall, these preliminary findings highlight a trade-off between purity and complexity, underscoring the importance of optimizing EV isolation protocols for clinical applications.