Impact of flow cytometry-based sorting on microRNA signature of extracellular vesicles derived from mesenchymal stromal cells

Extracellular vesicles are tiny packages released by cells that carry molecular signals such as microRNAs, which help regulate inflammation, tissue repair, and disease processes. Vesicles produced by mesenchymal stromal cells are of particular interest because of their potential use in regenerative medicine and conditions like osteoarthritis.

Researchers at IRCCS Istituto Ortopedico Galeazzi, Italy examined how a commonly used purification step, fluorescence based flow cytometry sorting, changes the molecular cargo of extracellular vesicles derived from adipose mesenchymal stromal cells. The goal was to understand whether increasing vesicle purity comes at the cost of losing biologically important information.

The team isolated vesicles from human donor cells using two approaches. One relied on standard ultracentrifugation, while the other applied fluorescence based sorting designed to enrich vesicles larger than 100 nanometers. Physical measurements showed that vesicle size and surface markers were largely preserved after sorting, suggesting that the overall vesicle population remained intact.

The biggest differences emerged when the researchers examined microRNA content using small RNA sequencing. Sorted vesicles contained far fewer detectable microRNAs than unsorted preparations. Despite this reduction, most of the core microRNAs were shared between both groups and showed similar abundance patterns. These shared microRNAs were linked to pathways involved in blood vessel formation, immune modulation, and gene regulation, all relevant to osteoarthritis biology.

A smaller set of microRNAs changed in abundance after sorting, with some detected only in the sorted vesicles. Importantly, even with these shifts, the overall balance of microRNAs associated with protective effects in osteoarthritis was maintained. This suggests that while sorting narrows molecular diversity, it preserves key functional signals.

Overall, the findings highlight a trade off between vesicle purity and molecular complexity. For clinical applications that rely on extracellular vesicles as therapeutic agents or biomarkers, optimizing isolation methods will be critical to ensure that important biological information is not lost.