Isolation of therapeutic extracellular vesicles using nanoporous membranes with uniform nanopores.

Extracellular vesicles (EVs) are nanosized particles secreted by most cells for information transmission, which affects the microenvironment. EVs are known to follow the characteristics and conditions of their mother cells and have attracted considerable attention for disease diagnosis and therapeutic effects. In particular, mesenchymal stem cell (MSC)-derived EVs have shown potential for facilitating regenerative wound healing, modulating immune responses, and inhibiting inflammatory diseases. However, previous isolation methods demonstrated limited EV yield, purity, and filter capacity. Here, we report a two-step tangential flow filtration (TFF) system using track-etched membranes with uniform cylindrical nanopores for effectively isolating EVs with high purity and yield. Using two different uniform nanoporous track-etched membranes (50 and 200 nm), only the particles in the small EV (sEV) size range were separated through a size-exclusion mechanism. Comparative analysis with the existing ultrafiltration membrane-based TFF system revealed that the nanoporous membrane-based TFF (Nano-TFF) system exhibited a separation efficiency (yield) exceeding twofold, achieving sEVs purity surpassing 90%. The efficacy of the highly purified sEVs was validated by incorporating them into wound dressing material and applying them to a wound animal model. Notably, the sEVs-loaded wound dressing group demonstrated enhanced wound recovery compared to control groups. The Nano-TFF system, which provides precise separation and high efficiency, can be applied to separate various bioactive agents, including sEVs, that require high-purity isolation.