Abstract

Extracellular vesicles (EVs), as critical mediators of intercellular communication, have emerged as promising biomarkers for disease diagnosis and therapeutics, making the acquisition of high-purity, high-yield EVs essential. However, traditional methods such as ultracentrifugation (UC) and magnetic-activated cell sorting (MACS), etc struggles to remove co-existing proteins in complex samples and faces limitations including low yield and high cost, restricting its large-scale clinical application. Hence, the development of a high-purity EVs preparation method is benefit for maximizing the biological functions of EVs and thereby replace targeted small molecule drugs for clinical applications. This study achieved high-purity isolation of umbilical cord mesenchymal stem cell-derived extracellular vesicles (UCMSC-EVs) using a combination of circular multicavity electrophoresis (CME) and ultrafiltration (UF). This method significantly improved EV purity and yield compared to conventional ultracentrifugation (UC), reducing protein contamination and enhancing the anti-tumor efficacy of UCMSC-EVs. Evaluation showed that CME-purified UCMSC-EVs maintained structural integrity and biological activity. High-purity UCMSC-EV therapy demonstrated greater safety and efficacy in inducing HepG2 cell apoptosis than sorafenib (SFB) therapy. Mechanistically, these purified EVs activate the p38 MAPK signaling pathway in hepatoma cells, promoting apoptosis. This method establishes a robust, universal platform for preparing high-purity extracellular vesicles (EVs) from diverse biological sources. It enables rapid, high-throughput separation and efficient purification through continuous operation, ensuring effective removal of contaminants. These capabilities offer substantial potential to advance both fundamental EV research and their clinical translation, significantly benefiting diagnostic applications and therapeutic development.