Rapid production method with increased yield of high‐purity extracellular vesicles obtained using extended mitochondrial targeting domain peptide

Extracellular vesicles (EVs) are nano‐sized membranous structures involved in intercellular communication and various physiological and pathological processes. Here, we present a novel method for rapid (within 15 min), large‐scale production of high‐purity EVs using eMTDΔ4, a peptide derived from Noxa. The treatment of mesenchymal stem cells derived from human Wharton's jelly after trypsinization and subsequent eMTDΔ4 stimulation in a chemically defined sucrose buffer with orbital shaking led to a substantial increase (approximately 30‐fold) in EV production with markedly high purity (approximately 45‐fold). These EVs (TS‐eEVs) showed higher regenerative and immunomodulatory potential than natural EVs obtained from the culture media after 48 h. The calcium chelator BAPTA‐AM and calpain inhibitor ALLM, but not the natural EV biogenesis inhibitor GW4869, blocked the TS‐eEV production induced by eMTDΔ4, indicating that the eMTDΔ4‐mediated regulation of intracellular calcium levels and calpain activity are closely associated with the rapid, mass production of TS‐eEVs. The present study may lead to considerable advances in EV‐based drug development and production of stem cell‐derived EVs for cell therapy.