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

Abstract

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.

Lycium barbarum polysaccharide inhibits ischemia-induced autophagy by promoting the biogenesis of neural stem cells-derived extracellular vesicles to enhance the delivery of miR-133a-3p

“…EVs exhibited similar EV marker expressions without changing the size and purity. And surprisingly, we were able to obtain LBP-pretreated EVs more than 2-fold higher quantity than those collected under standard culture conditions, but not accompanied by damage to the vitality of the source cells, which is contrary to the previously reported methods, including physical and chemical stimulation [ 30 – 32 ]. Therefore, we proposed a feasible method to promote the reproducible generation of NSC-EVs, which may render EVs suitable for disease treatment.…”

Biomimetic Nano-Drug Delivery System: An Emerging Platform for Promoting Tumor Treatment

“… 209 , 210 Nonetheless, drug delivery systems using EVs remain restricted in industrial production due to their insufficient quantities. 211 …”

“…209,210 Nonetheless, drug delivery systems using EVs remain restricted in industrial production due to their insufficient quantities. 211 Recently, EVs have been widely used to design actively targeted biomimetic nanomedicines. Zhao et al 212 constructed biomimetic NPs by wrapping an exosome membrane derived from autologous breast cancer cells onto cationic bovine serum albumin coupled with siS100A4.…”

Thermostable Basic Fibroblast Growth Factor Enhances the Production and Activity of Human Wharton’s Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles

“…Exosomes are small extracellular vesicles released by various cell types, including MSCs, and are involved in cell-cell communication in MSCs. Exosomes are crucial mediators of cell-cell communication because these vesicles contain various bioactive molecules, such as proteins, nucleic acids, and lipids, which can be transferred to recipient cells to modulate their function [5,13,14]. Cell culture conditions can influence the exosome secretion levels and functionality, thus enhancing their production yield and quality by optimizing culture conditions such as growth media composition [15][16][17][18].…”

“…However, despite their versatile applications in various fields, obtaining a sufficient quantity of exosomes remains a limitation. Various research efforts are underway to achieve a sufficient yield of EVs [5,10,13].…”