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ACS Biomaterials Science & Engineering

ACS Biomaterials Science & Engineering

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EGF, TNF-α, and Hypoxia Preconditioning Enhances the Production and Therapeutic Effects of Mesenchymal Stem Cell-Derived Extracellular Vesicles for Regenerative Medicine

Published:27 August 2025 DOI: 10.1021/acsbiomaterials.5c00898 PMID: 40864985
Yin-Hsueh Chen, Xu Duan, Qing Nie, Li-Jun Li, Gang Chen*, Ye Li* and Hong-Yu Yang*, 

Abstract

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSC-EVs) are promising therapeutic agents for various diseases. However, current methods to improve MSC-EV production are insufficient to meet the clinical demands. Although various strategies have been investigated to enhance MSC-EV production, they are often hampered by limited scalability, loss of stemness, or suboptimal therapeutic outcomes. Our study identified three key stimulators that significantly boosted MSC-EV production: epidermal growth factor (EGF), tumor necrosis factor-α (TNF-α), and hypoxia. Employing an orthogonal design, we developed an optimized cell culture condition, subsequently referred to as ETH (EGF 10 ng/mL, TNF-α 50 ng/mL, and a hypoxic environment of 1% O2) preconditioning. This approach led to a remarkable 4- to 5-fold increase in MSC-EV yield while preserving the stemness of MSCs. Through proteomic analysis, we elucidated the underlying mechanisms of ETH preconditioning, providing insight into the complex processes driving enhanced MSC-EV production. Notably, MSC-EVs generated through ETH preconditioning demonstrated enhanced therapeutic potential including superior angiogenesis, collagen deposition, and regulation of inflammation. These findings present a scalable and effective strategy for elevating MSC-EV production, paving the way for its broader clinical application in regenerative medicine.

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