AUTHOR=Calascibetta Filippo , Martorana Annalisa , Lo Pinto Margot , Carcione Claudia , D’Arpa Salvatore , Amico Giandomenico , Miceli Vitale , Cuscino Nicola , Iannolo Gioacchin , Volpe Lorenzo , Scilabra Simone Dario , Conaldi Pier Giulio , Chinnici Cinzia Maria 

TITLE=GMP-compliant, serum-free cultures preserve therapeutic potential of extracellular vesicles from human mesenchymal stromal cells

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=Volume 13 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2025.1633912

DOI=10.3389/fcell.2025.1633912

ISSN=2296-634X

ABSTRACT=The therapeutic potential of extracellular vesicles (EVs) derived from human mesenchymal stromal cells (MSCs) is limited by the lack of standardized, Good Manufacturing Practice (GMP)-compliant production protocols. This study investigates the effects of MSC-Brew, a commercially available GMP-grade medium, on MSC-derived EVs in comparison to those produced in conventional cultures with DMEM supplemented with 10% fetal bovine serum (FBS). MSCs from adult dermis were successfully isolated and expanded in Brew medium while retaining their characteristic surface marker expression. MSC-EVs derived from Brew cultures met the Minimal Information for Studies of Extracellular Vesicles (MISEV) criteria, including particle size, concentration, marker expression, and minimal inflammatory cytokine content. Notably, Brew-EVs exhibited a significantly higher particle-to-protein ratio compared to EVs produced in FBS-containing cultures, indicating improved purity. Proteomic analysis revealed a largely conserved composition between Brew-EVs and conventionally produced EVs, and microRNA (miRNA) profiling identified only four differentially expressed miRNAs. Brew-EVs were enriched in anti-fibrotic miRNAs and effectively reduced collagen secretion in transforming growth factor (TGF)-β1-activated LX-2 cells, a human hepatic stellate cell line used as a model of liver fibrosis. These findings support MSC-Brew medium as a standardized, serum-free platform for the consistent production of high-quality EVs suitable for therapeutic applications.