Abstract
Aim: Extracellular vesicles (EVs) released by mesenchymal stem cells (MSCs), known as MSC-EVs, have gained attention as potential treatments owing to their immunomodulatory functions. Despite growing clinical interest, donor-to-donor inconsistencies remain key challenges for standardizing MSC-EV production under good manufacturing practice (GMP) conditions. This work aimed to systematically compare the molecular and functional consistency of EVs derived from three independent human adipose tissue-MSC donors. Methods: GMP-grade EVs were initially isolated using tangential flow filtration on a large scale and then characterized by multiple biophysical analyses. To characterize the protein composition of EVs across batches, quantitative proteomic analysis was performed using tandem mass tags and mass spectrometry. For functional validation, an in vitro macrophage inflammation assay was conducted by treating natural lipopolysaccharide-stimulated cells with EVs, and cytokine levels were measured using enzyme-linked immunosorbent assays (ELISA). Results: Quantitative proteomic profiling identified 2,615 proteins, of which 84%-94% were not significantly changed across batches, highlighting a robust core proteome. Notably, 361 membrane-associated proteins were consistently conserved, including transporters, adhesion molecules, and signaling receptors, implicating these components in EV-mediated intercellular communication and immunomodulation. Functional analysis using an in vitro macrophage inflammation model demonstrated that all EV batches reproducibly suppressed pro-inflammatory cytokine production in a dose-dependent manner, with no significant inter-batch differences. Conclusion: Collectively, these findings indicate that MSC-EVs maintain both molecular and functional stability across different donors, and that a conserved proteomic signature underlies their reproducible anti-inflammatory activity. This study provides a foundation for establishing standardized quality criteria and advancing MSC-EVs toward clinical therapeutic applications.