Optimization of polyethylene glycol-based isolation of exosomes from mesenchymal stem cells for regenerative medicine applications

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BACKGROUND: Mesenchymal stem cells (MSCs) and their derived exosomes have gained significant attention in regenerative medicine due to their unique therapeutic properties, including immunomodulatory and regenerative capabilities. However, the development of a simple, scalable, and reproducible method for isolating exosomes from MSC-conditioned media remains a challenge. METHODS: We optimized a polyethylene glycol (PEG)-based precipitation method for exosome isolation by initially evaluating three different PEG molecular weights (1140, 3350, and 8000 Da). Protein quantification and nanoparticle tracking analysis (NTA) were performed for all three PEG types to assess yield and particle size. Based on these results, PEG 3350 and PEG 8000 were selected for further characterization by scanning electron microscopy (SEM), size exclusion chromatography (SEC), and Western blotting. Subsequently, proteomic and metabolomic analyses were conducted using exosomes isolated with PEG 3350. For functional assays, HeLa cells were exposed to increasing concentrations of MSC-derived exosomes under either 1% or 10% fetal bovine serum (FBS). Cell viability was evaluated at 24 h and 48 h using the the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. RESULTS: Our results demonstrated that PEG 3350 provided the highest yield and purity of MSC-derived exosomes. The isolated exosomes exhibited an average size below 200 nm, as confirmed by SEM and NTA. Western blotting validated the presence of exosome-specific markers CD63, CD9, and LGALS3BP, while liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified 357 proteins and 1,085 metabolites, confirming the molecular integrity of the exosomes. Additionally, SEC analysis revealed that repeated PEG-based enrichment cycles effectively improved exosome purity by removing non-vesicular contaminants. Functionally, exosomes isolated with PEG 3350 exhibited no cytotoxicity in HeLa cells and, at higher concentrations, promoted a condition-dependent increase in cell viability. CONCLUSIONS: This study presents an optimized PEG 3350-based method for exosome isolation, providing a scalable and reproducible approach for obtaining high-purity MSC-derived exosomes. These findings have significant implications for the development of exosome-based therapeutics in regenerative medicine.

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