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1- Department of Neuroscience, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
2- Epilepsy Research Center, Department of Neurosurgery, Münster University, Münster, Germany
3- Multiple Sclerosis Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran
2- Epilepsy Research Center, Department of Neurosurgery, Münster University, Münster, Germany
3- Multiple Sclerosis Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran
Abstract:
Background: Exosomes, nano-scale extracellular vesicles, hold transformative potential in regenerative medicine and neurodegenerative disease treatment. However, inconsistent isolation methods, contamination risks, and lack of standardization impede clinical translation. This study introduces a protocol, guided by the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines, for isolating high-concentration exosomes from human umbilical cord mesenchymal stem cells (UC-MSCs).
Methods: UC-MSCs were expanded in alpha-MEM with 10% fetal bovine serum, followed by serum-free conditioning. Phenotypic characterization via flow cytometry confirmed CD90/CD44 positivity and CD45/CD11b negativity. Exosomes were isolated via differential centrifugation, filtration, and dual ultracentrifugation. Characterization was performed using transmission electron microscopy (TEM), dynamic light scattering (DLS), and bicinchoninic acid (BCA) assay, and Western blot analysis for CD9 and CD63, with calnexin as a negative control.
Results: TEM confirmed exosome integrity with spherical or cup-shaped morphology and intact bilayers. DLS showed a monodisperse population (121.3 ± 23.7 nm, PDI < 0.3) and stable zeta potential (−37.3 to −43.8 mV). The BCA assay quantified exosomal protein at 1098.2 μg/mL, surpassing conventional yields. Western blot confirmed expression of CD9 and CD63 and absence of calnexin, indicating minimal contamination.
Conclusions: This standardized, reproducible protocol produces therapeutic-grade UC-MSC exosomes with high structural fidelity and colloidal stability, aligning with MISEV criteria. While scalability remains a challenge, the method provides a critical foundation for translational studies. Future work should prioritize functional assays in neurodegenerative models, cargo profiling, and comparative analyses with other MSC sources. This study advances exosome research toward clinical-grade applications, bridging gaps in regenerative medicine and therapeutic development.
Methods: UC-MSCs were expanded in alpha-MEM with 10% fetal bovine serum, followed by serum-free conditioning. Phenotypic characterization via flow cytometry confirmed CD90/CD44 positivity and CD45/CD11b negativity. Exosomes were isolated via differential centrifugation, filtration, and dual ultracentrifugation. Characterization was performed using transmission electron microscopy (TEM), dynamic light scattering (DLS), and bicinchoninic acid (BCA) assay, and Western blot analysis for CD9 and CD63, with calnexin as a negative control.
Results: TEM confirmed exosome integrity with spherical or cup-shaped morphology and intact bilayers. DLS showed a monodisperse population (121.3 ± 23.7 nm, PDI < 0.3) and stable zeta potential (−37.3 to −43.8 mV). The BCA assay quantified exosomal protein at 1098.2 μg/mL, surpassing conventional yields. Western blot confirmed expression of CD9 and CD63 and absence of calnexin, indicating minimal contamination.
Conclusions: This standardized, reproducible protocol produces therapeutic-grade UC-MSC exosomes with high structural fidelity and colloidal stability, aligning with MISEV criteria. While scalability remains a challenge, the method provides a critical foundation for translational studies. Future work should prioritize functional assays in neurodegenerative models, cargo profiling, and comparative analyses with other MSC sources. This study advances exosome research toward clinical-grade applications, bridging gaps in regenerative medicine and therapeutic development.
Keywords: Exosomes, Mesenchymal stem cells, Ultracentrifugation, Regenerative medicine, Neurodegenerative diseases, Translational research
Type of Study: Original |
Subject:
Cellular and molecular Neuroscience
Received: 2025/05/30 | Accepted: 2025/07/30
Received: 2025/05/30 | Accepted: 2025/07/30
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