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Background: Mesenchymal stem cells (MSCs)-derived exosomes can function similar to MSCs in repairing damaged tissues and modulating immune responses. They are considered as an effective tool in regenerative medicine as well as autoimmune diseases and cancer. Methods: MSCs were isolated from adipose tissue of mice, and characterized. Cell supernatant was used for extraction of exosomes using ultracentrifugation with 110000 g and polyethylene glycol (PEG). Dynamic light scattering (DLS) technique, transmission electron microscopy, and Bradford assay were used to evaluate the accuracy of the isolated exosomes. Findings: The results of isolation of exosomes derived from MSCs using PEG showed that most of the extracted spherical exosomes were in the range of 50-300 nm; but the results of isolation of exosomes derived from MSCs using ultracentrifuge showed the range of 30-100 nm. Using Bradford test, the concentration of exosomes extracted was recorded as 1296.7 mg/ml by ultracentrifugation and 1322.4 mg/ml by PEG. Conclusion: Comparison of two methods of separation of exosomes showed that the extracted exosomes were more purified by ultracentrifugation; but in PEG method, the exosomes were larger and less purified due to PEG deposition on the particles. However, because of less access to ultracentrifuge, PEG separation is also applicable.