Isolation and Characterization of Mesenchymal Stem Cells-Derived Exosomes by Method of Ultracentrifuge and Polyethylene Glycol

Document Type : Original Article (s)

Authors

1 PhD Student, Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

2 Professor, Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

3 Assistant Professor, Department of immunology, School of medical sciences, Tarbiat Modares University, Tehran, Iran

4 Assistant Professor, Cell-Based Therapies Research Center, Digestive Disease Research Institute, Tehran University of Medical Sciences, Tehran, Iran

Abstract

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.

Keywords

References

  1. Li P, Kaslan M, Lee SH, Yao J, Gao Z. Progress in exosome isolation techniques. Theranostics 2017; 7(3): 789-804.
  2. Batrakova EV, Kim MS. Development and regulation of exosome-based therapy products. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2016; 8(5): 744-57.
  3. Egea-Jimenez AL, Zimmermann P. Lipids in exosome biology. Handb Exp Pharmacol 2019. [Epub ahead of print].
  4. Shimasaki T, Yamamoto S, Arisawa T. Exosome research and co-culture study. Biol Pharm Bull 2018; 41(9): 1311-21.
  5. Jeppesen DK, Fenix AM, Franklin JL, Higginbotham JN, Zhang Q, Zimmerman LJ, et al. Reassessment of exosome composition. Cell 2019; 177(2): 428-45.
  6. Mincheva-Nilsson L, Baranov V, Nagaeva O, Dehlin E. Isolation and characterization of exosomes from cultures of tissue explants and cell lines. Curr Protoc Immunol 2016; 115(1): 14.
  7. Ludwig N, Hong CS, Ludwig S, Azambuja JH, Sharma P, Theodoraki MN, et al. Isolation and analysis of tumor-derived exosomes. Curr Protoc Immunol 2019; 127(1): e91.
  8. Helwa I, Cai J, Drewry MD, Zimmerman A, Dinkins MB, Khaled ML, et al. A comparative study of serum exosome isolation using differential ultracentrifugation and three commercial reagents. PLoS One 2017; 12(1): e0170628.
  9. Chang M, Chang YJ, Chao PY, Yu Q. Exosome purification based on PEG-coated Fe3O4 nanoparticles. PLoS One 2018; 13(6): e0199438.
  10. Rider MA, Hurwitz SN, Meckes DG, Jr. ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles. Sci Rep 2016; 6: 23978.
  11. Lai RC, Chen TS, Lim SK. Mesenchymal stem cell exosome: A novel stem cell-based therapy for cardiovascular disease. Regen Med 2011; 6(4): 481-92.
  12. Boelens MC, Wu TJ, Nabet BY, Xu B, Qiu Y, Yoon T, et al. Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways. Cell 2014; 159(3): 499-513.
  13. Yang T, Martin P, Fogarty B, Brown A, Schurman K, Phipps R, et al. Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio. Pharm Res 2015; 32(6): 2003-14.
  14. O'Loughlin AJ, Woffindale CA, Wood MJ. Exosomes and the emerging field of exosome-based gene therapy. Curr Gene Ther 2012; 12(4): 262-74.
  15. Lai RC, Yeo RW, Lim SK. Mesenchymal stem cell exosomes. Semin Cell Dev Biol 2015; 40: 82-8.
  16. Zhang B, Yin Y, Lai RC, Tan SS, Choo AB, Lim SK. Mesenchymal stem cells secrete immunologically active exosomes. Stem Cells Dev 2014; 23(11): 1233-44.
  17. Yu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. Int J Mol Sci 2014; 15(3): 4142-57.
  18. Kourembanas S. Exosomes: Vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Annu Rev Physiol 2015; 77: 13-27.
  19. Zhang S, Chu WC, Lai RC, Lim SK, Hui JH, Toh WS. Exosomes derived from human embryonic mesenchymal stem cells promote osteochondral regeneration. Osteoarthritis Cartilage 2016; 24(12): 2135-40.
  20. Yeo RW, Lai RC, Zhang B, Tan SS, Yin Y, Teh BJ, et al. Mesenchymal stem cell: An efficient mass producer of exosomes for drug delivery. Adv Drug Deliv Rev 2013; 65(3): 336-41.
  21. Lou G, Chen Z, Zheng M, Liu Y. Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases. Exp Mol Med 2017; 49(6): e346.
  22. Vakhshiteh F, Atyabi F, Ostad SN. Mesenchymal stem cell exosomes: A two-edged sword in cancer therapy. Int J Nanomedicine 2019; 14: 2847-59.
  23. Skottvoll FS, Berg HE, Bjorseth K, Lund K, Roos N, Bekhradnia S, et al. Comparison of ultracentrifugation and a commercial kit for isolation of exosomes derived from glioblastoma and breast cancer cells. bioRxiv 2018; 274910.
  24. Weng Y, Sui Z, Shan Y, Hu Y, Chen Y, Zhang L, et al. Effective isolation of exosomes with polyethylene glycol from cell culture supernatant for in-depth proteome profiling. Analyst 2016; 141(15): 4640-6.
  25. Del Fattore A, Luciano R, Pascucci L, Goffredo BM, Giorda E, Scapaticci M, et al. Immunoregulatory effects of mesenchymal stem cell-derived extracellular vesicles on T lymphocytes. Cell Transplant 2015; 24(12): 2615-27.
  26. Buzas EI, Gyorgy B, Nagy G, Falus A, Gay S. Emerging role of extracellular vesicles in inflammatory diseases. Nat Rev Rheumatol 2014; 10(6): 356-64.
  27. Chen W, Huang Y, Han J, Yu L, Li Y, Lu Z, et al. Immunomodulatory effects of mesenchymal stromal cells-derived exosome. Immunol Res 2016; 64(4): 831-40.
  28. Alexander M, Hu R, Runtsch MC, Kagele DA, Mosbruger TL, Tolmachova T, et al. Exosome-delivered microRNAs modulate the inflammatory response to endotoxin. Nat Commun 2015; 6: 7321.
  29. Du YM, Zhuansun YX, Chen R, Lin L, Lin Y, Li JG. Mesenchymal stem cell exosomes promote immunosuppression of regulatory T cells in asthma. Exp Cell Res 2018; 363(1): 114-20.
  30. Tavasolian F, Abdollahi E, Rezaei R, Momtazi-Borojeni AA, Henrotin Y, Sahebkar A. Altered expression of microRNAs in rheumatoid arthritis. J Cell Biochem 2018; 119(1): 478-87.
Journal of Isfahan Medical School
Volume 38, Issue 562 - Serial Number 562
January and February 2020
Pages 31-38

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History

  • Receive Date: 19 February 2020
  • Revise Date:
  • Accept Date: 06 April 2022

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