The exosome isolation and characterization from DLD-1 cell line media

Abstract

Exosomes, as extracellular organelles, are the most well-known vesicles among microvesicles. They are released from almost all body fluids as well as cells. Exosomes, which have gained popularity in biology studies with their nano-level dimensions (30-200nm) and membrane structures, are primarily used in drug encapsulation, regeneration, and cell-free cellular therapy. The fact that exosomes, which also function as biomarkers, have an ever-expanding area of use has led researchers to search for the isolation and characterization of this vesicle. In this study, the isolation of exosomes of the DLD-1 colorectal cancer line was carried out by ultracentrifugation method and characterized biophysically and biochemically by SEM, NTA, and flow cytometry, and the proliferative effect of these exosomes on HT29 cells, one of the other colorectal cancer lines, was investigated. As a result, it was proved that the isolated vesicles were morphological and size-appropriate to the literature and expressed CD63 and CD81 proteins. It was observed that DLD-1 exosomes increased proliferation on the HT29 cell line in a dose-dependent manner. Within the framework of the findings, this study could be an excellent reference for exosome isolation and characterization.

Keywords

• Exosome
• isolation
• characterization
• proliferation effect.

References

1. R.M. Johnstone, M. Adam, J.R. Hammond, L. Orr, C. Turbide, Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes), J. Biol. Chem., 262 (1987) 9412-9420.
2. Y. Zhang, Y. Liu, H. Liu, W.H. Tang, Exosomes: biogenesis, biologic function and clinical potential. Cell Biosci., 9 (2019) 1-18.
3. A.V. Vlassov, S. Magdaleno, R. Setterquist, R. Conrad, Exosomes: Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim. Biophys. Acta, 1820 (2012) 940-948.
4. V. Deschoolmeester, M. Baay, P. Specenier, F. Lardon, J.B. Vermorken, A review of the most promising biomarkers in colorectal cancer: one step closer to targeted therapy. Oncologist, 15 (2010) 699-731.
5. H. Chen, V. Chengalvala, H. Hu, D. Sun, Tumor-derived exosomes: Nanovesicles made by cancer cells to promote cancer metastasis. Acta Pharm. Sin. B, 11 (2021) 2136-2149.
6. L. Kotelevets, E. Chastre, Extracellular vesicles in colorectal cancer: From tumor growth and metastasis to biomarkers an nanomedications. Cancers, 15 (2023) 1-48.
7. Y. Umwali, CB. Yue, A.N.A. Gabriel, Y. Zhang, X. Zhang, Roles of exosomes in diagnosis and treatment of colorectal cancer. World J. Clin. Cases, 9 (2021) 4467-4479.
8. R.J. Lobb, M. Becker, S. Wen, C.S. Wong, A.P. Wiegmans, A. Leimgruber, A. Möller, Optimized exosome isolation protocol for cell culture supernatant and human plasma. J. Extracell. Vesicles, 4 (2015) 1-11.

9. G. Raposo, W. Stoorvogel, Extracellular vesicles: exosomes, microvesicles, and friends. J. Cell Biol., 200 (2013), 373-383.
10. B. Meehan, J. Rak, D. Di Vizio, Oncosomes–large and small: what are they, where they came from?, J. Extracell. Vesicles, 5 (2016) 1-2.
11. P. Fonseka, A.L. Marzan, S. Mathivanan, Introduction to the community of extracellular vesicles. New Frontiers: Extracellular Vesicles, Subcellular Biochemistry, Springer, Switzerland, 2021.
12. B. Wang, Y. Wang, Z. Yan, Y. Sun, C. Su, Colorectal cancer cell-derived exosomes promote proliferation and decrease apoptosis by activating the ERK pathway. Int. J. Clin. Exp. Pathol., 12 (2019) 2485-2495.
13. Y.J. Yoon, O.Y. Kim, Y.S. Gho, Extracellular vesicles as emerging intercellular communicasomes. BMB Rep., 47 (2014) 531-539.
14. H. Kalra, G.P. Drummen, S. Mathivanan, Focus on extracellular vesicles: introducing the next small big thing. Int J Mol Sci, 17 (2016) 1-30s.
15. R.J. Simpson, H. Kalra, S. Mathivanan, ExoCarta as a resource for exosomal research. J. Extracell. Vesicles, 1(2012) 1-6.
16. J. Lötvall, A.F. Hill, F. Hochberg, E.I. Buzás, D. Di Vizio, C. Gardiner, Y.S. Gho, I.V. Kurochkin, S. Mathivanan, P. Quesenberry, S. Sahoo, H. Tahara, M. H. Wauben, K. W. Witwer, C. Thery, Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J. Extracell. Vesicles, 3 (2014) 1-6.
17. K.W. Witwer, E.I. Buzás, L.T. Bemis, A. Bora, C. Lässer, J. Lötvall, E.N.N. Hoen, M.S. Piper, S. Sivaraman, J. Skog, C. Thery, M. H. Wauben, F. Hochberg, Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J. Extracell. Vesicles, 2 (2013) 1-25.
18. K.W. Witwer, C. Soekmadji, A.F. Hill, M.H. Wauben, E.I. Buzás, D. Di Vizio, J.M. Falcon-Perez, C. Gardiner, F. Hochberg, I.V. Kurochkin, J. Lötvall, S. Mathivanan, R. Nieuwland, S. Sahoo, H. Tahara, A.C Torrecilhas, A. M. Weaver, H. Yin, L. Zheng, Y. S. Gho, P. Quesenberry, C. Thery, Updating the MISEV minimal requirements for extracellular vesicle studies: building bridges to reproducibility. J. Extracell. Vesicles, 6 (2017) 1-8.
19. M. Zhang, K. Jin, L. Gao, Z. Zhang, F. Li, F. Zhou, L. Zhang, Methods and technologies for exosome isolation and characterization. Small Methods, 2 (2018) 1-10.
20. M. Yáñez-Mó, P.R.M. Siljander, Z. Andreu, A. Bedina Zavec, F.E. Borràs, EI. Buzas, Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles, 4 (2015) 1-60.
21. E. Van der Pol, A.N. Böing, E.L. Gool, R. Nieuwland, Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles. J. Thromb. Haemost., 14(2016) 48-56.
22. M.A. Ashraf, W. Peng, Y. Zare, K.Y. Rhee, Effects of size and aggregation/agglomeration of nanoparticles on the interfacial/interphase properties and tensile strength of polymer nanocomposites. Nanoscale Res. Lett., 13 (2018)1-7.