Volume 13, Issue 4 (12-2025)
Jorjani Biomed J 2025, 13(4): 1-2 |
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Khosravi A. Standardizing cell culture methods is critical for reliable and reproducible exosome isolation. Jorjani Biomed J 2025; 13 (4) :1-2
URL: http://goums.ac.ir/jorjanijournal/article-1-1123-en.html
URL: http://goums.ac.ir/jorjanijournal/article-1-1123-en.html
Stem Cell Research Center, Biomedical Research Institute, Golestan University of Medical Sciences, Gorgan, Iran; Department of Molecular Medicine, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran , ayyoobfarsian@yahoo.com
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Editorial
EVs are lipid bilayer-enclosed structures produced and released by living cells, including EXOs (30-150 nm), microvesicles (150-1000 nm), and apoptotic bodies (1-5000 nm) (1-3). EXOs are formed through the fusion of multivesicular bodies (MVBs) with the plasma membrane in the endosomal compartment and are released into the extracellular space by direct budding from the plasma membrane (1,4,5). Pan and Johnstone initially identified and named EXOs when they investigated the maturation of sheep reticulocytes (6). For many years, EXOs were thought to be responsible for carrying cellular metabolic waste (7). Zhang et al. confirmed the spherical shape of EXOs using electron microscopy (8). Their biological importance and relevance to disease were understood many years later, when researchers explored their roles in cell-to-cell communication and disease (9,10). Many cellular functions, such as apoptosis, cell growth, and cell division, are regulated by EXOs through the delivery of active biomolecules, including nucleic acids, proteins, and lipids, to target cells (11,12). EXOs have been found in various body fluids, such as blood, urine, saliva, sweat, tears, vaginal fluid, and cerebrospinal fluid (1), as well as in cell culture supernatant (13).
Several studies have now been designed on EXOs and have shown that EXOs play an important role in cancer biology and immunoregulation (14-18). One of the main challenges in EXO research is the lack of an efficient standardized isolation method for precise EXO subpopulations because of their small size and heterogeneity (19-22). In addition, the isolation of intact and pure EXOs is necessary, especially when they are intended to be used as vehicles for drug delivery in the treatment of human disease (4). Biofluid-isolated EXOs unavoidably have a mixed cellular origin (20). Pure EXOs can be obtained from the conditioned medium (CM) of a cell line. However, flow cytometry analysis has shown that not all components of a cell line have the same phenotype (17). Therefore, it may be necessary to use a subpopulation of a cell line with specific surface CD marker. EXOs are usually isolated from culture medium conditioned by specific cells for 24-48 h (20), although conditioning times of 15-60 min have also been reported (23,24). Hence, the conditioning time of culture medium in specific cells must be standardized.
The use of serum supplements that contain their own EXOs in cell culture medium can contaminate EXOs secreted by specific cells. Ultracentrifugation and serum-free media (SFM) are recommended to eliminate serum-derived EXOs (25); however, both approaches have limitations, such as the need for efficient elimination of serum-derived vesicles and serum protein quantification in ultracentrifugation, and the secretion of EXOs with different compositions in the SFM strategy (20). To overcome external serum-derived EXOs, it is mandatory to develop precise and robust antibody-based methods for the complete elimination of external EXOs or defined SFM that mimic human serum composition. Dead or dying cells release different vesicles that contaminate live cell-derived EXOs. Also, the composition of EXOs may change due to the uptake of soluble materials released from dying cells (20). It seems that separating live cell-derived EXOs from dead cell-derived EXOs is a challenge that requires the development of methods that can detect and isolate live cell-derived EXOs. However, standardization of conditioning time and washing dead cells before EXO isolation may be helpful. Any microbial contamination, such as mycoplasma or viruses, can contaminate the exosomal content of conditioned medium (20). Hence, cell lines should be checked for microbial contamination before EXO isolation.
Acknowledgement
None.
Funding sources
None.
Ethical statement
Not applicable.
Conflicts of interest
None.
Author contributions
All parts of the manuscript were written and submitted by Ayyoob Khosravi.
Data availability statement
Not applicable.
EVs are lipid bilayer-enclosed structures produced and released by living cells, including EXOs (30-150 nm), microvesicles (150-1000 nm), and apoptotic bodies (1-5000 nm) (1-3). EXOs are formed through the fusion of multivesicular bodies (MVBs) with the plasma membrane in the endosomal compartment and are released into the extracellular space by direct budding from the plasma membrane (1,4,5). Pan and Johnstone initially identified and named EXOs when they investigated the maturation of sheep reticulocytes (6). For many years, EXOs were thought to be responsible for carrying cellular metabolic waste (7). Zhang et al. confirmed the spherical shape of EXOs using electron microscopy (8). Their biological importance and relevance to disease were understood many years later, when researchers explored their roles in cell-to-cell communication and disease (9,10). Many cellular functions, such as apoptosis, cell growth, and cell division, are regulated by EXOs through the delivery of active biomolecules, including nucleic acids, proteins, and lipids, to target cells (11,12). EXOs have been found in various body fluids, such as blood, urine, saliva, sweat, tears, vaginal fluid, and cerebrospinal fluid (1), as well as in cell culture supernatant (13).
Several studies have now been designed on EXOs and have shown that EXOs play an important role in cancer biology and immunoregulation (14-18). One of the main challenges in EXO research is the lack of an efficient standardized isolation method for precise EXO subpopulations because of their small size and heterogeneity (19-22). In addition, the isolation of intact and pure EXOs is necessary, especially when they are intended to be used as vehicles for drug delivery in the treatment of human disease (4). Biofluid-isolated EXOs unavoidably have a mixed cellular origin (20). Pure EXOs can be obtained from the conditioned medium (CM) of a cell line. However, flow cytometry analysis has shown that not all components of a cell line have the same phenotype (17). Therefore, it may be necessary to use a subpopulation of a cell line with specific surface CD marker. EXOs are usually isolated from culture medium conditioned by specific cells for 24-48 h (20), although conditioning times of 15-60 min have also been reported (23,24). Hence, the conditioning time of culture medium in specific cells must be standardized.
The use of serum supplements that contain their own EXOs in cell culture medium can contaminate EXOs secreted by specific cells. Ultracentrifugation and serum-free media (SFM) are recommended to eliminate serum-derived EXOs (25); however, both approaches have limitations, such as the need for efficient elimination of serum-derived vesicles and serum protein quantification in ultracentrifugation, and the secretion of EXOs with different compositions in the SFM strategy (20). To overcome external serum-derived EXOs, it is mandatory to develop precise and robust antibody-based methods for the complete elimination of external EXOs or defined SFM that mimic human serum composition. Dead or dying cells release different vesicles that contaminate live cell-derived EXOs. Also, the composition of EXOs may change due to the uptake of soluble materials released from dying cells (20). It seems that separating live cell-derived EXOs from dead cell-derived EXOs is a challenge that requires the development of methods that can detect and isolate live cell-derived EXOs. However, standardization of conditioning time and washing dead cells before EXO isolation may be helpful. Any microbial contamination, such as mycoplasma or viruses, can contaminate the exosomal content of conditioned medium (20). Hence, cell lines should be checked for microbial contamination before EXO isolation.
Acknowledgement
None.
Funding sources
None.
Ethical statement
Not applicable.
Conflicts of interest
None.
Author contributions
All parts of the manuscript were written and submitted by Ayyoob Khosravi.
Data availability statement
Not applicable.
Editorial: Editorial |
Subject:
Molecular Sciences
Received: 2025/11/11 | Accepted: 2025/12/10 | Published: 2025/12/20
Received: 2025/11/11 | Accepted: 2025/12/10 | Published: 2025/12/20
References
1. Kim T, Hong JW, Lee LP. Correction: Efficient methods of isolation and purification of extracellular vesicles. Nano Converg. 2025;12(1):64. [View at Publisher] [DOI] [PMID] [Google Scholar]
2. Safdar A, Saleem A, Tarnopolsky MA. The potential of endurance exercise-derived exosomes to treat metabolic diseases. . Nat Rev Endocrinol. 2016;12:504-17. [View at Publisher] [DOI] [PMID] [Google Scholar]
3. Murk JLAN, Humbel BM, Ziese U, Griffith JM, Posthuma G, Slot JW, et al. Endosomal Compartmentalization in Three Dimensions: Implications for Membrane Fusion. . Proc Natl Acad Sci U S A. 2003;100(23):13332-7. [View at Publisher] [DOI] [PMID] [Google Scholar]
4. Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular organelles important in intercellular communication. . J Proteomics. 2010;73(10):1907-20. [View at Publisher] [DOI] [PMID] [Google Scholar]
5. Dilsiz N. A comprehensive review on recent advances in exosome isolation and characterization: Toward clinical applications. Transl Oncol. 2024:50:102121. [View at Publisher] [DOI] [PMID] [Google Scholar]
6. Pan BT, Johnstone RM. Fate of the Transferrin Receptor during Maturation of Sheep ReticulocytesIn Vitro: Selective Externalization of the Receptor. Cell. 1983;33(3):967-78. [View at Publisher] [DOI] [PMID] [Google Scholar]
7. Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. Vesicle Formation during Reticulocyte Maturation. Association of Plasma Membrane Activities with Released Vesicles (Exosomes). J Biol Chem. 1987;262(19):9412-20. [View at Publisher] [DOI] [PMID] [Google Scholar]
8. Zhang P, Zhou X, He M, Shang Y, Tetlow AL, Godwin AK,etal. Ultrasensitive Detection of Circulating Exosomes with a 3D-Nanopatterned Microfluidic Chip. Nat Biomed Eng. 2019;3(6):438-451. [View at Publisher] [DOI] [PMID] [Google Scholar]
9. Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002;2(8):569-79. [View at Publisher] [DOI] [PMID] [Google Scholar]
10. Van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R. Classifcation, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev. 2012;64(3):676-705. [View at Publisher] [DOI] [PMID] [Google Scholar]
11. Khoshsirat Sh, Keramatinia A, Khoramgah MS, Vafaei-Nezhad S, Niknazar S, Darabi Sh et al. Exosome therapy in spinal cord injury: a review. J Otorhinolaryngol Facial Plastic Surg. 2019;5(2):1-8. [View at Publisher] [DOI] [Google Scholar]
12. Lu M, Yuan S, Li S, 2, Li L, Liu M, Wan S. The exosome-derived biomarker in atherosclerosis and its clinical application. J Cardiovasc Transl Res. 2019;12(1):68-74. [View at Publisher] [DOI] [PMID] [Google Scholar]
13. Logozzi M, Di Raimo R, Mizzoni D, Fais S. Immunocapture-based ELISA to characterize and quantify exosomes in both cell culture supernatants and body fuids. Methods Enzymol. 2020:645:155-180. [View at Publisher] [DOI] [PMID] [Google Scholar]
14. Zhao Y, Weber SR, Lease J, Russo M, Siedlecki CA, Xu L-C, et al. Liquid Biopsy of Vitreous Reveals an Abundant Vesicle Population Consistent with the Size and Morphology of Exosomes. Transl Vis Sci Technol. 2018;7(3):6. [View at Publisher] [DOI] [PMID] [Google Scholar]
15. Mateescu B, Kowal EJ, van Balkom BWM, Bartel S, Bhattacharyya SN, Buzás EI, et al. Obstacles and Opportunities in the Functional Analysis of Extracellular Vesicle RNA - an ISEV Position Paper. J Extracell Vesicles. 2017;6:1286095. [View at Publisher] [DOI] [PMID] [Google Scholar]
16. Fallah A, Hosseinzadeh Colagar A, Khosravi A, Saeidi M. Exosomes from SHED-MSC regulate polarization and stress oxidative indexes in THP-1 derived M1 macrophages. Archives of Biochemistry and Biophysics 2024(755 ):109987. [View at Publisher] [DOI] [PMID] [Google Scholar]
17. Fallah A, Hosseinzadeh Colagar A, Khosravi A, Mohammad-Hasani A, Saeidi M. The role of natural exosomes from SHED-MSC in immunoregulation of M0/M1polarized macrophage cells. Front Immunol. 2025:16:1550280. [View at Publisher] [DOI] [PMID] [Google Scholar]
18. Mohammad-Hasani A, Mohammadi S, Saeidi M, Fallah A, Khosravi A. The secretome derived from serum-free media of SHED-MSCs can modulate THP-1-derived macrophage cells. .J Cell Mol Med. 2026;30(9):e71063. [View at Publisher] [DOI] [PMID] [Google Scholar]
19. Araldi E, Krämer-Albers E-M, Nolte-'t Hoen E, Peinado H, Psonka-Antonczyk KM, Rao P, et al. . International society for extracellular vesicles: first annual meeting, April 17-21: ISEV-2012. J Extracell Vesicles. 2012;1:19995. [View at Publisher] [DOI] [PMID] [Google Scholar]
20. Witwer KW , Buzás EI, Bemis LT, Bora A, Lässer C, Lötvall J, et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013;2:20360. [View at Publisher] [DOI] [PMID] [Google Scholar]
21. Chen J, Li P, Zhang T, Xu Z, Huang X, Wang R ,et al. Review on Strategies and Technologies for Exosome Isolation and Purification. Front Bioeng Biotechnol. 2022:9:811971. [View at Publisher] [DOI] [PMID] [Google Scholar]
22. Suresh PS, Zhang Q. Comprehensive Comparison of Methods for Isolation of Extracellular Vesicles from Human Plasma. J Proteome Res. 2025;24(6):2956-67. [View at Publisher] [DOI] [PMID] [Google Scholar]
23. Valadi H, Ekström K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9(6):654-9. [View at Publisher] [DOI] [PMID] [Google Scholar]
24. Montecalvo A, Shufesky WJ, Stolz DB, Sullivan MG, Wang Z, Divito SJ, et al. Exosomes as a short-range mechanism to spread alloantigen between dendritic cells during T cell allorecognition. J Immunol. 2008;180(5):3081-90 [View at Publisher] [DOI] [PMID] [Google Scholar]
25. Thery C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006:Chapter 3:Unit 3.22. [View at Publisher] [DOI] [PMID] [Google Scholar]
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