Abstract
Mesenchymal stem cell–derived extracellular vesicles (MSC-EVs) have shown great promise for regeneration and immunomodulation. However, efficient and scalable methods for their preparation are still lacking. In this study, we present the adoption of a label-free technique known as “EXODUS” to isolate and purify MSC-EVs from the conditioned medium. Our findings indicate that EXODUS can rapidly isolate EVs from 10 mL of conditioned medium with a 5-fold higher yield compared to conventional approaches, including ultracentrifugation (UC) and polyethylene glycol precipitation (PEG) methods. Additionally, pre-storing the conditioned medium at 4°C for 1 week resulted in a ~2-fold higher yield of MSC-EVs compared to the freshly prepared medium. However, storing the purified EV particles at 4°C for 1 month led to a 2-fold reduction in particle concentration. Furthermore, we found that MSC-EVs isolated using EXODUS exhibit higher expression levels of EV markers such as Alix, Flotillin1, CD81, and TSG101 in comparison to PEG and UC methods. We also discovered that MSC-EVs isolated using EXODUS are enriched in response to cytokine, collagen-containing extracellular matrix, and calcium ion binding compared to PEG method and enriched in extracellular structure organization, extracellular matrix, and extracellular matrix structure constituents compared to UC. Finally, we demonstrated that MSC-EVs isolated using EXODUS exhibit greater potential in animal organ development, tissue development, and anatomical structure morphogenesis compared to the UC. These findings suggest that EXODUS is a suitable method for the large-scale preparation of high-quality MSC-EVs for various clinical applications.
Data availability
All data generated or analyzed during this study have been included in this published article and its supplementary information files.
References
Ding DC, Shyu WC, Lin SZ. Mesenchymal stem cells. Cell Transplant. 2011. https://doi.org/10.3727/096368910x.
Bian D, Wu Y, Song G, Azizi R, Zamani A. The application of mesenchymal stromal cells (MSCs) and their derivative exosome in skin wound healing: a comprehensive review. Stem Cell Res Ther. 2022. https://doi.org/10.1186/s13287-021-02697-9.
Ahn SY. The role of MSCs in the tumor microenvironment and tumor progression. Anticancer Res. 2020. https://doi.org/10.21873/anticanres.14284
Pan D, Chang X, Xu M, Zhang M, Zhang S, Wang Y, et al. UMSC-derived exosomes promote retinal ganglion cells survival in a rat model of optic nerve crush. J Chem Neuroanat. 2019. https://doi.org/10.1016/j.jchemneu.2019.01.006.
Bruno S, Grange C, Deregibus MC, Calogero RA, Saviozzi S, Collino F, et al. Mesenchymal stem cell-derived microvesicles protect against acute tubular injury. J Am Soc Nephrol. 2009. https://doi.org/10.1681/ASN.2008070798.
Shao L, Zhang Y, Lan B, Wang J, Zhang Z, Zhang L, et al. MiRNA-sequence indicates that mesenchymal stem cells and exosomes have similar mechanism to enhance cardiac repair. BioMed Res Int. 2017. https://doi.org/10.1155/2017/4150705.
Baek G, Choi H, Kim Y, Lee H-C, Choi C. Mesenchymal stem cell-derived extracellular vesicles as therapeutics and as a drug delivery platform. Stem Cells Transl Med. 2019. https://doi.org/10.1002/sctm.18-0226.
Chen Y, Shen H, Ding Y, Yu Y, Shao L, Shen Z. The application of umbilical cord-derived MSCs in cardiovascular diseases. J Cell Mol Med. 2021. https://doi.org/10.1111/jcmm.16830.
Zhang J, Buller BA, Zhang ZG, Zhang Y, Lu M, Rosene DL, et al. Exosomes derived from bone marrow mesenchymal stromal cells promote remyelination and reduce neuroinflammation in the demyelinating central nervous system. Exp Neurol. 2022. https://doi.org/10.1016/j.expneurol.2021.113895.
Hua T, Yang M, Song H, Kong E, Deng M, Li Y, et al. Huc-MSCs-derived exosomes attenuate inflammatory pain by regulating microglia pyroptosis and autophagy via the miR-146a-5p/TRAF6 axis. J Nanobiotechnol. 2022. https://doi.org/10.1186/s12951-022-01522-6.
Zhu Q, Huang Y, Yang Q, Liu F. Recent technical advances to study metabolomics of extracellular vesicles. Microchem J. 2021. https://doi.org/10.1016/j.microc.2021.106816.
Mol EA, Goumans M-J, Doevendans PA, Sluijter JP, Vader P. Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation. Nanomed Nanotechnology Biol Med. 2017;6:2061–5. https://doi.org/10.1016/j.nano.2017.03.011.
Kim JY, Rhim W-K, Yoo Y-I, Kim D-S, Ko K-W, Heo Y, et al. Defined MSC exosome with high yield and purity to improve regenerative activity. J Tissue Eng. 2021. https://doi.org/10.1177/2041731421100862.
Abramowicz A, Widlak P, Pietrowska M. Proteomic analysis of exosomal cargo: the challenge of high purity vesicle isolation. Mol Biosyst. 2016. https://doi.org/10.1039/C6MB00082G.
Li M, Lou D, Chen J, Shi K, Wang Y, Zhu Q, et al. Deep dive on the proteome of salivary extracellular vesicles: comparison between ultracentrifugation and polymer-based precipitation isolation. Anal Bioanal Chem. 2021. https://doi.org/10.1007/s00216-020-03004-w.
Zhu Q, Cheng L, Deng C, Huang L, Li J, Wang Y, et al. The genetic source tracking of human urinary exosomes. Proc National Acad Sci. 2021. https://doi.org/10.1073/pnas.2108876118.
Chen Y, Zhu Q, Cheng L, Wang Y, Li M, Yang Q, et al. Exosome detection via the ultrafast-isolation system: EXODUS. Nat Methods. 2021. https://doi.org/10.1038/s41592-020-01034-x.
Zhu Q, Li H, Ao Z, Xu H, Luo J, Kaurich C, et al. Lipidomic identification of urinary extracellular vesicles for non-alcoholic steatohepatitis diagnosis. J Nanobiotechnol. 2022. https://doi.org/10.1186/s12951-022-01540-4.
Zhu Q, Huang L, Yang Q, Ao Z, Yang R, Krzesniak J, et al. Metabolomic analysis of exosomal-markers in esophageal squamous cell carcinoma. Nanoscale. 2021. https://doi.org/10.1039/d1nr04015d.
Zhu Q, Luo J, Li HP, Ye W, Pan R, Shi KQ, et al. Robust acute pancreatitis identification and diagnosis: RAPIDx. ACS Nano. 2023. https://doi.org/10.1021/acsnano.3c00922.
Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006. https://doi.org/10.1002/0471143030.cb0322s30.
Kordelas L, Rebmann V, Ludwig A, Radtke S, Ruesing J, Doeppner T, et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leuk. 2014. https://doi.org/10.1038/leu.2014.41.
Reza-Zaldivar EE, Hernández-Sapiéns MA, Minjarez B, Gutiérrez-Mercado YK, Márquez-Aguirre AL, Canales-Aguirre AA. Potential effects of MSC-derived exosomes in neuroplasticity in Alzheimer’s disease. Front Cell Neurosci. 2018. https://doi.org/10.3389/fncel.2018.00317.
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. https://doi.org/10.1038/emm.2017.63.
Harrell CR, Simovic Markovic B, Fellabaum C, Arsenijevic A, Djonov V, Arsenijevic N, et al. Therapeutic potential of mesenchymal stem cell-derived exosomes in the treatment of eye diseases. Cell Biol Transl Med. 2018. https://doi.org/10.1007/5584_2018_219.
Kim M, Shin DI, Choi BH, Min B-H. Exosomes from IL-1β-primed mesenchymal stem cells inhibited IL-1β- and TNF-α-mediated inflammatory responses in osteoarthritic SW982 cells. Tissue Eng Regen Med. 2021. https://doi.org/10.1007/s13770-020-00324-x.
Katsuda T, Ochiya T. Molecular signatures of mesenchymal stem cell-derived extracellular vesicle-mediated tissue repair. Stem Cell Res Ther. 2015. https://doi.org/10.1186/s13287-015-0214-y.
Born LJ, Harmon JW, Jay SM. Therapeutic potential of extracellular vesicle-associated long noncoding RNA. Bioeng Transl Med. 2020. https://doi.org/10.1002/btm2.10172.
Webber J, Clayton A. How pure are your vesicles? J Extracell Ves. 2013. https://doi.org/10.3402/jev.v2i0.19861.
Patel GK, Khan MA, Zubair H, Srivastava SK, Khushman M, Singh S, et al. Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications. Sci Rep. 2019. https://doi.org/10.1038/s41598-019-41800-2.
Hancock R, Duong P, Chung A, Bouchareychas L, Raffai RL. Cushioned-density gradient ultracentrifugation (C-DGUC) improves the isolation efficiency of extracellular vesicles. Plos One. 2019;14:e0215324. https://doi.org/10.1371/journal.pone.0215324.
Paolini L, Zendrini A, Di Noto G, Busatto S, Lottini E, Radeghieri A, et al. Residual matrix from different separation techniques impacts exosome biological activity. Sci Rep. 2016. https://doi.org/10.1038/srep23550.
Gupta S, Rawat S, Arora V, Kottarath SK, Dinda AK, Vaishnav PK, et al. An improvised one-step sucrose cushion ultracentrifugation method for exosome isolation from culture supernatants of mesenchymal stem cells. Stem Cell Res Ther. 2018. https://doi.org/10.1186/s13287-018-0923-0.
Sakai-Kato K, Yoshida K, Takechi-Haraya Y, Izutsu KI. Physicochemical characterization of liposomes that mimic the lipid composition of exosomes for effective intracellular trafficking. Langmuir. 2020. https://doi.org/10.1021/acs.langmuir.0c02491.
Bosch S, de Beaurepaire L, Allard M, Mosser M, Heichette C, Chrétien D, et al. Trehalose prevents aggregation of exosomes and cryodamage. Sci Rep. 2016. https://doi.org/10.1038/srep36162.
Cheng Y, Zeng Q, Han Q, Xia W. Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes. Protein Cell. 2019. https://doi.org/10.1007/s13238-018-0529-4.
Simbari F, McCaskill J, Coakley G, Millar M, Maizels RM, Fabrias G, et al. Plasmalogen enrichment in exosomes secreted by a nematode parasite versus those derived from its mouse host: implications for exosome stability and biology. J Extracell Ves. 2016. https://doi.org/10.3402/jev.v5.30741.
Honary S, Zahir F. Effect of zeta potential on the properties of nano-drug delivery systems - a review (Part 1). Trop J Pharm Res. 2013. https://doi.org/10.4314/tjpr.v12i2.19.
Kim K, Park J, Sohn Y, Oh CE, Park JH, Yuk JM, et al. Stability of plant leaf-derived extracellular vesicles according to preservative and storage temperature. Pharm. 2022. https://doi.org/10.3390/pharmaceutics14020457.
Wang ZG, He ZY, Liang S, Yang Q, Cheng P, Chen AM. Comprehensive proteomic analysis of exosomes derived from human bone marrow, adipose tissue, and umbilical cord mesenchymal stem cells. Stem Cell Res Ther. 2020. https://doi.org/10.1186/s13287-020-02032-8.
Wiklander OP, Brennan MÃ, Lotvall J, Breakefield XO, El Andaloussi S. Advances in therapeutic applications of extracellular vesicles. Sci Transl Med. 2019. https://doi.org/10.1126/scitranslmed.aav8521.
Li Z, Xiao J, Xu X, Li W, Zhong R, Qi L, et al. M-CSF, IL-6, and TGF-β promote generation of a new subset of tissue repair macrophage for traumatic brain injury recovery. Sci Adv. 2021. https://doi.org/10.1126/sciadv.abb6260.
Kitahashi T, Kogawa R, Nakamura K, Sekiya I. Integrin beta1, PDGFRbeta, and type II collagen are essential for meniscus regeneration by synovial mesenchymal stem cells in rats. Sci Rep. 2022. https://doi.org/10.1038/s41598-022-18476-2.
Johnston JM, Connizzo BK, Shetye SS, Robinson KA, Huegel J, Rodriguez AB, et al. Collagen V haploinsufficiency in a murine model of classic Ehlers-Danlos syndrome is associated with deficient structural and mechanical healing in tendons. J Orthop Res. 2017. https://doi.org/10.1002/jor.23571.
Cappuzzello C, Doni A, Dander E, Pasqualini F, Nebuloni M, Bottazzi B, et al. Mesenchymal stromal cell-derived PTX3 promotes wound healing via fibrin remodeling. J Investig Dermatol. 2016. https://doi.org/10.1038/JID.2015.346.
Wang J, Karra R, Dickson AL, Poss KD. Fibronectin is deposited by injury-activated epicardial cells and is necessary for zebrafish heart regeneration. Dev Biol. 2013. https://doi.org/10.1016/j.ydbio.2013.08.012.
Funding
This study was funded by the Wenzhou Municipal Science & Technology Bureau, Zhejiang Provincial Department of Science and Technology, and the Ministry of Science and Technology (China).
The work was supported by the Zhejiang Provincial Leading Health Talent Project (to Hao Chen); the Zhejiang Provincial Natural Science Foundation (LY22H120002 to Qingfu Zhu); and the Wenzhou Basic Research Projects (Y2020916 to Qingfu Zhu).
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Fei Liu and Hao Chen conceived the project, designed the experiments, and contributed to data interpretation, manuscirpt editing, and data analysis. Fangfang Ni, Qingfu Zhu, and Hengrui Li performed the experiments, conducted data analysis, and drafted the manuscript. All authors have approved the final version of the manuscript.
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Ni, F., Zhu, Q., Li, H. et al. Efficient preparation of high-purity and intact mesenchymal stem cell–derived extracellular vesicles. Anal Bioanal Chem 416, 1797–1808 (2024). https://doi.org/10.1007/s00216-024-05193-0
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DOI: https://doi.org/10.1007/s00216-024-05193-0