Skip to main content

Advertisement

SpringerLink
Log in

Efficient preparation of high-purity and intact mesenchymal stem cell–derived extracellular vesicles

  • Communication
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Data availability

All data generated or analyzed during this study have been included in this published article and its supplementary information files.

References

  1. Ding DC, Shyu WC, Lin SZ. Mesenchymal stem cells. Cell Transplant. 2011. https://doi.org/10.3727/096368910x.

    Article  PubMed  Google Scholar 

  2. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Ahn SY. The role of MSCs in the tumor microenvironment and tumor progression. Anticancer Res. 2020. https://doi.org/10.21873/anticanres.14284

  4. 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.

    Article  PubMed  Google Scholar 

  5. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  6. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  7. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  8. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  9. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  10. 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.

    Article  Google Scholar 

  11. 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.

    Article  Google Scholar 

  12. 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.

    Article  CAS  Google Scholar 

  13. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  14. 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.

    Article  PubMed  Google Scholar 

  15. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  16. 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.

    Article  Google Scholar 

  17. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  18. 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.

    Article  Google Scholar 

  19. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  20. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  21. 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.

    Article  PubMed  Google Scholar 

  22. 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.

    Article  Google Scholar 

  23. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  24. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  25. 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.

    Article  Google Scholar 

  26. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  27. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  28. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Webber J, Clayton A. How pure are your vesicles? J Extracell Ves. 2013. https://doi.org/10.3402/jev.v2i0.19861.

    Article  Google Scholar 

  30. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  31. 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.

    Article  CAS  Google Scholar 

  32. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  33. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  34. 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.

    Article  PubMed  Google Scholar 

  35. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  36. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  37. 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.

    Article  Google Scholar 

  38. 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.

    Article  Google Scholar 

  39. 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.

    Article  Google Scholar 

  40. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  41. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  42. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  43. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  44. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  45. 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.

    Article  PubMed  Google Scholar 

  46. 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.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

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).

Author information

Author notes

  1. Fangfang Ni and Qingfu Zhu share equal contribution.

Authors and Affiliations

  1. National Engineering Technology Research Center for Ophthalmology and Optometry, School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, China

    Fangfang Ni, Qingfu Zhu, Hengrui Li, Fei Liu & Hao Chen

  2. Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, USA

    Fei Liu

Authors
  1. Fangfang Ni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qingfu Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hengrui Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fei Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hao Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding authors

Correspondence to Fei Liu or Hao Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 428 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-024-05193-0

Keywords

Search

Navigation