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Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables

Nature Protocols volume 16, pages 603–633 (2021)Cite this article

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Abstract

The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant EV (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics to sample EV and function as an internal quantitative and qualitative control throughout analysis. Spiking rEV in bodily fluids prior to EV analysis maps technical variability of EV applications and promotes intra- and inter-laboratory studies. This protocol, which is an Extension to our previously published protocol (Tulkens et al., 2020), describes the production, separation and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on complementary fluorescence, nucleic acid and protein measurements. We demonstrate the use of rEV for method development, data normalization and assessment of pre-analytical variables. The protocol can be adopted by researchers with standard laboratory and basic EV separation/characterization experience and requires ~4–5 d.

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Fig. 1: Comparison of rEV with other reported reference materials.
Fig. 2: Visual guidelines showing the outcomes at various Steps during the protocol for rEV production and characterization.
Fig. 3: Setup of AF4–MALS–FLD.
Fig. 4: Characterization of rEV using complementary methods.
Fig. 5: The use of exogenous fusion protein (gag-EGFP) or nucleic acids (EGFP mRNA) to detect rEV in EV-comprising samples.
Fig. 6: Proteomic analysis of urine and blood plasma, spiked or not with increasing numbers of rEV.
Fig. 7: Diverse applications of rEV.

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Data availability

Proteomic data were submitted to the PRIDE database (PXD017542; Username: reviewer18821@ebi.ac.uk, Password: 0oqaE1p8). The source data underlying Figs. 4, 5, 6 and 7 are provided as Supplementary Data files 1, 2, 3 and 4, respectively. All other relevant data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by Ghent University (Concerted Research Actions and Industrial Research Fund), Ghent University Hospital, Kom Op Tegen Kanker, and research project (A.H.) and PhD position (E.G.) strategic basic research from the Fund for Scientific Research Flanders (FWO).

Author information

Authors and Affiliations

  1. Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium

    Edward Geeurickx, Lien Lippens, Olivier De Wever & An Hendrix

  2. Cancer Research Institute Ghent, Ghent, Belgium

    Edward Geeurickx, Lien Lippens, Bruno G. De Geest, Olivier De Wever & An Hendrix

  3. Department of Medical Oncology, Ghent University Hospital, Ghent, Belgium

    Lien Lippens

  4. Department of Biochemistry, University of Turku, Turku, Finland

    Pekka Rappu

  5. Department of Pharmaceutics, Ghent University, Ghent, Belgium

    Bruno G. De Geest

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  1. Edward Geeurickx
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Contributions

E.G. planned, designed and performed the experiments and co-wrote the manuscript. L.L and B.G.D.G. performed rEV separation and characterization using AF4–MALS. P.R. performed proteomics experiments. O.D.W. and A.H. initiated the project, designed and interpreted experiments and co-wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to An Hendrix.

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Competing interests

A.H., O.D.W. and E.G. are inventors on the patent application covering the rEV technology (WO2019091964). The remaining authors declare no competing interests.

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Peer review information Nature Protocols thanks Bernd Giebel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Related links

Key references using this protocol

Geeurickx, E. et al. Nat. Commun. 10, 3288 (2019): https://doi.org/10.1038/s41467-019-11182-0

Tulkens, J., De Wever, O. & Hendrix, A. Nat. Protoc. 15, 40–67 (2020): https://doi.org/10.1038/s41596-019-0236-5

Dhondt, B. et al. J. Extracell. Vesicles 9, 1736935 (2020): https://doi.org/10.1080/20013078.2020.1736935

This protocol is an extension to: Nat. Protoc. 15, 40–67 (2020): https://doi.org/10.1038/s41596-019-0236-5

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Data 1

Source data of Fig. 4.

Supplementary Data 2

Source data of Fig. 5.

Supplementary Data 3

Source data of Fig. 6.

Supplementary Data 4

Source data of Fig. 7.

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Geeurickx, E., Lippens, L., Rappu, P. et al. Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables. Nat Protoc 16, 603–633 (2021). https://doi.org/10.1038/s41596-020-00446-5

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