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Generation and Characterization of Cell-Derived Microvesicles from HUVECs

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Abstract

Microvesicles (MVs) are of great interest for the biomedical field as novel diagnostics biomarkers. MV research, however, is still impaired by the lack of standardization of the analysis methods. In this study, our goals were to develop a method for the reproducible generation of an MV population from cell culture, to compare the sizing of MVs by atomic force microscopy (AFM) and nanoparticle tracking analysis (NTA) and to develop an MV array allowing the sizing and phenotyping of MVs simultaneously with the AFM as a read-out. MVs were isolated from apoptotic human umbilical vein endothelial cells (HUVECs) and had a mean size of 115 nm as measured by AFM and of 197 nm as measured by NTA. MVs isolated from apoptotic and control HUVECs could not be distinguished by their size (p > 0.05). HUVECs and their released MVs shared the same phenotype (positive for endothelial markers CD31 and CD146 and negative for platelet marker CD42b). Our method generated MVs with a concentration which was reproducible within a range of 20%. This is a relevant model system for medical applications. The size of the MVs was larger as measured using NTA compared to that using AFM. The MV array was efficient to characterize the surface markers of MVs and has the potential to identify the cellular origins of MVs in patient samples.

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Acknowledgements

The author would like to thank Mark Holm Olsen for the preparation of the gold substrates and proof-reading, as well as Julie Kirkegaard for proof-reading and Christian Engelbrekt for providing training and access to the NTA. This work has been supported by the Technical University of Denmark.

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Authors and Affiliations

  1. Department of Micro- and Nanotechnology, Technical University of Denmark, Produktionstorvet 423, 2800 Kgs, Lyngby, Denmark

    Solène Cherre, Mathilde Granberg & Noemi Rozlosnik

  2. Statens Serum Institut, Artillerivej 5, 2300, Copenhagen, Denmark

    Ole Østergaard & Niels H. H. Heegaard

  3. Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, University of Southern Denmark, Søndre Blvd. 29, 5000, Odense C, Denmark

    Niels H. H. Heegaard

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  1. Solène Cherre
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  2. Mathilde Granberg
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Correspondence to Solène Cherre.

Appendix: Characterization of the Complete Growth Medium for HUVEC Culture

Appendix: Characterization of the Complete Growth Medium for HUVEC Culture

The different components of the complete growth medium of HUVECs were processed like the cell culture supernatants (centrifugation protocol described in Section 2.5) to assess if MVs or MV like particles were present in the complete growth medium before culture with the cells. Figure 7a shows the concentration of MVs detected in the processed aliquots of the medium components as measured with NTA. In the complete growth medium, 1.8 × 109 MVs/ml (SD + / − 5 × 108, corresponding to 28%) were detected. The levels of MVs measured in Ham’s F12K and the penicillin/streptomycin solution were below 10% of the level of MVs in the complete growth medium. However, the level of MVs in the FBS reached 1.1 × 1010 MVs/ml (SD + / − 3.6 × 108, corresponding to 3%), that was approximately ten times more than that in the complete medium. The size distributions of the MV from the complete growth medium and from the FBS (Fig. 7b) both show a main peak at 160 nm. The buffer used (phosphate-buffered saline, PBS) was filtered through a 0.1- μ m syringe filter prior of use and also had a low level of MVs (1.1 × 108 MVs/ml).

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Cherre, S., Granberg, M., Østergaard, O. et al. Generation and Characterization of Cell-Derived Microvesicles from HUVECs. BioNanoSci. 8, 140–153 (2018). https://doi.org/10.1007/s12668-017-0438-7

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