University of Oulu

Van Dorpe, S., Lippens, L., Boiy, R. et al. Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility. J Nanobiotechnol 21, 157 (2023).

Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility

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Author: Van Dorpe, Sofie1,2,3; Lippens, Lien1,2; Boiy, Robin1,2;
Organizations: 1Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium
2Cancer Research Institute Ghent, Ghent, Belgium
3Department of Gynecology, Ghent University Hospital, Ghent, Belgium
4Department of Life Technologies, University of Turku, Turku, Finland
5Biocenter Oulu, University of Oulu, Oulu, Finland
6Department of Medical Oncology, Ghent University Hospital, Ghent, Belgium
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 8.2 MB)
Persistent link:
Language: English
Published: Springer Nature, 2023
Publish Date: 2023-09-21


Background: Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers.

Results: Automated versus manual density-based separation of trackable recombinant extracellular vesicles (rEV) spiked in PBS significantly reduces variability in rEV recovery as quantified by fluorescent nanoparticle tracking analysis and ELISA. To validate automated density-based EV separation from complex body fluids, including blood plasma and urine, we assess reproducibility, recovery, and specificity by mass spectrometry-based proteomics and transmission electron microscopy. Method reproducibility is the highest in the automated procedure independent of the matrix used. While retaining (in urine) or enhancing (in plasma) EV recovery compared to manual liquid handling, automation significantly reduces the presence of body fluid specific abundant proteins in EV preparations, including apolipoproteins in plasma and Tamm-Horsfall protein in urine.

Conclusions: In conclusion, automated liquid handling ensures cost-effective EV separation from human body fluids with high reproducibility, specificity, and reduced hands-on time with the potential to enable larger-scale biomarker studies.

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Series: Journal of nanobiotechnology
ISSN: 1477-3155
ISSN-E: 1477-3155
ISSN-L: 1477-3155
Volume: 21
Issue: 1
Article number: 157
DOI: 10.1186/s12951-023-01917-z
Type of Publication: A1 Journal article – refereed
Field of Science: 1182 Biochemistry, cell and molecular biology
Funding: This work was supported by a project grant and starting PhD fellowship (SVD) from Stand up to Cancer (Kom Op Tegen Kanker), the Flemish cancer society; and a project grant and PhD fellowship (SVD) from Fund for Scientific Research Flanders (FWO).
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