Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material

André Görgens; Michel Bremer; Rita Ferrer-Tur; Florian Murke; Tobias Tertel; Peter A Horn; Sebastian Thalmann; Joshua A Welsh; Christine Probst; Coralié Guerin; Chantal M Boulanger; Jennifer C Jones; Helmut Hanenberg; Uta Erdbrügger; Joanne Lannigan; Franz L Ricklefs; Samir El-Andaloussi; Bernd Giebel

doi:10.1080/20013078.2019.1587567

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material

J Extracell Vesicles. 2019 Mar 21;8(1):1587567. doi: 10.1080/20013078.2019.1587567. eCollection 2019.

Authors

André Görgens^{1

2

3}, Michel Bremer¹, Rita Ferrer-Tur¹, Florian Murke¹, Tobias Tertel¹, Peter A Horn¹, Sebastian Thalmann⁴, Joshua A Welsh⁵, Christine Probst⁶, Coralié Guerin^{7

8}, Chantal M Boulanger^{7

9}, Jennifer C Jones⁵, Helmut Hanenberg¹⁰, Uta Erdbrügger¹¹, Joanne Lannigan¹², Franz L Ricklefs¹³, Samir El-Andaloussi^{2

3

14}, Bernd Giebel¹

Affiliations

¹ Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
² Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Stockholm, Sweden.
³ Evox Therapeutics Limited, Oxford, UK.
⁴ Luminex B.V., 's-Hertogenbosch, Netherlands.
⁵ Translational Nanobiology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
⁶ Amnis/Luminex, Seattle, WA, USA.
⁷ Paris Descartes University, Paris, France.
⁸ Institut Curie, cytometry core, PSL University, Paris, France.
⁹ INSERM, U970, Paris Cardiovascular Research Center-PARCC, Paris, France.
¹⁰ Department of Pediatrics III, University Children's Hospital Essen, University Duisburg-Essen, Essen, Germany.
¹¹ Department of Medicine, Nephrology Division, University of Virginia, Charlottesville, VA, USA.
¹² Flow Cytometry Core, University of Virginia School of Medicine, Charlottesville, VA, USA.
¹³ Department of Neurological Surgery, University Medical Center Hamburg Eppendorf, Hamburg, Germany.
¹⁴ Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

Abstract

Extracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases.

Keywords: CD63; Extracellular vesicles; exosomes; flow cytometry; imaging flow cytometry; microvesicles; reference material; standardisation; submicron particle analysis.

Grants and funding

B.G. received support from the Stem Cell Network North Rhine Westphalia, the LeitmarktAgentur.NRW and the European Union (European Regional Development Fund 2014–2020; ERA-NET EuroTransbio 11: EVtrust [031B0332B]; EU COST programme ME-HaD [BM1202]). S.E.A. is supported by the Swedish Research Council (VR-Med and EuroNanoMedII), Evox Therapeutics, Karolinska Institutet Faculty, Wibergs Stiftelse, SSF-IRC, Vinnova and the Swedish Society of Medical Research (SSMF). A.G. and J.A.W. are International Society for Advancement of Cytometry (ISAC) Marylou Ingram Scholars.