Development of a Sampling and Storage Protocol of Extracellular Vesicles (EVs)—Establishment of the First EV Biobank for Polytraumatized Patients

Author:

Weber Birte1,Ritter Aileen1,Han Jiaoyan1,Schaible Inna1,Sturm Ramona1,Relja Borna2,Huber-Lang Markus3ORCID,Hildebrand Frank4,Pallas Christiane5,Widera Marek5ORCID,Henrich Dirk1ORCID,Marzi Ingo1ORCID,Leppik Liudmila1ORCID

Affiliation:

1. Department of Trauma-, Hand- and Reconstructive Surgery, University Hospital Frankfurt, Goethe University, 60486 Frankfurt am Main, Germany

2. Translational and Experimental Trauma Research, Department of Trauma, Hand, Plastic and Reconstructive Surgery, University Hospital Ulm, 89081 Ulm, Germany

3. Institute of Clinical and Experimental Trauma-Immunology, University Hospital Ulm, 89081 Ulm, Germany

4. Department of Trauma and Reconstructive Surgery, University Hospital RWTH Aachen, 52074 Aachen, Germany

5. Institute for Medical Virology, University Hospital Frankfurt, Goethe University, 60596 Frankfurt am Main, Germany

Abstract

In the last few years, several studies have emphasized the existence of injury-specific EV “barcodes” that could have significant importance for the precise diagnosis of different organ injuries in polytrauma patients. To expand the research potential of the NTF (network trauma research) biobank of polytraumatized patients, the NTF research group decided to further establish a biobank for EVs. However, until now, the protocols for the isolation, characterization, and storage of EVs for biobank purposes have not been conceptualized. Plasma and serum samples from healthy volunteers (n = 10) were used. Three EV isolation methods of high relevance for the work with patients’ samples (ultracentrifugation, size exclusion chromatography, and immune magnetic bead-based isolation) were compared. EVs were quantified using nanoparticle tracking analysis, EV proteins, and miRNAs. The effects of different isolation solutions; the long storage of samples (up to 3 years); and the sensibility of EVs to serial freezing–thawing cycles and different storage conditions (RT, 4/−20/−80 °C, dry ice) were evaluated. The SEC isolation method was considered the most suitable for EV biobanking. We did not find any difference in the quantity of EVs between serum and plasma-EVs. The importance of particle-free PBS as an isolation solution was confirmed. Plasma that has been frozen for a long time can also be used as a source of EVs. Serial freezing–thawing cycles were found to affect the mean size of EVs but not their amount. The storage of EV samples for 5 days on dry ice significantly reduced the EV protein concentration.

Funder

Deutsche Forschungsgemeinschaft

Publisher

MDPI AG

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