Working Group 1 - Guidelines for nomenclature and analysis

WG leader: Villem Stoorvogel

Co-WG leader: Clotilde Thery

Aims:

The various defining terms and methods for ME isolation, verification and analysis will be considered by all interested members of this Action to reach a consensus on the most appropriate terminology and methods moving forward. As detailed more below, this challenge is of fundamental importance and due consideration will be given to this when interpreting data related to Challenges 2-4 below. Furthermore, if and when new techniques for ME isolation and analysis become available, knowledge of these will be exchanged at the earliest stage possible among members of the COST Action.

Microvesicles and exosomes for analysis are typically isolated from medium conditioned by cultured cells, serum, plasma, saliva and other body fluids, based on size; through a range of methods, selected on the given Researcher’s preference but not standardised between European laboratories. These include series of filtration and/or ultracentrifugation techniques to separate nano-sized and larger vesicles, sometimes followed by flotation into sucrose-gradients to further purify sub-populations of similarly-sized small vesicles based on differential density and separate out non-membranous macromolecular complexes. What has yet to be determined is whether size is really a key differentiating characteristics between ME populations. What is also not currently understood is whether ME increase their size under certain pathological conditions that may confer stress on cells (link to WG3). Additionally, besides the commonly used filtration and centrifugation methods, flow cytometry (FC), chromatography, immunoadsorption/bead capture, microfluidics, and other commercially-available polymer-based extraction such as ExoQuickTM are reported.

Furthermore, basic quantification and characterisation of the resulting ME populations typically includes some combination of the following analysis i.e. protein content/surface proteins by Western blotting, transmission and/or scanning electron microscopy, FC, Dynamic Light Scatter (DLS), Nanoparticle Tracking Analysis based on Brownian motion (NTA), atomic force microscopy, and Mass Spectrometry (MS). A comparative evaluation of experience and success with all methods assessed and results generated will be of substantial help to both experienced and new researchers in this field in selecting the optimal combination(s) of methods for subsequent collaborative studies.

Tasks:

Evaluating currently used terminology for ME

Collecting current knowledge on terminology used in describing and defining microvesicles and/or exosomes and subsequently integrating, critically evaluating data;
Summarising and distribution of this data within ME-HAD to derive harmonisation and a consensus on what European researchers believe to be the most relevant terminology;
Dissemination of this information to all international researchers active in ME research.

Collating and evaluating methods used for ME isolation and analysis

Collating of current knowledge and expertise on methods and technologies for isolating microvesicles and/or exosomes (with emphasis not only on successful outcome, but also considering “negative” data to ensure we collectively learn from this);
Collating of information on methods and technologies for analysing ME; and integrating, critically-evaluating data;
Summarising and distribution this data within ME-HAD, in order to derived harmonisation and a consensus on ME-related methodology;
Dissemination of this information to international researchers active in ME research;
Develop and publish a Compendium “Handbook of Terminology &Techniques in Microvesicles & Exosomes Research”

Establishing principles for ME large-scale production

Advancing from the outcome of the above described two tasks, ME-HAD will establish principles for ME large-scale production, identified as necessary for future clinical applications.