Table 2.

Major factors to consider when isolating EVs from sources relevant to cardiovascular studies

Source of EVs	Major factors to consider	Potential solutions
Cell culture conditioned medium containing serum	Risk of contamination from serum components including animal-derived EVs coming from serum	Contaminating EVs can be pre-removed from serum Consider using serum-free mediuma
Cell culture conditioned medium without serum	Risk of cell phenotypic changes/death contaminating EVs with intracellular or apoptotic vesicles	Use short-term culture Quantify levels of cell death
Plasma	Care must be taken not to activate platelets during collection and handling Platelets disrupt during a freeze–thaw cycle and hamper EV isolation Challenging to remove contaminating blood proteins and lipoproteins	Carefully define suitable pre-analytical procedures Isolate EVs using a combination of orthogonal techniques
Serum	EVs are released from activated platelets Challenging to remove contaminating blood proteins and lipoproteins EVs lost in the fibrin clot	Carefully define suitable pre-analytical procedures. Isolate EVs using a combination of orthogonal techniques.
Tissue (e.g. myocardium)	Challenging to disrupt tissue without damaging the cell membrane Risk of shaving epitopes from EVs when using proteolytic enzymes	Perform control experiments to ensure cells are not disrupted Titrate enzyme quantity and use the minimum

The importance of these points will vary depending on the intended use of the EVs, and must be evaluated separately for each experiment.

As noted in the main text, these solutions can introduce problems of their own.For example, EV removal from serum also removes other components, and it is probably not possible to remove 100% of the EVs. Serum-free medium may negatively affect cell health and EV quality.