Current Methods for Analysing Mesenchymal Stem Cell-Derived Extracellular Vesicles.

The use of extracellular vesicles (EVs) generated by mesenchymal stem cells (MSCs) holds great promise as a novel therapeutic approach. Although their immunomodulatory and regeneration potential has been reported to be similar to that of MSCs, the use of MSC-derived EVs in clinical settings will require several problems to be resolved. It is necessary to develop a standardised and widely accepted isolation technology and to improve methods such as the quantification and characterisation of MSC-derived EVs. In this way, EV studies can be compared, the acquired knowledge can be safely transferred to clinical platforms and the clinical results can be evaluated appropriately. There are many procedures for the collection and analysis of vesicles derived from different cells; however, this review provides an overview of methods for the determination of the total protein amount, specific proteins, particle number, non-protein markers like lipids and RNA, microscopy and other methods focusing on MSC-derived EVs.

Platelet activation and blood extracellular vesicles: The influence of venepuncture and short blood storage.

Extracellular vesicles (EVs) as membrane-bound particles released by various cells are potential tools for diagnosis and treatment. Blood cells, particularly platelets, are the source of circulating EVs. MATERIAL: EVs were enriched with gradient ultracentrifugation and measured by nanoparticle tracking assay. A flow cytometric multiplex assay was used for cellular source determination. Activation of platelets was measured as a percentage of CD62p+/CD61+ platelets and correlated with the concentration and size of released EVs. RESULTS: In general there was no statistically significant correlation between EVs` concentration and degree of platelet activation. EVs from different cellular sources were detected. Comparing different needle thicknesses, there was a decrease in the EVs concentration for the 16G needle versus the 21G needle, but no difference was observed for EVs` size and phenotype or platelets activation. During blood storage, platelet activation increased, but there was no effect on the EVs` concentration, size, or phenotype. CONCLUSIONS: Preanalytical factors like needle thickness and storage time can affect the MVs' properties. Activation of platelets during blood collection or blood storage occurs; however, it is difficult to determine its effect on the physiological properties of EVs since the mechanisms of EVs` biogenesis and especially clearness are not precisely known.