Technical challenges of working with extracellular vesicles

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review

Erythrocytes derived microvesicles : their characterisation and potential role as delivery vectors

Extracellular vesicles (EV) are heterogeneous populations of small vesicles released by virtually all cells and are classified depending on their biogenesis and size. Microvesicles (MV) are a subtype of EV and are released under optimal cell conditions and/or upon stimuli. In this study, the influence of erythrocyte derived MV (eMV) on the growth of THP-1 and Jurkat cell lines was observed. HeLa cells and those infected with Human Rhinovirus type 16 derived MV (HRV16iHMV) were used as parallel samples for all experiments. This is because virus infected cell derived EV have been extensively studied and it is understood that viral-MV uses the virus mechanism of entry to communicate with neighbouring cells. Whereas the eMV mechanism is still not fully understood. Therefore, to understand the mechanism of eMV action on the growth of cell lines, their characterisation was explored. Flow cytometry analysis using fluorescence sub-micron particle size reference indicates that the samples acquired during this PhD research meet the ‘current requirements’ in the field, to be classified as MV according to their sizes (100-1000 nm), and that the amount of release of eMV increases depending on the stimulus used (CaCl2, Normal Human Serum), due to remodelling of the cell membrane. Immunoblotting and flow cytometry surface marker profiling data shows that eMV are composed of key molecules that may allow their survival in the extracellular environment and communication with neighbouring cells. CD235 surface marker confirms their parent cells and the presence of CD47 suggests how eMV may avoid phagocytosis. Surface receptors including CD36, CD58 and CD63 indicate how they may communicate and enter the neighbouring cells through endocytosis. The identification of proteases suggests how eMV may be capable of tissue remodelling and degradation, whereas the presence of miRNAs may indicate how eMV can have an impact on the recipient cell by downregulating gene expression. Fluorescence microscopy shows eMV interacting with the cell membrane and the flow cytometry data suggests that eMV inhibit the growth of THP-1. The mechanism which clarifies this impact is unknown. Nevertheless, this PhD project has identified key biological molecules which constitute biologically active entities and might therefore, help eMV to transfer information and substances to recipient cells displaying their potential as delivery vectors. Thus, data obtained here can positively contribute towards the EV field