Abstract
Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4–10), osmolarity (50–1000 mOsm/L), temperature (15–60 °C), and surfactant Triton X-100 (10–500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius (Rh), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS–citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50–1000 mOsm/L, pH 4–10) had no significant effect on the Rh (=100–130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.
Highlights
Extracellular vesicles (EVs) are a heterogeneous group of nano- to micro-sized membraneenclosed particles that are found in almost every sample of biological origin [1]
The hbEVs were isolated by a differential centrifugation protocol (Methods) from an erythrocyte suspension that had been stored at 4 ◦ C for 6 days (SEM images of the pellets in selected steps of the isolation are provided in Appendix A)
The results shown in this work (Figure 1), the results of previous studies of erythrocyte blebbing in the presence amphiphilic molecules [73], and the lack of evidence for any other mechanism leading to the formation of small particles in this system indicate that the particles in the supernatants of the suspensions of the washed erythrocytes were formed by the outward budding of the plasma membrane
Summary
Extracellular vesicles (EVs) are a heterogeneous group of nano- to micro-sized membraneenclosed particles that are found in almost every sample of biological origin [1]. They function as a means of intercellular communication [1,2,3,4] and are involved in several physiological and pathological contexts, such as in embryogenesis [5,6,7,8,9], neuronal communication [10], blood coagulation [11,12], inflammation [13,14], tumorigenesis [1,15], and horizontal gene transfer [7,8,16]. Even when the same type of equipment and protocols are used, the reproducibility of isolation tends to be poor [22,29]
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