Abstract
Exosome-like small-extracellular vesicles (sEVs) are extracellular vesicles that act in intercellular communication and are involved in several biologic and pathologic processes. While sEVs increase the stability of their cargo molecules, there is still a need for standardization of sampling and isolation of these microvesicles. We aimed to determine the best sampling method for isolation of sEVs from peripheral blood from reproductive-aged women. Material and Methods: We included samples of plasma from our biobank collected in 2014 by venipuncture in heparin tubes and stored at -80°C. We also included blood samples collected in heparin tubes and Ethylenediamine tetraacetic acid (EDTA) tubes and stored at -80°C for one to two weeks prior processing. All blood samples were collected from the same nine reproductive-aged female volunteers. sEVs were isolated from plasma by ultracentrifugation and filtration and indirectly quantified using Pierce BCA Protein Assay kit. Transmission electron microscopy (TEM) and Nano Tracking Analysis (NTA) were performed to confirm the isolation of sEVs. Results and Discussion: TEM and NTA confirmed the isolation of sEVs. Protein concentration of short-time stored heparin samples was not statistically different from long-time stored heparin samples (1847.2 ± 651.4 vs. 2363.2 ± 1025.1, p = 0.14). There was no difference between heparin and EDTA plasma samples recently collected (2363.2 ± 1025.1 vs. 2044.8 ± 653.2, p = 0.44). In conclusion, blood samples may be collected using heparin or EDTA for isolation of sEVs. Long-time stored plasma samples maintain sEVs integrity and may be used, especially in comparative studies.
Highlights
Exosome-like small-extracellular vesicles are doubled-membrane vesicles with a diameter that varies between 30 to 150 nm that along with microvesicles and apoptotic bodies form the heterogeneous group of extracellular vesicles [1] [2]
Exosome-like small-extracellular vesicles are extracellular vesicles that act in intercellular communication and are involved in several biologic and pathologic processes
We included blood samples collected in heparin tubes and Ethylenediamine tetraacetic acid (EDTA) tubes and stored at −80 ̊C for one to two weeks prior processing
Summary
Exosome-like small-extracellular vesicles (sEVs) are doubled-membrane vesicles with a diameter that varies between 30 to 150 nm that along with microvesicles and apoptotic bodies form the heterogeneous group of extracellular vesicles [1] [2]. SEVs are important regulators of immune response and act as crucial mediators of cellular communication in several physiologic and pathologic processes by serving as vehicles for transfer of different molecules between cells [4] [5]. They have been described to carry a variety of molecules such as membrane and cytosolic proteins, lipids and nucleic acids [6]. A third biogenesis mechanism that has been proposed is tetraspanin-mediated organization of specific proteins such as the amyloidogenic protein pre-melanosome protein [7]
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