Comparison of Different Isolation Methods for Plasma-Derived Extracellular Vesicles in Patients with Hyperlipidemia

Abstract

Extracellular vesicles are commonly found in human body fluids and can reflect current physiological conditions of human body and act as biomarkers of disease. The quality of isolated extracellular vesicles facilitates the early diagnosis of various diseases accompanied by hyperlipidemia. Nonetheless, there are no reports on which special methods are suitable for isolating extracellular vesicles from the plasma of patients with hyperlipidemia. Thus, this study compared three different research-based extracellular vesicle isolation approaches, namely ultracentrifugation (UC), polyethylene glycol (PEG) precipitation, and size exclusion chromatography (SEC), and determined which of them was the most effective method. We selected blood samples from 12 patients with clinically diagnosed hyperlipidemia and isolated plasma-derived extracellular vesicles using three methods. The morphology of the isolated extracellular vesicles was observed using transmission electron microscopy, while the concentration was detected by asymmetric flow field-flow fractionation and multi-angle light scattering. Marker proteins were identified by Western blotting, and protein composition was evaluated by silver staining. Both determined the contaminations in the extracellular vesicle samples. The results showed that the three methods can be successfully used for the isolation of extracellular vesicles. The extracellular vesicles isolated by UC were larger in size, and the yield was much lower. Although the yield of extracellular vesicles isolated by PEG precipitation was greatly improved, the contamination was increased. Of the three methods, only the SEC-isolated extracellular vesicles were characterized by high yield and low contamination. Therefore, our data suggested that the SEC was a more ideal method for isolating extracellular vesicles from the plasma of patients with hyperlipidemia.

Effect of the 35 nm and 70 nm Size Exclusion Chromatography (SEC) Column and Plasma Storage Time on Separated Extracellular Vesicles

“…For the frozen sample data (n = 6), the 70 nm results of the comparison of the two types of columns were used. Based on our previous comparison of the 35 nm and 70 nm columns, which showed more efficient sEV isolation with less lipoprotein contamination in the 70 nm column, we isolated EVs collecting fractions 1-8 using the 70 nm column for this comparison [51].…”

Emerging Roles of Using Small Extracellular Vesicles as an Anti-Cancer Drug

“…Several comparative studies suggested that purification methods affect yield, population, and the therapeutic efficacy of sEVs [ 124 , 125 , 126 , 127 , 128 ]. A position paper from the International Society for Extracellular Vesicles categorized the purification methods of sEVs in terms of point of recovery and specificity [ 6 ].…”