Generation and Characterization of Cell-Derived Microvesicles from HUVECs

Abstract

Microvesicles (MVs) are of great interest for the biomedical field as novel diagnostics biomarkers. MV research, however, is still impaired by the lack of standardization of the analysis methods. In this study, our goals were to develop a method for the reproducible generation of an MV population from cell culture, to compare the sizing of MVs by atomic force microscopy (AFM) and nanoparticle tracking analysis (NTA) and to develop an MV array allowing the sizing and phenotyping of MVs simultaneously with the AFM as a read-out. MVs were isolated from apoptotic human umbilical vein endothelial cells (HUVECs) and had a mean size of 115 nm as measured by AFM and of 197 nm as measured by NTA. MVs isolated from apoptotic and control HUVECs could not be distinguished by their size (p > 0.05). HUVECs and their released MVs shared the same phenotype (positive for endothelial markers CD31 and CD146 and negative for platelet marker CD42b). Our method generated MVs with a concentration which was reproducible within a range of 20%. This is a relevant model system for medical applications. The size of the MVs was larger as measured using NTA compared to that using AFM. The MV array was efficient to characterize the surface markers of MVs and has the potential to identify the cellular origins of MVs in patient samples.