Isolation of Human Small Extracellular Vesicles and Tracking of Their Uptake by Retinal Pigment Epithelial Cells In Vitro

Abstract

Small extracellular vesicles (EVs) are among the most frequently investigated EVs and play major roles in intercellular communication by delivering various cargo molecules to target cells. They could potentially represent an alternative delivery strategy to treat ocular toxoplasmosis, a parasitosis affecting the retinal pigment epithelium (RPE). To date, the uptake of human small EVs by RPE cells has never been reported. In this study, we report on the intracellular uptake of fluorescently labelled human urine and fibroblast-derived small EVs by human RPE cells. In summary, both dye-labelled urinary small EVs and small EVs obtained from fibroblasts stably expressing membrane-bound green fluorescent protein were successfully internalized by RPE cells as revealed by immunohistochemistry. In recipient ARPE19 cells, BODIPY-labelled small EVs were found in close vicinity to the parasite Toxoplasma gondii. Additionally, an ultrastructural method was enabled to distinguish between labelled exogenous and endogenous small EVs within target cells.

Characterization of bovine uterine fluid extracellular vesicles proteomic profiles at follicular and luteal phases of the oestrous cycle

“…In the current study, we measured the proportion of particles labelled with CMG resulting to lower particle counts compared to measurements without the dye, which indicate that only a proportion of particles measured with the conventional NTA could be EV. This result is in line with previous studies, which have measured proportionately lower fluorescently labelled particles compared to total nanoparticles in cell culture media (Midekessa et al 2021 ) and human urine samples (Marcu et al 2020 ). Hence, alternative methods to conventional NTA and standardization of these measurements are necessary to accurately measure EV counts needed for biomedical function and for the understanding their biological properties (Hartjes et al 2019 ).…”

Characterization of Extracellular Vesicles Labelled with a Lipophilic Dye Using Fluorescence Nanoparticle Tracking Analysis

“…Different strategies have been developed for membrane labeling of EVs using fluorescent dye molecules to examine EV uptake into target cells and for tracking of EVs [9][10][11] and to follow their fate once taken up by cells [12]. Various commercially available fluorescent dyes, such as PKH67 [13], DiO [14], DiI [15], DiD [16], DiR [17], CFSE [18], and CellMask™ Green (CMG), have been extensively used for the membrane labeling of EVs.…”