Population Analysis of Extracellular Vesicles in Microvolumes of Biofluids

Abstract

Extracellular Vesicles (EVs), membrane vesicles released by all cells, are emerging mediators of cell-cell communication. By carrying biomolecules from tissues to biofluids, EVs have attracted attention as non-invasive sources of clinical biomarkers in liquid biopsies. Although frequently employed for content characterization of EVs, the study of bulk preparations lacks information on sub-populations and the intrinsic heterogeneity of vesicles. Importantly, these strategies also difficult the characterization of EVs from small quantities of samples. We here present a Flow Cytometry strategy that enables detailed population analysis of EVs, at the same time decreasing sample volume requirements and accelerating the overall processing time. We show its unique application for quality control of isolates of EVs by comparing the proportion of vesicular and non-vesicular particles in samples prepared by different protocols. In addition, we demonstrate its suitability for the study of populations of EVs from samples characterized by challenging small volumes. To illustrate that, we perform longitudinal non-lethal analysis of EVs in mouse plasma and in single-animal collections of murine vitreous humor. By allowing for the analysis of EVs from minimal amounts of sample, our Flow Cytometry strategy has an unexplored potential in the study of EVs in clinical samples with intrinsically limited volumes. When compared to conventional methods, it also multiplies by several times the number of different analytes that can be studied from a single collection of biofluid.

Extracellular Vesicles Enriched in hsa-miR-301a-3p and hsa-miR-1293 Dynamics in Clear Cell Renal Cell Carcinoma Patients: Potential Biomarkers of Metastatic Disease

“…A transmission electron microscopy (TEM) image ( Figure 1 B) shows the variability of sizes and morphology present in EVs from purified PFP. We also utilized EVs flow cytometry to confirm the purity of our EV isolates [ 39 ]. In all cases, more than 80% of particles present in our isolates corresponded to CFSE + vesicular structures ( Figure 1 C).…”

“…We employed EVs flow cytometry procedure for quantification of vesicular structures in our EV isolates, as recently described by Maia and colleagues [ 39 ]. Briefly, 2 × 10 9 particles of purified EVs were mixed with 40 µL of PBS containing Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE – Thermo Fisher Scientific-LTI C34554, Waltham, MA, USA) in a final concentration of 40 µM and incubated for 90 min at 37 °C.…”

Plasma Extracellular Vesicle-Derived TIMP-1 mRNA as a Prognostic Biomarker in Clear Cell Renal Cell Carcinoma: A Pilot Study

“…We employed EVs flow cytometry for quantification of vesicular structures in our EV isolates, as recently described by Maia et al [ 47 ]. In total, 2 × 10 9 particles of purified EVs were mixed with 40 µL of PBS containing Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE—Thermo Fisher Scientific—LTI C34554, Waltham, MA, USA) in a final concentration of 40 µM and incubated for 90 min at 37 °C.…”

New strategies of neurodegenerative disease treatment with extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs)

“…The uptake of EVs by cells is another method of entry. Endothelial cells and pancreatic cells internalize a greater proportion of lymph node stroma-derived EVs in Tspan8, while parental lymph node stroma cells internalize a lesser percentage [134]. Furthermore, peritoneal exudate cells are more efficient at internalizing EVs derived from pancreatic adenocarcinoma than granulocytes or T cells.…”