Detection and phenotyping of extracellular vesicles by size exclusion chromatography coupled with on-line fluorescence detection

Diána Kitka,Tamás Beke-Somfai,Judith Mihály,Zoltán Varga,Jean-Luc Fraikin

doi:10.1038/s41598-019-56375-1

Abstract

New methods for quantifying extracellular vesicles (EVs) in complex biofluids are critically needed. We report the development of a new technology combining size exclusion chromatography (SEC), a commonly used EV purification technique, with fluorescence detection of specifically labelled EVs. The resulting platform, Flu-SEC, demonstrates a linear response to concentration of specific EVs and could form the basis of a system with phenotyping capability. Flu-SEC was validated using red blood cell derived EVs (REVs), which provide an ideal EV model with monodisperse size distribution and high EV concentration. Microfluidic Resistive Pulse Sensing (MRPS) was used to accurately determine the size distribution and concentration of REVs. Anti-CD235a antibody, specific to glycophorin A, and the more general wheat germ agglutinin (WGA), were selected to label REVs. The results show the quantitative power of Flu-SEC: a highly linear fluorescence response over a wide range of concentrations. Moreover, the Flu-SEC technique reports the ratio of EV-bound and free-antibody molecules, an important metric for determining optimal labelling conditions for other applications. Flu-SEC represents an orthogonal tool to single-particle fluorescent methods such as flow cytometry and fluorescent NTA, for the quantification and phenotyping of EVs.

Highlights

New methods for quantifying extracellular vesicles (EVs) in complex biofluids are critically needed
Morphological characterization of red blood cell derived EVs (REVs) was performed by freeze-fracture combined transmission electron microscopy (FF-TEM), which preserves the native structure of EVs
The position of the EV peak confirms that REVs are completely excluded from the pores of the Sepharose CL-2B gel, which has a size exclusion limit of 40 × 106 Da for globular proteins: The total volume of the column is VT≈4 mL and EVs elute at 1.5 mL, which corresponds to the void volume (V0≈VT/3), and the EV size measured by Microfluidic Resistive Pulse Sensing (MRPS) (163 nm) exceeds the equivalent particle size exclusion limit

Summary

Introduction

New methods for quantifying extracellular vesicles (EVs) in complex biofluids are critically needed. We report the development of a new technology combining size exclusion chromatography (SEC), a commonly used EV purification technique, with fluorescence detection of labelled EVs. The resulting platform, Flu-SEC, demonstrates a linear response to concentration of specific EVs and could form the basis of a system with phenotyping capability. Microfluidic Resistive Pulse Sensing (MRPS) was used to accurately determine the size distribution and concentration of REVs. Anti-CD235a antibody, specific to glycophorin A, and the more general wheat germ agglutinin (WGA), were selected to label REVs. The results show the quantitative power of Flu-SEC: a highly linear fluorescence response over a wide range of concentrations. Red blood cell- (RBC-) derived EVs (REVs) were chosen to validate Flu-SEC, because REVs have a monodisperse size distribution from a single-cell origin and can be isolated in high concentration without other contaminating biological nanoparticles. This initial validation of the Flu-SEC technique paves the way for Flu-SEC-based phenotyping of EVs in complex body fluids

Methods

Results

Discussion

Conclusion