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Size Separation of Exosomes and Microvesicles Using Flow Field-Flow Fractionation/Multiangle Light Scattering and
Young Beom Kim1, Gwang Bin Lee1, Myeong Hee Moon1
1Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seoul 03722, South Korea.
Analytical Chemistry
|June 13, 2022
Summary
This study shows ultrafiltration (UF) combined with flow field-flow fractionation (FlFFF) and multiangle light scattering (MALS) effectively separates exosomes and microvesicles by size. This method enhances detection and lipidomic profiling of extracellular vesicles (EVs).
Area of Science:
- Biochemistry
- Cell Biology
- Nanotechnology
Background:
- Extracellular vesicles (EVs), including exosomes and microvesicles, are crucial for intercellular communication.
- Current isolation methods like ultracentrifugation (UC) and ultrafiltration (UF) have limitations in efficiency and size-based separation.
- Understanding EV heterogeneity is vital for their application in diagnostics and therapeutics.
Purpose of the Study:
- To develop and validate a method for simultaneous size-based separation of exosomes and microvesicles.
- To improve the detection sensitivity of low-concentration EV species.
- To analyze the size-dependent lipidomic profiles of separated EV subpopulations.
Main Methods:
- Comparison of ultracentrifugation (UC) and ultrafiltration (UF) for EV isolation from cell culture media.
- Application of flow field-flow fractionation (FlFFF) with field programming for simultaneous size separation.
- Utilized multiangle light scattering (MALS) for enhanced detection and size determination (root-mean-square radius).
- Western blotting and transmission electron microscopy (TEM) for EV characterization.
- Nanoflow ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS) for lipidomic analysis.
Main Results:
- Ultrafiltration (UF) yielded higher EV recovery compared to ultracentrifugation (UC).
- FlFFF successfully separated exosomes and microvesicles into distinct small and large size fractions.
- MALS significantly improved the detection of low-abundance EV populations.
- Lipidomic analysis revealed size-dependent lipid enrichment patterns, with larger exosomes showing higher enrichment and microvesicles showing the opposite trend.
- Distinct lipid profiles were observed between small and large exosomes and microvesicles.
Conclusions:
- UF followed by FlFFF-MALS provides an efficient, sequential centrifugation-free approach for size-based separation of exosomes and microvesicles.
- This integrated technique enhances EV detection and enables detailed size-dependent lipidomic profiling.
- The method is valuable for monitoring EV size distribution changes in different biological states and for biomarker discovery.
