The impact of storage on extracellular vesicles: A systematic study

Area of Science:

• Biotechnology
• Analytical Chemistry
• Cell Biology

Background:

• Extracellular vesicles (EVs) are crucial biomarkers, but their properties can be affected by storage conditions.
• Standard -80°C storage is common, yet its effects and potential improvements remain debated.
• Understanding storage impacts is vital for reliable EV-based research and diagnostics.

Purpose of the Study:

• To systematically evaluate the impact of -80°C storage and freeze-thaw cycles on extracellular vesicle (EV) characteristics.
• To investigate potential EV fusion phenomena induced by storage conditions.
• To compare eight different storage strategies for their effects on EV integrity.

Main Methods:

• EVs isolated from human plasma and murine microglia using size exclusion chromatography and ultracentrifugation.
• Analysis of concentration, size, zeta potential, and purity using tunable resistive pulse sensing and contaminant protein assays.
• Flow cytometry used to track fluorescently tagged EV populations (GFP and mCherry) to detect fusion.

Main Results:

• -80°C storage decreased EV concentration and purity over time, increasing particle size and variability.
• Storage altered EV zeta potential, shifting their position on size-charge plots.
• Freeze-thaw cycles reduced EV numbers and increased particle size, with observed double-positive EVs suggesting fusion.

Conclusions:

• Storage significantly impacts EV sample integrity, leading to particle loss, reduced purity, and fusion.
• Current storage methods, including -80°C and freeze-thaw cycles, induce artefactual changes in EVs.
• Processing fresh, non-archival samples is advisable for most extracellular vesicle analyses.