Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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The impact of storage on extracellular vesicles: A systematic study.

Author(s): Gelibter S, Marostica G, Mandelli A, Siciliani S, Podini P, Finardi A, Furlan R

Publication: J Extracell Vesicles, 2022, Vol. 11, Page e12162

PubMed ID: 35102719 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare concentration, size, protein levels and zeta potential of EVs after frozen storage of plasma or isolated EVs in 8 different additives. The authors also investigated the effects of both the number and temperature of freeze-thaw cycles of isolated EVs on particle concentration, size, and protein levels.  EDTA blood was collected from five fasting donors and platelet poor plasma (PPP) was immediately separated by centrifugation at 3000 g for 15 min. Platelet free plasma (PFP) was then obtained by recentrifugation at 15000 g for 40 min at 4°C and filtration through a 0.22 µm filter. EVs were then isolated immediately or after storage of PFP at -80°C for 4 weeks and 6 months.  EVs were then isolated from PFP using qEV2 and qEVoriginal size exclusion chromatography columns and ultracentrifugation. To investigate the effects of EV storage, EVs from fresh PFP were analyzed after 4 weeks or 6 months of storage at −80°C without any preservative, at -80°C after the addition of a single preservative (25 mM Trehalose, 6% DMSO, 10% DMSO, 30% glycerol, Protease Inhibitor Cocktail, or 25 mM Trehalose and lyophilized), or at 4°C after the addition of 0.02% Sodium Azide.   Effects of freeze-thaw cycling were investigated by subjecting EVs to 1-3 cycles of snap freezing in liquid nitrogen and thawing at 37°C (snap) or freezing in a -80°C freezer and thawing on wet ice (slow). EV size distribution, concentration and ζ (zeta) potential were measured by Tunable Resistive Pulse Sensing on a qNano instrument. Altx, ANXA1, Flottlin-1, H3, Lamp1, ApoE, and GM130 levels were quantified by Western Blot. Contaminant protein concentrations were measured by Micro BCA Protein Assay Kit. EVs were visualized by transmission electron microscopy (TEM).

   Summary of Findings:

   TEM analysis of isolated EVs confirmed the presence of CD63-positive cup-shaped particles of the appropriate size; and Western blot analysis confirmed the presence of the EV proteins lysosomal associated membrane protein 1 (LAMP1), ALG-2-interacting protein X (ALIX), Flotillin-1, and Annexin A1 (ANXA-1) and the absence of the non-EV proteins apolipoprotein E (ApoE), histone H3 and golgi matrix protein 130 (GM130). EV concentration was significantly lower in EVs stored at -80°C for 4 weeks compared to fresh EVs (P=0.0085), and in EVs stored at -80°C for 6 months compared to those stored for 4 weeks (P=0.0044), but there was no effect of additives. EV concentration was significantly lower when plasma was stored at -80°C for 4 weeks compared to fresh plasma (P=0.0114) and in EVs stored at -80°C for 6 months compared to those stored for 4 weeks (P=0.0269).  Although the median zeta potential of EVs was unaffected by frozen storage of plasma or isolated EVs, the Zeta potential of a subset of particles noticeably shifted toward higher values when plasma or EVs were stored at -80C for 6 months.  Total protein concentrations (contaminant proteins) in EVs showed a non-significant increase with storage of either EVs (from 4.20 μg/mL in fresh to 14.28 μg/mL after 6 months, P=0.0934) or plasma (from 4.20 μg/mL in fresh to 24.72 μg/mL after 6 months, P=0.0934), but were unaffected by EV additive. Although unaffected by plasma storage, median EV size increased with storage of EVs (84.77 nm for fresh versus 10.7.40 after 6 months, P= 0.0239) as did  size distribution (P=0.0114), which occurred regardless of additive. Particle size was negatively associated with particle concentration (τb=−0.41, P = 0.033) but positively associated with contaminant protein concentration (τb = 0.371, P = 0.05) using Kendall’s tau-b correlation.

   EV concentration was significantly reduced when EVs underwent one freeze-thaw cycle (P=0.0003) but were not further reduced with additional cycles. The reduction in EV concentration following freeze-thaw cycling was comparable in specimens which were snap-cycled (frozen in liquid nitrogen and thawed at 37°C) and those that were slow-cycled (frozen at -80°C and thawed on wet ice). Particle size increased progressively with the number freeze-thaw cycles (P=0.0002 for snap-cycled and P<0.0001 for slow-cycled), with significant differences observed for each additional cycle; although no differences in particle size were observed between snap- and slow-cycled specimens. No change in protein concentration was observed after ≤2 cycles, and morphology by TEM was unaffected by freeze-thaw cycling. Finally, the authors observed EV during freeze-thaw cycling in cell lines.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen
   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Morphology	Electron microscopy
   Cell count/volume	Light scattering
   Protein	Western blot
   Protein	Colorimetric assay
   Cell count/volume	Electron microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Blood and blood products	Extracellular vesicles Platelet free plasma
   Storage	Short-term storage solution	25 mM Trehalose 6% DMSO 10% DMSO 30% Glycerol Protease Inhibitor Cocktail 25 mM Trehalose and lyophilized 0.02% Sodium Azide None
   Biospecimen Preservation	Type of fixation/preservation	Frozen Lyophilized None (fresh)
   Western blot Specific	Targeted peptide/protein	LAMP1 ALIX Flotillin-1 ANXA-1 ApoE Histone H3 GM130.
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles
   Storage	Thaw temperature/condition	37°C for specimens frozen in liquid nitrogen Wet ice for specimens frozen at -80°C
   Storage	Storage duration	0 days 4 weeks 6 months

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