Development of a Sampling and Storage Protocol of Extracellular Vesicles (EVs)-Establishment of the First EV Biobank for Polytraumatized Patients.
Author(s): Weber B, Ritter A, Han J, Schaible I, Sturm R, Relja B, Huber-Lang M, Hildebrand F, Pallas C, Widera M, Henrich D, Marzi I, Leppik L
Publication: Int J Mol Sci, 2024, Vol. 25, Page
PubMed ID: 38891833 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of extracellular vesicle (EV) isolation method (ultracentrifugation, immune magnetic bead-based kit, or size exclusion chromatography (SEC) with three different Dulbecco’s phosphate buffered saline (dPBS) types), use of serum versus plasma, plasma storage (≤3 years at -80°C), storage of isolated EV’s (≤5 days at room temperature, 4°C, -20°C, -80°C, or on dry ice), and freeze-thaw cycling of isolated EVs (0-5 cycles) on EV particle count, and EV protein and miRNA concentration.
Conclusion of Paper
EVs suitable for miRNA extraction and detection of EV markers by Western blot were obtained using all three EV isolation methods, but the authors chose SEC for all further analysis because of its scalability and suitability for all downstream analyses. Although no significant effects of dPBS type on particle count, or protein or miRNA concentration were identified, use of unfiltered dPBS or dPBS with Ca/Mg resulted in higher background than use of filtered dPBS without Ca/Mg. Although there was a tendency toward fewer EVs and lower protein and miRNA concentrations from serum than plasma, the differences were not significant. Particle count was higher in EVs after a single (but not 2-5) freeze-thaw cycle(s) compared to freshly isolated EVs, but particle count was unaffected by storage of plasma for up to 3 years at -80°C or storage of isolated EVs at room temperature, 4°C, -20°C, -80°C, or on dry ice for up to 5 days. Protein concentration was higher in EVs from plasma stored for 3 years than a few weeks at -80°C (P<0.01) and lower in EVs store on dry ice for 5 days than in fresh EVs, but protein concentrations were comparable between freshly isolated EVs and those freeze-thaw cycled up to five times, and those stored at room temperature, 4°C, -20°C, or-80°C for ≤5 days or on dry ice for ≤4 days. miRNA concentration was lower in EVs freeze-thaw cycled once (but not 2-5 times) than in fresh EVs. miRNA concentration was not altered after storage plasma for up to 3 years at -80°C, or storage of isolated EVs at room temperature, 4°C, -20°C, -80°C, or on dry ice for ≤3 days. Mean EV size was larger in EV samples that underwent 2-4 (but not 5) freeze-thaw cycles than in fresh EVs. Particle counts were modestly correlated with protein content as measured by Bradford Assay (r=0.42, P<0.05).
Studies
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Study Purpose
This study investigated the effects of EV isolation method (ultracentrifugation, immune magnetic bead-based kit, or SEC with three different dPBS types), use of serum versus plasma, plasma storage (≤3 years at -80°C), and isolated EV storage (≤5 days at room temperature, 4°C, -20°C, -80°C, or on dry ice) and freeze-thaw cycling (0-5 cycles) on EV particle count, and EV protein and miRNA concentrations. Blood was collected from ten healthy volunteers and stored on ice until plasma/serum isolation (no other details of blood collection were provided). EDTA plasma and serum were separated by centrifugation at 3500 rpm and 4°C for 15 min. To investigate the effects of extraction method, EVs were isolated from serum and plasma using ultracentrifugation or the size exclusion chromatography (SEC) with Exo-Spin columns and the immune magnetic bead-based Exosome Isolation Kit Pan. The ultracentrifugation procedure included debris removal by centrifugation of plasma/serum at 4°C for 10 min at 2000 × g, followed by a 30 min centrifugation at 10,000 × g, then a 2 h 100,000 × g centrifugation at 4°C to pellet EVs, EV washing in filtered Dulbecco’s phosphate buffered saline (dPBS) without Ca/Mg and precipitation by centrifugation at 4°C for 1 h at 100,000× g, then suspension in filtered Dulbecco PBS without Ca/Mg. To test the effects of dPBS type, SEC was performed using unfiltered dPBS without Ca/Mg, filtered dPBS without Ca/Mg, and unfiltered dPBS with Ca/Mg. Unless otherwise specified, EVs were isolated by SEC. To investigate the effects of plasma/serum storage, EVs were isolated from five plasma and five serum specimens stored at -80°C for 1, 2, and 3 years and compared to EVs isolated after a few weeks. To investigate the effects of freeze-thaw cycling on isolated EVs, EVs were isolated by SEC from plasma and were analyzed after each of five cycles of freezing at -80°C for >24 h and thawing at room temperature. EVs were quantified and analyzed for size distribution by nanoparticle tracking analysis. EV protein content was quantified by the Coomassie Plus Bradford Assay. miRNAs were extracted using the miRNeasy Serum/Plasma Kit and quantified by spectrophotometer.
Summary of Findings:
While EVs suitable for miRNA extraction and detection of EV markers by Western blot were obtained using all three EV isolation methods, the authors found ultracentrifugation required expensive equipment and was not scalable, and that magnetic beads remained in EVs isolated using the kit that could interfere with downstream assays. Consequently, SEC was used for all further analysis. Although no significant effect of dPBS type was identified on particle count or protein or miRNA concentration, use of unfiltered dPBS or dPBS with Ca/Mg resulted in a higher background than use of filtered dPBS without Ca/Mg. Although there was a tendency toward fewer EVs and lower protein and miRNA concentrations from serum than plasma, the differences were not significant. Particle count and miRNA concentration were not significantly affected by storage of plasma for up to 3 years at -80°C, but the protein concentration was higher from plasma stored for 3 years than plasma stored for a few weeks (P<0.01). While EVs freeze-thaw cycled once had more particles and less miRNA than freshly isolated EVs (P<0.05, both), neither mean size nor protein concentration was affected. Mean EV size was larger in EV samples that underwent 2-4 (but not 5) freeze-thaw cycles than in fresh EVs (P<0.001 for 2 cycles, P<0.01 for 3 cycles, and P<0.05 after 4 cycles), but there were no differences in particle count or protein or miRNA concentration. Protein concentration was lower in EVs stored on dry ice for 5 days than in fresh EVs (P<0.01), but particle and protein concentration were otherwise unaffected by storage of isolated EVs at room temperature, 4°C, -20°C, -80°C, or on dry ice for up to 5 days. Similarly, storage of isolated EVs at room temperature, 4°C, -20°C, -80°C, or on dry ice for up to 3 days (longest timepoint examined) did not affect miRNA concentration. Particle counts were modestly correlated with protein content as measured by Bradford Assay (r=0.42, P<0.05).
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Spectrophotometry Cell count/volume Light scattering Protein Colorimetric assay Morphology Light scattering Protein Western blot Protein Flow cytometry RNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma vesicles
Small extracellular vesicles
Analyte Extraction and Purification Analyte isolation method Ultracentrifugation
Size exclusion chromatography
Immune magnetic bead-based isolation
Storage Storage temperature Room temperature
4°C
-20°C
-80°C
On dry ice
Storage Storage duration 1 day
2 days
3 days
4 days
5 days
A few weeks
1 year
2 years
3 years
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
3 cycles
4 cycles
50 cycles
Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)