Ann Lab Med.  2020 May;40(3):253-258. 10.3343/alm.2020.40.3.253.

Isolation of Small Extracellular Vesicles From Human Serum Using a Combination of Ultracentrifugation With Polymer-Based Precipitation

• Ryu KJ
• Lee JY
• Park C
• Cho D
• Kim SJ

• KMID: 2466019
• DOI: http://doi.org/10.3343/alm.2020.40.3.253

Abstract

Methods for reproducibly isolating and enriching small extracellular vesicles (EVs) from blood are essential for clinical utilization of small EVs in cancer patients. We combined ultracentrifugation (UC) with polymer-based precipitation (ExoQuick [EQ] or Total Exosome Isolation [TEI] kit) to isolate small EVs (diameter, 30-150 nm) from the serum of breast cancer patients. We compared the performance of four cycles of UC (UC4x) with that of two cycles of UC followed by enrichment using the EQ (UC2x→EQ) or TEI (UC2x→TEI) kits. The mean concentration of small EVs isolated from 1 mL of serum using UC2x→EQ (139.0±29.1 µg) and UC2x→TEI (140.4±5.0 µg) did not differ from that obtained using UC4x (141.8±26.9 µg). The mean number of EV particles obtained using UC4x was 29.2±9.9×109 per mL of serum, whereas UC2x→EQ and UC2x→TEI yielded higher numbers of EVs (50.7±17.0×10⁹ and 59.3±20.6×10⁹, respectively). Concentrations of EV microRNAs, including miR-21 and miR-155, did not differ between the three methods. In conclusion, performing UC prior to the use of polymer-based precipitation kits could be feasible for isolating small EVs from human serum in large sample-based translational researches.

Keyword

Extracellular vesicle; Precipitation; Ultracentrifugation; Isolation; Performance

MeSH Terms

Breast Neoplasms
Extracellular Vesicles*
Humans*
MicroRNAs
Translational Medical Research
Ultracentrifugation*
MicroRNAs

Figure

• 

  Fig. 1 Comparison of EV isolation methods. (A) Study overview: three methods for isolating EVs from serum samples of breast cancer patients. (B) Silver-stained polyacrylamide gel of supernatants from the first to the final EV enrichment steps. (C) Silver-stained polyacrylamide gel of small EVs enriched after the final step. (D) Western blotting analysis for CD63 and albumin in serum and small EVs isolated using the three methods (UC4x: four cycles of UC; UC2x→EQ: two cycles of UC followed by enrichment using the EQ kit; UC2x→TEI: two cycles of UC followed by enrichment using the TEI kit). Ten micrograms of protein were used from each sample. Abbreviations: EV, extracellular vesicle; UC, ultracentrifugation; EQ, ExoQuick; TEI, Total Exosome Isolation kit; PBS, phosphate-buffered saline; IgGH, immunoglobulin G heavy chain; IgGL, immunoglobulin G light chain.

• 

  Fig. 2 Characterization of extracellular vesicle. (A) Protein concentrations in small EVs isolated using the three methods. (B) Transmission electron microscopy images of small EVs; size bar=100 nm. (C) Western blotting analysis for Alix, CD63, TSG101, and CD81 using 10 µg of small EVs isolated using the three methods (UC4x: four cycles of UC; UC2x→EQ: two cycles of UC followed by enrichment using the EQ kit; UC2x→TEI: two cycles of UC followed by enrichment using the TEI kit). (D) Size distribution of small EVs and comparison of three methods using nanoparticle tracking analysis. (E-G) Comparison of mean number of EV particles, mean size, and mode size among the three methods. The data represent the mean±SD of three independent experiments. Abbreviations: see Fig. 1.

• 

  Fig. 3 Analysis of miRNA cargo in EV. (A) The quality of EV RNA was evaluated using an Agilent2100 bioanalyzer with an RNA 6000 Pico kit. (B) miR-21 and miR-155 concentrations in the small EVs did not differ among the three methods. (C) Serum concentrations of miR-21 and miR-155. (D-F) miR-101, miR-223, and miR-451a concentrations did not differ among the three methods. The data represent the mean±SD of three independent experiments. Boxes and whiskers indicate the minimum–maximum, and the lines represent the median of three independent experiments (B, D, E, and F). Abbreviations: miRNA, microRNA; EV, extracellular vesicle; UC, ultracentrifugation; EQ, ExoQuick; TEI, Total Exosome Isolation kit; UC4x, four cycles of UC; UC2x-EQ, two cycles of UC followed by enrichment using the EQ kit; UC2x-TEI, two cycles of UC followed by enrichment using the TEI kit.

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