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. 2018 Jun 26;7(1):1490145.
doi: 10.1080/20013078.2018.1490145. eCollection 2018.

Enrichment of Extracellular Vesicles From Human Synovial Fluid Using Size Exclusion Chromatography

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Free PMC article

Enrichment of Extracellular Vesicles From Human Synovial Fluid Using Size Exclusion Chromatography

Andrew D Foers et al. J Extracell Vesicles. .
Free PMC article

Abstract

As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesicle (EV) enrichment using standard methods. In this study of human SF, a size exclusion chromatography (SEC)-based method of EV enrichment is shown to deplete contaminants that remain after standard ultracentrifugation-based enrichment methods. Specifically, considerable levels of serum albumin, the high-density lipoprotein marker, apolipoprotein A-I, fibronectin and other extracellular proteins and debris are present in EVs prepared by differential ultracentrifugation. While the addition of a sucrose density gradient purification step improved purification quality, some contamination remained. In contrast, using a SEC-based approach, SF EVs were efficiently separated from serum albumin, apolipoprotein A-I and additional contaminating proteins that co-purified with high-speed centrifugation. Finally, using high-resolution mass spectrometry analysis, we found that residual contaminants which remain after SEC, such as fibronectin and other extracellular proteins, can be successfully depleted by proteinase K. Taken together, our results highlight the limitations of ultracentrifugation-based methods of EV isolation from complex biological fluids and suggest that SEC can be used to obtain higher purity EV samples. In this way, SEC-based methods are likely to be useful for identifying EV-enriched components and improving understanding of EV function in disease.

Keywords: Synovial fluid; extracellular matrix; extracellular vesicles; fibronectin; high-density lipoprotein; rheumatoid arthritis; serum albumin; size exclusion chromatography; sucrose density gradient ultracentrifugation; ultracentrifugation.

Figures

Figure 1.
Figure 1.
Workflow of SF sample preparation and EV enrichments by differential ultracentrifugation, sucrose density gradient ultracentrifugation and SEC.
Figure 2.
Figure 2.
Analysis of EV enrichments from SF by differential ultracentrifugation. (a) EV pellets isolated by differential ultracentrifugation were assessed for the presence of canonical EV markers (syntenin, FLOT1, TSG101, Rab 27b, HSP70 and annexin 1) and specific contaminating proteins (serum albumin, ApoA-I and fibronectin) by western blot. Results are from a single SF donation obtained from a patient with inflammatory arthritis, and are representative of results observed with other donors. (b) Negative staining TEM analysis of differential ultracentrifugation EV isolations from two separate donors. EVs (black arrows) and amorphous material (white arrows) are indicated. Scale bars = 200 nm.
Figure 3.
Figure 3.
Analysis of EV enrichments from synovial fluid by sucrose density gradient ultracentrifugation. (a) Sucrose density gradient ultracentrifugation fractions were assessed for the presence of EV markers and specific contaminating proteins by Western blot with equal fraction volumes loaded. Results are from the same SF sample that was used for Figure 2b, and are representative of results observed with other donors. (b) Negative staining TEM analysis of EV containing fractions from two separate donors showing the presence of EVs (black arrows) and unspecified material (white arrows). Scale bars = 200 nm.
Figure 4.
Figure 4.
Analysis of EV enrichment from synovial fluid by SEC. (a) Chromatogram of absorbance at 280 nm versus elution time. SEC fractions were concentrated by ultracentrifugation at 100,000 x g and the resulting pellets assessed for: (b) protein abundance by SDS PAGE plus Coomassie staining, with loading proportional to fraction volume, (c) the presence and abundance of EV markers, serum albumin, ApoA-I and Fibronectin by Western blot, with loading proportional to fraction volume, and (d) the purity of the EV enrichment by Western blot with equal protein amount loaded into each well (fractions 1, 7 and 8 were not assessed due to insufficient amounts of protein). Results are from the same SF sample that was used for Figures 2b and 3b, and are representative of results observed with other donors. (e) Negative staining TEM analysis of non-concentrated SEC eluent from two separate donors, containing a mixture of EVs (black arrows) and contaminating material (white arrows). Scale bars = 200 nm.
Figure 5.
Figure 5.
Proteomic analysis of EVs isolated from synovial fluid of a rheumatoid arthritis patient. (a) Venn diagram comparing proteins identified by mass spectrometry analysis of SEC EVs in the presence or absence of proteinase K. About 652 unique proteins were identified without proteinase K treatment, and this number reduced to 270 following the addition of proteinase K. (b–d) GO analysis of the proteins identified from mass spectrometry analysis of proteinase K treated sample including cellular function and biological processes.

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Grant support

Supported by the National Health and Medical Research Council (GNT1064591) and the Reid Family Trust.

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