Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2024 Aug;48(4):2775-2782.
doi: 10.1007/s11259-024-10405-0. Epub 2024 May 8.

Comparative characterisation of extracellular vesicles from canine and human plasma: a necessary step in biomarker discovery

Stephanie Marie Bollard  1   2   3 , J Howard  4   5 , C Casalou  6 , L Mooney  7 , S Peters  7 , C Sweeney  7 , A Ajaykumar  8 , K Triana  8 , A McCann  4   5 , P A Kelly  7 , S M Potter  4   9
Affiliations
Comparative Study

Comparative characterisation of extracellular vesicles from canine and human plasma: a necessary step in biomarker discovery

Stephanie Marie Bollard et al. Vet Res Commun. 2024 Aug.

Abstract

Extracellular Vesicles (EV) have become an interesting focus as novel biomarkers of disease and are increasingly reported upon in humans and other species. The Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines were published to improve rigor and standardisation within the EV field and provide a framework for the reliable isolation and characterisation of EV populations. However, this rigor and standardisation has been challenging in the area of comparative medicine. Herein we present the successful isolation of EVs from human and canine plasma using Size Exclusion Chromatography and characterise these EVs according to best international practice. This study provides evidence for the reliable comparison of human and canine EVs isolated by this approach, and a baseline description of the EVs from healthy dogs to inform future biomarker studies. This work also demonstrates that the MISEV2018 guidelines can be successfully applied to EVs isolated from canine plasma.

Keywords: Canine; Comparative medicine; Extracellular vesicles; OneHealth; Size exclusion chromatography.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Comparison of the elution profile of canine and human EVs isolated using SEC. Nanoparticle Tracking Analysis (NTA) and BCA protein assay were used to compare the elution profile of EVs isolated from 500µL plasma from the pooled plasma of n  = 10 dogs and a single healthy human, each across n  = 3 technical replicates. NTA demonstrates the concentration of EVs in each fraction isolated using the qEV Gen2 70 nm Izon column in (A) canine and (B) human plasma, with the majority of EVs eluting in the Purified Collection Volume (PCV) consisting of 4 fractions, following a Buffer Volume (BV) of 2500µL. Subsequent fractions showed an increase in protein concentration, indicating contamination. Purity examination using particle per µg protein concentration ratio, confirm the selected fractions are the least contaminated in (C) canine and (D) human samples
Fig. 2
Fig. 2
Size Exclusion Chromatography successfully isolates and enriches for small EVs (<200nm) from canine and human plasma.  The size distribution curves of the numbered fractions from the purified collection volume as measured using NTA are shown from (A) canine and (B) human plasma. Both species demonstrate a similar size distribution profile across the selected fractions, though a higher volume of small EVs (< 200 nm) was eluted in Fraction 2, after a buffer volume of 2500µL in the canine EVs (A) whereas the majority of small EVs (200 nm) were eluted in Fraction 3 and 4 for the human sample (B). Similarly, (C) the modal size of the EVs isolated within the purified collection volume was similar ( p = 0.10, Mann-Whitney test ) and < 200 nm in both species. TEM images taken at 87000x demonstrate the expected cup shape morphology of the EVs isolated from both (D) dogs and (E) human. In these images, the EVs as indicated by the black arrows are of the expected size, approximately 200 nm. Lipoproteins are indicated by red arrows and can be seen in the preparations from both species. Wide-field images of the EVs of both species are seen in Supplemental Fig. 2
Fig. 3
Fig. 3
Comparative Western Blot analysis of isolated canine and human EVs showing similar EV marker profile. EVs isolated from pooled plasma of dogs (n  = 10) in triplicate and individual human (n  = 3) donors were lysed, concentrated, and analysed using western blot according to the MISV2018 guidelines. All EVs were positive for HSP70, CD63 and ApoA1, and negative for calnexin, as expected for small EVs < 200 nm isolated using SEC. Representative images are shown
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Almiñana C, Rudolf Vegas A, Tekin M, Hassan M, Uzbekov R, Fröhlich T, Bollwein H, Bauersachs S (2021) Isolation and characterization of equine uterine extracellular vesicles: a comparative methodological study. Int J Mol Sci 22. 10.3390/ijms22020979 - PMC - PubMed
    1. Brennan K, Martin K, FitzGerald SP, O’Sullivan J, Wu Y, Blanco A, Richardson C, Mc Gee MM (2020) A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum. Sci Rep 10(1):1039 10.1038/s41598-020-57497-7 - DOI - PMC - PubMed
    1. da Silva Nunes PC, Mazzarella R, da Silveira JC, Dellova DCAL (2022) Evaluation of circulating extracellular vesicles and miRNA in neutered and obese female dogs. Sci Rep 12(1):16439 10.1038/s41598-022-20523-x - DOI - PMC - PubMed
    1. Eirin A, Ferguson CM, Zhu X-Y, Saadiq IM, Tang H, Lerman A, Lerman LO (2020) Extracellular vesicles released by adipose tissue-derived mesenchymal stromal/stem cells from obese pigs fail to repair the injured kidney. Stem Cell Res 47:101877 10.1016/j.scr.2020.101877 - DOI - PMC - PubMed
    1. Gordon I, Paoloni M, Mazcko C, Khanna C (2009) The comparative oncology trials consortium: using spontaneously occurring cancers in dogs to inform the cancer drug development pathway. PLoS Med 6(10):e1000161 - PMC - PubMed

Publication types

LinkOut - more resources

  NODES
INTERN 2
twitter 2