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The separation and characterization of extracellular vesicles from medium conditioned by bovine embryos

Krishna Pavani (UGent) , Xiaoyuan Lin (UGent) , Joachim Hamacher, Wim Van Den Broeck (UGent) , Liesbeth Couck (UGent) , Luc Peelman (UGent) , An Hendrix (UGent) and Ann Van Soom (UGent)
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Abstract
Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst embryos. They are secreted into the embryo culture medium and, as such, represent a source of novel biomarkers for identifying the quality of cells and embryos. However, only small amounts of embryo-conditioned medium are available, which represents a challenge for EV enrichment. Our aim is to assess the suitability of different EV separation methods to retrieve EVs with high specificity and sufficient efficiency. Bovine embryo-conditioned medium was subjected to differential ultracentrifugation (DU), OptiPrep(TM) density gradient (ODG) centrifugation, and size exclusion chromatography. Separated EVs were characterized by complementary characterization methods, including Western blot, electron microscopy, and nanoparticle tracking analysis, to assess the efficiency and specificity. OptiPrep(TM) density gradient centrifugation outperformed DU and SEC in terms of specificity by substantial removal of contaminating proteins such as ribonucleoprotein complexes (Argonaute-2 (AGO-2)) and lipoproteins (ApoA-I) from bovine embryo-derived EVs (density: 1.02-1.04, 1.20-1.23 g/mL, respectively). In conclusion, ODG centrifugation is the preferred method for identifying EV-enriched components and for improving our understanding of EV function in embryo quality and development.
Keywords
Physical and Theoretical Chemistry, Inorganic Chemistry, Organic Chemistry, Spectroscopy, Molecular Biology, Catalysis, General Medicine, Computer Science Applications, extracellular vesicles, isolation, protocols, ultracentrifugation, size exclusion chromatography, Optiprep(TM) density gradient, CHROMATOGRAPHY

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MLA
Pavani, Krishna, et al. “The Separation and Characterization of Extracellular Vesicles from Medium Conditioned by Bovine Embryos.” INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 21, no. 8, 2020, doi:10.3390/ijms21082942.
APA
Pavani, K., Lin, X., Hamacher, J., Van Den Broeck, W., Couck, L., Peelman, L., … Van Soom, A. (2020). The separation and characterization of extracellular vesicles from medium conditioned by bovine embryos. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 21(8). https://doi.org/10.3390/ijms21082942
Chicago author-date
Pavani, Krishna, Xiaoyuan Lin, Joachim Hamacher, Wim Van Den Broeck, Liesbeth Couck, Luc Peelman, An Hendrix, and Ann Van Soom. 2020. “The Separation and Characterization of Extracellular Vesicles from Medium Conditioned by Bovine Embryos.” INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 21 (8). https://doi.org/10.3390/ijms21082942.
Chicago author-date (all authors)
Pavani, Krishna, Xiaoyuan Lin, Joachim Hamacher, Wim Van Den Broeck, Liesbeth Couck, Luc Peelman, An Hendrix, and Ann Van Soom. 2020. “The Separation and Characterization of Extracellular Vesicles from Medium Conditioned by Bovine Embryos.” INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 21 (8). doi:10.3390/ijms21082942.
Vancouver
1.
Pavani K, Lin X, Hamacher J, Van Den Broeck W, Couck L, Peelman L, et al. The separation and characterization of extracellular vesicles from medium conditioned by bovine embryos. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. 2020;21(8).
IEEE
[1]
K. Pavani et al., “The separation and characterization of extracellular vesicles from medium conditioned by bovine embryos,” INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 21, no. 8, 2020.
@article{8665570,
  abstract     = {Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst embryos. They are secreted into the embryo culture medium and, as such, represent a source of novel biomarkers for identifying the quality of cells and embryos. However, only small amounts of embryo-conditioned medium are available, which represents a challenge for EV enrichment. Our aim is to assess the suitability of different EV separation methods to retrieve EVs with high specificity and sufficient efficiency. Bovine embryo-conditioned medium was subjected to differential ultracentrifugation (DU), OptiPrep(TM) density gradient (ODG) centrifugation, and size exclusion chromatography. Separated EVs were characterized by complementary characterization methods, including Western blot, electron microscopy, and nanoparticle tracking analysis, to assess the efficiency and specificity. OptiPrep(TM) density gradient centrifugation outperformed DU and SEC in terms of specificity by substantial removal of contaminating proteins such as ribonucleoprotein complexes (Argonaute-2 (AGO-2)) and lipoproteins (ApoA-I) from bovine embryo-derived EVs (density: 1.02-1.04, 1.20-1.23 g/mL, respectively). In conclusion, ODG centrifugation is the preferred method for identifying EV-enriched components and for improving our understanding of EV function in embryo quality and development.},
  articleno    = {2942},
  author       = {Pavani, Krishna and Lin, Xiaoyuan and Hamacher, Joachim and Van Den Broeck, Wim and Couck, Liesbeth and Peelman, Luc and Hendrix, An and Van Soom, Ann},
  issn         = {1422-0067},
  journal      = {INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES},
  keywords     = {Physical and Theoretical Chemistry,Inorganic Chemistry,Organic Chemistry,Spectroscopy,Molecular Biology,Catalysis,General Medicine,Computer Science Applications,extracellular vesicles,isolation,protocols,ultracentrifugation,size exclusion chromatography,Optiprep(TM) density gradient,CHROMATOGRAPHY},
  language     = {eng},
  number       = {8},
  pages        = {12},
  title        = {The separation and characterization of extracellular vesicles from medium conditioned by bovine embryos},
  url          = {http://dx.doi.org/10.3390/ijms21082942},
  volume       = {21},
  year         = {2020},
}

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