Journal of Animal Science and Technology

Korean Society of Animal Sciences and Technology (한국축산학회)
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Exosomes isolation from bovine serum: qualitative and quantitative comparison between ultracentrifugation, combination ultracentrifugation and size exclusion chromatography, and exoEasy methods

  • Eun-Yeong Bok (Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration) ;
  • Sang Young Seo (Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration) ;
  • Han Gyu Lee (Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration) ;
  • Sudu Hakuruge Madusha Pramud Wimalasena (Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration) ;
  • Eunju Kim (Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration) ;
  • Ara Cho (Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration) ;
  • Young-Hun Jung (Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration) ;
  • Tai-Young Hur (Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration) ;
  • Kyoung-Min So (Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration) ;
  • Sung-Lim Lee (College of Veterinary Medicine, Gyeongsang National University) ;
  • Yoon Jung Do (Division of Animal Diseases & Health, National Institute of Animal Science, Rural Development Administration)
  • Received : 2023.11.24
  • Accepted : 2024.04.18
  • Published : 2024.09.30
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Abstract

Exosomes have been extensively studied as disease biomarker in humans, given their role in transporting bioactive molecules. However, despite the great potential of exosomes as noninvasive diagnostic markers and therapeutic nanocarriers for bovine diseases, few studies have been conducted on bovine exosome. Thus, this study aimed to quantitatively and qualitatively compare three isolation methods to identify a suitable method for bovine serum. Exosomes were isolated using ultracentrifugation alone (UC), a combination of ultracentrifugation and size exclusion chromatography (US), or membrane affinity-based exoEasy kit (EE). Isolated particles were evaluated using a range of complementary techniques. Transmission electron microscopy showed that all three isolation methods resulted in particles with a cup-shaped morphology. The particle concentration measured by nanoparticle trafficking analyzer of US was lower compared to those of UC and EE method. As a result of immunoblotting, exosome markers including TSG101, CD81, and HSP70 were detected in US particles, while in UC and EE, only TSG101 expression was confirmed. Particles isolated from UC and EE showed a contamination with the blood protein albumin, whereas particles from US did not show albumin contamination. In addition, to evaluate the possibility of using exosomes as biomarkers, the profiles of the small RNA in the exosomes were compared using the bioanalyzer 2100. As a result, in the EE method, the band of small RNA (25-200 nt) was most prominent, and in the US methods, a distinct band was observed in the small RNA range. Collectively, the purity of exosomes without non-exosomal contamination was highest in the US method. However, for the detection of small RNA, the EE method was found to be the most suitable. Therefore, the results suggest that the optimal isolation method varies depending on the specific purpose of exosome isolation.

Keywords

Acknowledgement

This study was supported by the 2023 RDA Fellowship Program of National Institute of Animal Science, Rural Development Administration. It was carried out with the support of the "Cooperative Research Program for Agriculture Science and Technology Development (Project title: Searching immune regulation gene for Johne's disease and building a biobank system, Project No. PJ01681403), Rural Development Administration, Korea.

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