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Paper-based Devices for Isolation and Characterization of Extracellular Vesicles
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Efficient Isolation and Characterization Methods for Chicken Serum Extracellular Vesicles.

Hatsune Mukohda1, Muhammet Mustafa Binici1, Katsuya Miyake2

  • 1Graduate School of Science and Technology, Niigata University, Niigata, Japan.

Animal Science Journal = Nihon Chikusan Gakkaiho
|August 8, 2025
PubMed
Summary

This study optimized methods for isolating and identifying extracellular vesicles (EVs) from chicken serum. Filtration (FL) proved most effective for obtaining highly purified chicken EVs, meeting international standards.

Keywords:
chickenextracellular vesiclesisolationmarker proteinserum

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Area of Science:

  • Veterinary Science
  • Biochemistry
  • Cell Biology

Background:

  • Extracellular vesicles (EVs) are crucial for cell-to-cell communication.
  • Standardized methods for chicken serum EV isolation and characterization are lacking.
  • Current methods do not fully align with Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines.

Purpose of the Study:

  • To establish efficient methods for isolating and characterizing chicken serum EVs.
  • To evaluate and compare four conventional isolation techniques: ultracentrifugation (UC), two polymer-based precipitations (PP), and filtration (FL).
  • To ensure isolated EVs meet MISEV guidelines.

Main Methods:

  • EV isolation using UC, PP (two types), and FL from chicken serum.
  • Characterization via Western Blotting (WB) for EV markers (CD9, PDCD6IP, TSG101, calnexin).
  • Nanoparticle tracking analysis (NTA) and Transmission Electron Microscopy (TEM) for size, morphology, and structure.
  • Quantitative PCR (qPCR) for microRNA (miR-451) expression.

Main Results:

  • All methods yielded samples with EV marker proteins, particles sized 30-150 nm, and typical EV morphology.
  • Filtration (FL) demonstrated the most distinct EV marker protein expression via WB.
  • FL-isolated EVs exhibited the highest expression of miR-451, a stable EV-associated microRNA.

Conclusions:

  • An optimal characterization method for chicken EVs was established.
  • Filtration (FL) is proposed as a superior method for isolating highly purified EVs from chicken serum.
  • This study provides a foundation for standardized chicken EV research.