Comprehensive Method for Exosome Isolation and Proteome Analysis for Detection of CCN Factors in/on Exosomes

Takanori Eguchi, Yuka Okusya, Yanyin Lu, Kisho Ono, Eman A. Taha, Shiro Fukuoka

Research output: Chapter in Book/Report/Conference proceedingChapter


Cellular Communication Network (CCN) proteins are secretory growth factors often associated with extracellular matrix (ECM) and extracellular vesicles (EVs) such as exosomes or matrix-coated vesicles. CCN factors and fragments loaded on/in EVs may play key roles in cell communication networks in cancer biology, bone and cartilage metabolism, wound healing, and tissue regeneration. CCN proteins and EVs/exosomes are found in body fluids, such as blood, urine, milk, and supernatants of the two-dimensionally (2D) cultured cells and three-dimensionally (3D) cultured tissues, such as spheroids or organoids. More than ten methods to isolate exosomes or EVs have been developed with different properties. Here, we introduce comprehensive protocols for polymer-based precipitation, affinity purification, ultracentrifugation methods combined with the ultrafiltration method for isolating CCN-loaded exosomes/EVs from 2D and 3D cultured tissues, and proteome analysis using mass spectrometry for comprehensive analysis of CCN proteins.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages18
Publication statusPublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • CCN factors
  • Exosome isolation methods
  • Extracellular vesicles
  • Mass spectrometry
  • Polymer-based precipitation
  • Proteome analysis
  • Spheroid
  • Tumoroid
  • Ultracentrifugation
  • Ultrafiltration

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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