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Comprehensive Method for Exosome Isolation and Proteome Analysis for Detection of CCN Factors in/on Exosomes
Takanori Eguchi1, Yuka Okusha2, Yanyin Lu3,4
1Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. eguchi@okayama-u.ac.jp.
Abstract:
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.
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