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

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CCN Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2582))

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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Acknowledgments

T.E. was supported by JSPS Kakenhi, grant numbers 17K11642-TE, 20K09904-CS, 19H03817-MT, 20H03888-HN, 20K20611-MT, 20H03888-HN, 21H03119-TY, and 21K08902-HY. Y.O. was supported by JSPS Overseas Research Fellowship. K.O. was supported by JSPS Kakenhi, grant number 21K17115. S.F. was supported by JSPS Kakenhi, grant number 21K20992. Y.L was supported by Rotary Yoneyama Memorial Foundation Fellowship. E.A.T. was supported by the Egypt-Japan Education Partnership Grant. The authors thank Heeyoun Bunch, Junya Futagawa, Kazuko Kobayashi, Kuniaki Okamoto, Chiharu Sogawa, Masaharu Takigawa, Masaki Matsumoto, Seiji Tamaru, Koji Ueda, and Haruo Urata for useful information, discussion, materials, or experimentation.

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Authors and Affiliations

  1. Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

    Takanori Eguchi, Yanyin Lu & Shiro Fukuoka

  2. Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Yuka Okusha

  3. Department of Dental Anesthesiology and Special Care Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Yanyin Lu

  4. Department of Oral and Maxillofacial Surgery, Okayama University Hospital, Okayama, Japan

    Kisho Ono

  5. Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan

    Eman A. Taha

  6. Department of Biochemistry, Ain Shams University Faculty of Science, Cairo, Egypt

    Eman A. Taha

  7. Department of Orthopaedic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Shiro Fukuoka

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  1. Takanori Eguchi
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  2. Yuka Okusha
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  3. Yanyin Lu
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  4. Kisho Ono
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  5. Eman A. Taha
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  6. Shiro Fukuoka
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Corresponding author

Correspondence to Takanori Eguchi .

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Editors and Affiliations

  1. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Masaharu Takigawa

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Eguchi, T., Okusha, Y., Lu, Y., Ono, K., Taha, E.A., Fukuoka, S. (2023). Comprehensive Method for Exosome Isolation and Proteome Analysis for Detection of CCN Factors in/on Exosomes. In: Takigawa, M. (eds) CCN Proteins. Methods in Molecular Biology, vol 2582. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2744-0_6

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  • DOI: https://doi.org/10.1007/978-1-0716-2744-0_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2743-3

  • Online ISBN: 978-1-0716-2744-0

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