2023 Fiscal Year Final Research Report
Biophysical and Biochemical Characterization of Novel Membrane Vesicles Responsible for Intercellular Biological Signaling
| Project/Area Number |
20K20297
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| Project/Area Number (Other) |
17H06242 (2017-2019)
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| Research Category |
Grant-in-Aid for Challenging Research (Pioneering)
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| Allocation Type | Multi-year Fund (2020)
Single-year Grants (2017-2019) |
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| Research Field |
Biomolecular chemistry and related fields
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| Research Institution | National Institute of Advanced Industrial Science and Technology |
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Principal Investigator |
Ishii Noriyuki 国立研究開発法人産業技術総合研究所, 生命工学領域, 主任研究員 (10261174)
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| Project Period (FY) |
2020-04-01 – 2024-03-31
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| Keywords | 細胞外小胞 / エクソソーム／エキソソーム / マイクロベシクル / 生体分子 / ナノコロイド / 透過型電子顕微鏡 / ランタノイド酢酸塩 / 動的光散乱 |
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| Outline of Final Research Achievements |
Exosomes, important extracellular vesicles (EVs) for intercellular communication, are difficult to distinguish from microvesicles. This study focused on multivesicular bodies (MVBs) and optimized the isolation, fractionation, and preparation of exosomes through stepwise direct observation using transmission electron microscopy (TEM). Fractionation under low-salt Bis-Tris buffer conditions revealed a specific population of approximately 50 nm. Furthermore, the presence of CD63, known to be one of the exosome markers, was confirmed by immunoelectron microscopy. Additionally, an attempt was made to transfer EV isolation and preparation from density gradient ultracentrifugation to tangential flow filtration, demonstrating the effectiveness of a continuous filtration method using regenerated cellulose membranes with different molecular weight cut-offs (MWCO). The isolation and characterization of intact EVs under optimal conditions are critical for biomedical applications.
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| Free Research Field |
生物物理学、物性基礎、電子顕微鏡解析
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| Academic Significance and Societal Importance of the Research Achievements |
細胞外小胞（EVs）をナノコロイドとして認識し、医学生物学分野で見過ごされていた物理化学的課題を明らかにしました。電子顕微鏡法による条件最適化で、EVsを細胞から放出されたインタクトな状態で分離・調製する条件を確立しました。エクソソーム（small EVs）研究の共通基盤として幅広く活用が期待されます。さらに、限外ろ過法の連続的適用を考案し、その過程でsmall EVsにも赤血球のような変形能が示唆されました。レオロジー的な解明が望まれます。EVsに適した電子染色試薬の開発も進めました。EVsの研究は、細胞間通信の理解、疾患の診断・治療、薬物送達、生態系への影響等に革新的な貢献が期待されます。
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