2021 Fiscal Year Final Research Report

Development of new cell preservation technologies utilizing antifreeze protein

Research Project

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Project/Area Number	19H02529
Research Category	Grant-in-Aid for Scientific Research (B)
Allocation Type	Single-year Grants
Section	一般
Review Section	Basic Section 27040:Biofunction and bioprocess engineering-related
Research Institution	National Institute of Advanced Industrial Science and Technology
Principal Investigator	Tsuda Sakae 国立研究開発法人産業技術総合研究所, 生命工学領域, 上級主任研究員 (70211381)
Co-Investigator(Kenkyū-buntansha)	大山恭史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 (80356675) 近藤英昌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 (80357045)
Project Period (FY)	2019-04-01 – 2022-03-31
Keywords	不凍タンパク質 / ナノ氷結晶 / 過冷却現象 / 凍結濃縮現象 / 分子構造 / 熱ヒステリシス
Outline of Final Research Achievements	The present study revealed that (1) a phenomenon called "freeze-induced concentration" is strongly inhibited by using liquid nitrogen or AFP, among which the latter consumes quite a few energy cost, (2) the survival rate (%) is not so improved when rat insulinoma cells (RIN-5F) were cooled to be filled with relatively small ice crystals, and (3) the survival rate (%) is dramatically improved when the RIN-5F cells were cooled to make it supercooling state with AFP, or make it to be filled with ultimately small "nano" ice crystals. In this study supercooling state was achieved by using insect-derived AFP, which realized significantly high (~53%) survival rate (%) of RIN-5F cells even after 20 days of preservation at -5 deg C. These new findings were opened to public though many journals, seminars, and invited lectures. Dr. Tsuda received "2019 Society Award" from Japanese society for cryobiology and cryotechnology.
Free Research Field	生物物理学、低温生物学
Academic Significance and Societal Importance of the Research Achievements	冷凍庫で保存中の細胞、組織、食品等の内部に大きな氷結晶が生成する現象が「氷の再結晶化」である。本研究はエネルギーコストのかからない不凍タンパク質(Antifreeze Protein, AFP)を使うことで再結晶化が止まることを見出し、さらにマイナス温度域に冷却した水をナノ氷結晶で埋め尽くす「準ガラス化状態」にする技術によって細胞組織を低温下で長期間生かし続けられることを明らかにした。特に、昆虫由来AFPには水をマイナス5°Cで準ガラス化状態にする能力があること、その状態が細胞の生存率を大幅に改善することを初めて明らかにした。得られた成果をオープンアクセス誌やセミナー・講演等で広く公開した。