{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T02:25:45Z","timestamp":1788402345439,"version":"build-2803163510"},"reference-count":51,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2021,11,24]],"date-time":"2021-11-24T00:00:00Z","timestamp":1637712000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["19H02529"],"award-info":[{"award-number":["19H02529"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["19K22989"],"award-info":[{"award-number":["19K22989"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cells for therapeutic use are often preserved at +4 \u00b0C, and the storage period is generally limited to 2\u20133 days. Here, we report that the survival rate (%) of mammalian cells is improved to 10\u201320 days when they are preserved with a subzero supercooled solution containing the antifreeze protein (AFP), for which an ability to stabilize both supercooled water and cell membrane integrity has been postulated. We chose adherent rat insulinoma (RIN-5F) cells as the preservation target, which were immersed into \u22125 \u00b0C-, \u22122 \u00b0C-, or +4 \u00b0C-chilled \u201cunfrozen\u201d solution of Euro-Collins or University of Washington (UW) containing the AFP sample obtained from insect or fish. Our results show that the survival rate of the cells preserved with the solution containing insect AFP was always higher than that of the fish AFP solution. A combination of the \u22125 \u00b0C-supercooling and insect AFP gave the best preservation result, namely, UW solution containing insect AFP kept 53% of the cells alive, even after 20 days of preservation at \u22125 \u00b0C. The insect AFP locates highly organized ice-like waters on its molecular surface. Such waters may bind to semiclathrate waters constructing both embryonic ice crystals and a membrane\u2013water interface in the supercooled solution, thereby protecting the cells from damage due to chilling.<\/jats:p>","DOI":"10.3390\/ijms222312680","type":"journal-article","created":{"date-parts":[[2021,11,30]],"date-time":"2021-11-30T04:48:37Z","timestamp":1638247717000},"page":"12680","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Subzero Nonfreezing Hypothermia with Insect Antifreeze Protein Dramatically Improves Survival Rate of Mammalian Cells"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8973-4443","authenticated-orcid":false,"given":"Akari","family":"Yamauchi","sequence":"first","affiliation":[{"name":"Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ai","family":"Miura","sequence":"additional","affiliation":[{"name":"Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2799-1775","authenticated-orcid":false,"given":"Hidemasa","family":"Kondo","sequence":"additional","affiliation":[{"name":"Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810, Japan"},{"name":"Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2328-9519","authenticated-orcid":false,"given":"Tatsuya","family":"Arai","sequence":"additional","affiliation":[{"name":"Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3403-237X","authenticated-orcid":false,"given":"Yuji C.","family":"Sasaki","sequence":"additional","affiliation":[{"name":"Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Japan"},{"name":"OPERANDO Open Innovation Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8563, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1399-0619","authenticated-orcid":false,"given":"Sakae","family":"Tsuda","sequence":"additional","affiliation":[{"name":"Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810, Japan"},{"name":"Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan"},{"name":"OPERANDO Open Innovation Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8563, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,11,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1023\/A:1024734003814","article-title":"Principles of low temperature cell preservation","volume":"8","author":"Rubinsky","year":"2003","journal-title":"Heart Fail. 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