{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:13:22Z","timestamp":1785413602451,"version":"3.56.0"},"reference-count":41,"publisher":"American Society for Microbiology","issue":"4","license":[{"start":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T00:00:00Z","timestamp":1777334400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T00:00:00Z","timestamp":1777334400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001700","name":"Ministry of Education, Culture, Sports, Science and Technology","doi-asserted-by":"publisher","award":["JPNBRP202228"],"award-info":[{"award-number":["JPNBRP202228"]}],"id":[{"id":"10.13039\/501100001700","id-type":"DOI","asserted-by":"publisher"}]},{"name":"JST [FOREST Program]","award":["JPMJFR240M"],"award-info":[{"award-number":["JPMJFR240M"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mSphere"],"published-print":{"date-parts":[[2026,4,28]]},"abstract":"ABSTRACT<\/jats:title>\n \n \n \n Entamoeba histolytica<\/jats:italic>\n , a protozoan parasite, causes amebiasis, which is a global public health problem. Clinical manifestation and pathogenesis of amebiasis are closely associated with the proliferation and tissue invasion abilities of\n E. histolytica<\/jats:italic>\n trophozoites. At the same time,\n E. histolytica<\/jats:italic>\n trophozoites are studied in a broad range of biology research fields. As such, the development of\n in vitro<\/jats:italic>\n culture of\n E. histolytica<\/jats:italic>\n trophozoites greatly facilitated amebic research. Now, a standard method for cryopreservation of\n E. histolytica<\/jats:italic>\n trophozoites is required because available methods either do not give a high enough revival rate, which can significantly delay projects, or they are not widely adopted. Here, we attempted to optimize the conditions for\n E. histolytica<\/jats:italic>\n trophozoite cryopreservation, including cell density, cooling rate, cell freezing reagent, and freezing profile. We found an optimized condition that reproducibly yielded >30% revival, regardless of storage period in liquid nitrogen (up to 365 days). This optimized condition is that\n E. histolytica<\/jats:italic>\n trophozoites are suspended in 0.5 mL CELLBANKER 2 in a 1 mL cryotube at 2 \u00d7 10\n 6<\/jats:sup>\n cells\/mL and frozen from 4 to \u221240\u00b0C at a rate of \u22120.2\u00b0C\/min using VIA Freeze Uno. This cryopreservation method can minimize the risk of losing\n E. histolytica<\/jats:italic>\n trophozoite lines required to continue projects, facilitating amebic research.\n <\/jats:p>\n <\/jats:sec>\n \n IMPORTANCE<\/jats:title>\n \n Amebiasis, which is caused by\n Entamoeba histolytica<\/jats:italic>\n infection, is the third deadliest parasitic disease globally. Proliferation of\n E. histolytica<\/jats:italic>\n trophozoites and their invasion into the host tissues cause amebiasis symptoms and pathogenesis.\n E. histolytica<\/jats:italic>\n trophozoites are also important in multiple biology research topics. Therefore,\n E. histolytica<\/jats:italic>\n trophozoites are a common subject in academic as well as clinical fields. A standard method for\n in vitro<\/jats:italic>\n culture of\n E. histolytica<\/jats:italic>\n trophozoites is well established. By contrast, a widely adopted practical method for cryopreservation of\n E. histolytica<\/jats:italic>\n trophozoites is not yet available. This hampers the advancement of amebic research, as the required\n E. histolytica<\/jats:italic>\n trophozoite lines sometimes cannot be revived from cryopreservation. In this study, we varied parameters critical to the revival rate, namely, cell density, cooling rate, freezing reagent, and freezing profile, and present an optimized cryopreservation method for\n E. histolytica<\/jats:italic>\n trophozoites, which gives reproducibly high revival rates.\n <\/jats:p>\n <\/jats:sec>","DOI":"10.1128\/msphere.00889-25","type":"journal-article","created":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T12:00:10Z","timestamp":1774872010000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Cryopreservation method for\n Entamoeba histolytica<\/i>\n trophozoites"],"prefix":"10.1128","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9528-3001","authenticated-orcid":true,"given":"Makoto","family":"Kazama","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03ppx1p25","id-type":"ROR","asserted-by":"publisher"}],"name":"NEKKEN Bio-Resource Center, 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