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A novel culture design aimed at promoting uniform maturation of epithelial cells using liquid materials is described. Culturing Madin\u2013Darby canine kidney (MDCK) cells at the liquid\u2013liquid interface yielded reduced migration and stimulated active cell growth. Similar to solid\u2013liquid interfaces, cells cultured on a fibronectin-coated liquid\u2013liquid interface exhibited active migration and growth, ultimately reaching a confluent state. These cells exhibited reduced stress fiber formation and adopted a cobblestone-like shape, which led to their even distribution in the culture vessel. To inhibit stress fiber formation and apoptosis, the exposure of cells on liquid\u2013liquid interfaces to Y27632, a specific inhibitor of the Rho-associated protein kinase (ROCK), facilitated tight junction formation (frequency of ZO-2-positive cells, <jats:italic>F<\/jats:italic><jats:sub>Z<\/jats:sub>\u2009=\u20090.73). In Y27632-exposed cells on the liquid\u2013liquid interface, the value obtained by subtracting the standard deviation of the ratio of nucleus densities in each region that compartmentalized a culture vessel from 1, denoted as <jats:italic>H<\/jats:italic><jats:sub>LN<\/jats:sub>, was 0.93\u00a0\u00b1\u00a00.01, indicated even cell distribution in the culture vessel at <jats:italic>t<\/jats:italic>\u2009=\u200972\u00a0h. The behavior of epithelial cells on liquid\u2013liquid interfaces contributes to the promotion of their uniform maturation.<\/jats:p>","DOI":"10.1038\/s41598-024-63115-7","type":"journal-article","created":{"date-parts":[[2024,5,29]],"date-time":"2024-05-29T09:02:28Z","timestamp":1716973348000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["A novel strategy to facilitate uniform epithelial cell maturation using liquid\u2013liquid interfaces"],"prefix":"10.1038","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8597-8942","authenticated-orcid":false,"given":"Rie","family":"Sonoi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masamichi","family":"Kamihira","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,5,29]]},"reference":[{"key":"63115_CR1","doi-asserted-by":"publisher","first-page":"1987","DOI":"10.1021\/acsbiomaterials.1c01500","volume":"8","author":"LT Hung","year":"2022","unstructured":"Hung, L. 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