{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,6]],"date-time":"2025-11-06T12:28:12Z","timestamp":1762432092701,"version":"build-2065373602"},"reference-count":61,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2021,6,24]],"date-time":"2021-06-24T00:00:00Z","timestamp":1624492800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cultured keratinocytes are desirable models for biological and medical studies. However, primary keratinocytes are difficult to maintain, and there has been little research on lingual keratinocyte culture. Here, we investigated the effect of Y-27632, a Rho kinase (ROCK) inhibitor, on the immortalization and characterization of cultured rat lingual keratinocyte (RLKs). Three Y-27632\u2013supplemented media were screened for the cultivation of RLKs isolated from Sprague\u2013Dawley rats. Phalloidin staining and TUNEL assay were applied to visualize cytoskeleton dynamics and cell apoptosis following Y-27632 removal. Label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies were conducted to characterize the cultured cells. Results showed that RLKs could be conditionally immortalized in a high-calcium medium in the absence of feeder cells, although they did not exhibit normal karyotypes. The removal of Y-27632 from the culture medium led to reversible cytoskeletal reorganization and nuclear enlargement without triggering apoptosis, and a total of 239 differentially expressed proteins were identified by proteomic analysis. Notably, RLKs derived from the non-taste epithelium expressed some molecular markers characteristic of taste bud cells, yet calcium imaging revealed that they rarely responded to tastants. Collectively, we established a high-calcium and feeder-free culture method for the long-term maintenance of RLKs. Our results shed some new light on the immortalization and differentiation of lingual keratinocytes.<\/jats:p>","DOI":"10.3390\/ijms22136782","type":"journal-article","created":{"date-parts":[[2021,6,24]],"date-time":"2021-06-24T11:01:38Z","timestamp":1624532498000},"page":"6782","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9313-5312","authenticated-orcid":false,"given":"Zixing","family":"Chen","sequence":"first","affiliation":[{"name":"School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenmeng","family":"He","sequence":"additional","affiliation":[{"name":"Division of Science and Technology, Beijing Normal University-Hong Kong Baptist University United International College, Zhuhai 519087, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4145-1540","authenticated-orcid":false,"given":"Thomas Chun Ning","family":"Leung","sequence":"additional","affiliation":[{"name":"School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China"},{"name":"State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6776-8151","authenticated-orcid":false,"given":"Hau Yin","family":"Chung","sequence":"additional","affiliation":[{"name":"School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,6,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1111\/j.1445-2197.1989.tb01615.x","article-title":"Review of keratinocyte culture techniques: Problems of growing skin","volume":"59","author":"Breidahl","year":"1989","journal-title":"Aust. 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