Details of Research Outputs

Cultured keratinocytes are desirable models for biological and medical studies. How-ever, 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–supplemented media were screened for the cultivation of RLKs isolated from Sprague–Dawley 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 cytoge-netic 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.