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–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 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.

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Although the technique for taste cell culture has been reported, cultured taste cells have remained poorly validated. This study systematically compared the cultured cells derived from both taste and non-taste tissues. Fourteen cell lines established from rat circumvallate papillae (RCVs* or RCVs), non-taste lingual epithelia (RVEs), and tail skins (RTLs) were analyzed by PCR, immunocytochemistry, proteomics, and calcium imaging. The cell lines were morphologically indistinguishable, and all expressed some taste-related molecules. Of the tested RCVs*, RCVs, RVEs, and RTLs (%), 84.7 ± 7.8, 63.9 ± 22.8, 46.8 ± 0.3, and 40.8 ± 15.1 of them were responsive to at least one tastant or ATP, respectively. However, the calcium signaling pathways in the responding cells differed from the canonical taste transduction pathways in the taste cells in vivo, suggesting that they were not genuine taste cells. In addition, the growth medium intended for taste cell culture did not prevent the proliferation of non-gustatory epithelial cells regardless of supplementation of Y-27632 and EGF. In conclusion, the current method for taste cell culture is susceptible to pseudotaste cells that may lead to overinterpretation. Thus, biosensors that rely on calcium responses of cultured taste cells should be applied with extreme caution.