Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632

Abstract

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.

Pseudo-Taste Cells Derived from Rat Taste and Non-Taste Tissues: Implications for Cultured Taste Cell-Based Biosensors

“…Cell culture was conducted as described in the previous literature. ,− In brief, circumvallate papillae taste tissues, non-taste ventral lingual epithelia, and tail skins were isolated from Sprague–Dawley rats (6 weeks). Mechanical dissociation was used to dissociate the circumvallate papillae taste tissues, whereas enzymatic digestion was used to obtain lingual and tail skin epithelial cells.…”

“…Calcium imaging was conducted as previously described . Briefly, cells seeded on coverslips were stained with 5 μM Fluo-4 AM for about 30 min at 37 °C.…”

“…Stimuli were dissolved in Tyrode’s solution and bath-applied to the cells for 1 min, followed by a 4 min rinse period of Tyrode’s solution. Individual stimuli used were 50 μM ATP, sweet stimulus (10 mM sucralose plus 10 mM acesulfame K), , sour stimulus (20 mM citric acid), umami stimulus (100 mM MSG plus 1 mM IMP), , bitter stimulus (2 mM denatonium benzoate), and 50 mM KCl . Signal transduction inhibitors, including 10 μΜ U73122 and 10 mM ZnCl 2 , were dissolved in corresponding taste stimuli to examine the signaling pathways.…”

“…Previously, we found that rat lingual keratinocytes could be effectively immortalized and expanded in the presence of a Rho kinase (ROCK) inhibitor Y-27632 and an epidermal growth factor (EGF) (hereinafter abbreviated as YE) . Interestingly, non-taste lingual epithelia were able to give rise to cells that expressed some of the taste-signaling elements, suggesting the necessity of comparing the taste and non-taste cells in vitro .…”

“…Previously, we found that rat lingual keratinocytes could be effectively immortalized and expanded in the presence of a Rho kinase (ROCK) inhibitor Y-27632 and an epidermal growth factor (EGF) (hereinafter abbreviated as YE) . Interestingly, non-taste lingual epithelia were able to give rise to cells that expressed some of the taste-signaling elements, suggesting the necessity of comparing the taste and non-taste cells in vitro . In addition, considering that taste cells are specialized cells derived from basal lingual keratinocytes and mature taste cells express various kinds of keratins characteristic of keratinocytes, it is highly important and meaningful to further investigate the effect of YE on the establishment of rat taste cell lines.…”