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.
Highlights
We found that Y-27632 enabled conditional immortalization of rat lingual keratinocytes in a modified growth medium that was originally designed for the culture of taste bud cells
To screen a growth medium suitable for the long-term expansion of rat lingual keratinocytes (RLKs), we tried two different commercially available media—EpiLife and Keratinocytes SFM (K-SFM)—that were widely used for keratinocyte culture and one high-calcium taste cell culture medium (MTCM)
We found that RLKs could attach and grow in all three types of media during primary culture, and these cells adopted a polygonal appearance typical of epithelial cells (Figure 1B)
Summary
Many attempts have been made to enhance the proliferative capabilities of cultured keratinocytes, which include co-culturing with feeder cells [7,8], using serum-free and low-calcium media [9,10,11], utilizing tissues from neonatal animals for primary culture [4,5], and using conditioned medium [5]. These methods managed to extend the propagation of keratinocytes to a certain degree, the complexity and availability of the specialized culture conditions are still substantial hurdles to starting keratinocyte culture
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