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Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632
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Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System
Zixing Chen1, Wenmeng He2, Thomas Chun Ning Leung1,3
1School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.
International Journal of Molecular Sciences
|July 2, 2021
Summary
Researchers developed a new method for culturing rat lingual keratinocytes (RLKs) using Y-27632, a Rho kinase inhibitor. This feeder-free approach enables long-term maintenance of RLKs for biological studies.
Area of Science:
- Cell Biology
- Tissue Engineering
- Dermatology
Background:
- Cultured keratinocytes are valuable models, but primary keratinocyte culture is challenging.
- Limited research exists on lingual keratinocyte culture, hindering their use in studies.
Purpose of the Study:
- To investigate the effect of Y-27632 on immortalization and characterization of cultured rat lingual keratinocytes (RLKs).
- To establish a feeder-free culture method for long-term RLK maintenance.
Main Methods:
- Screening of Y-27632-supplemented media for RLK cultivation.
- Phalloidin staining, TUNEL assay, label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies.
- Conditional immortalization in high-calcium medium without feeder cells.
Main Results:
- RLKs were conditionally immortalized in a high-calcium, feeder-free medium.
- Y-27632 removal induced reversible cytoskeletal changes and nuclear enlargement without apoptosis.
- Proteomic analysis identified 239 differentially expressed proteins; RLKs showed limited taste response.
Conclusions:
- A high-calcium, feeder-free culture method for long-term RLK maintenance was established.
- The study provides insights into lingual keratinocyte immortalization and differentiation.
- This method supports the use of RLKs as models in biological and medical research.
