Characterization of a temperature-sensitive mouse middle ear epithelial cell line

Abstract

Conclusion. Temperature-sensitive mouse middle ear epithelial cells have been successfully established and characterized. Objective. Temperature-sensitive middle ear epithelial cell lines are essential for pathophysiologic studies of otitis media. They are useful for studying the pathogen–host interaction, receptor identification, signal transduction, cytokine/mucin production and cellular responses, especially for cell proliferation and differentiation. The purpose of this study was to establish a large T-antigen [simian virus 40 A-gene (SV40)] mutant-immortalized mouse middle ear epithelial cell line for otitis media studies. Material and methods. Primary culture of middle ear epithelial cells was established from the middle ear mucosa of an Immortomouse. The cells were transduced by a temperature-sensitive large T-antigen mutant and cultured for >50 passages. The expression of mRNA transcripts and proteins for epithelial cells was characterized by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The temperature-sensitive properties of cells cultured at 33°C and 39°C were evaluated using ³H-thymidine incorporation, Trypan blue exclusion and peroxisome proliferator-activated receptor-γ activity. Results. Immortalized middle ear epithelial cells demonstrated a cobblestone-like monolayer culture. The cells expressed mucosal cell markers such as mucins, keratins and collagens. They proliferated at 33°C when the SV40 antigen was active and differentiated at 39°C when the SV40 antigen was inactive.

• Immortalization
• luciferase assay
• middle ear epithelial cells
• mouse
• reverse transcriptase polymerase chain reaction
• tissue culture