Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells

Kazuhiro Ikeura,Kazuo Tsubota,Tetsuya Kawakita,Kazuyuki Tsunoda,Taneaki Nakagawa,Robert Oshima

doi:10.1371/journal.pone.0147407

Abstract

PurposeHuman and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs).MethodsSGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis.ResultsSG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype.ConclusionsEpithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium.

Highlights

Saliva is essential for maintaining oral health, alimentary bolus formation, and protection of the oral mucous membranes
Epithelial cells originating from wild type murine submandibular glands (SGs) could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium
Dissociated SG cells were cultured in low calcium, serum-free medium supplemented with epithelial growth factor (EGF) and cholera toxin (CT), which are known to inhibit fibroblast growth and accelerate epithelial cell growth, respectively

Summary

Introduction

Saliva is essential for maintaining oral health, alimentary bolus formation, and protection of the oral mucous membranes. Salivary gland atrophy caused by Sjogren’s syndrome or following radiation therapy for head and neck cancers can result in hyposalivation and xerostomia that can significantly affect the patient’s quality of life. Xerostomia increases with age and polypharmacy; this condition may be more prevalent than originally expected.[1]. Artificial saliva, and muscarinic-3 receptor stimulants are often prescribed to patients with mild-to-moderate xerostomia.[2] these treatments have poor efficacy in patients with severe salivary gland atrophy where reduced salivary flow has much more detrimental effects, including erosion of oral mucous membrane, infections, and dysphagia, which can dramatically impair quality of life. The development of more effective medical treatments is necessary.[2]

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