title: Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells.
abstract: OBJECTIVE
  Human 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).
  
  
  METHODS
  SGs 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.
  
  
  RESULTS
  SG 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.
  
  
  CONCLUSIONS
  Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium.
authors.affiliation: Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.|Department of Dentistry and Oral Surgery, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Ikeura
authors.affiliation: Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kawakita
authors.affiliation: Department of Dentistry and Oral Surgery, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Tsunoda
authors.affiliation: Department of Dentistry and Oral Surgery, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Taneaki
authors.initials: T
authors.lastname: Nakagawa
authors.affiliation: Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tsubota
mesh_terms: D000199:Actins / Q000032:analysis; D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D004848:Epithelium / Q000737:chemistry; D005260:Female; D053547:Keratin-14 / Q000032:analysis; D053538:Keratin-18 / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D010750:Phosphoproteins / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction; D013363:Submandibular Gland / Q000737:chemistry / Q000166:cytology*; D015534:Trans-Activators / Q000032:analysis
chemical_list: D000199:Actins; D053547:Keratin-14; D053538:Keratin-18; C507446:Krt14 protein, mouse; D010750:Phosphoproteins; D015534:Trans-Activators; C486419:Trp63 protein, mouse; C544013:alpha-smooth muscle actin, mouse