title: Structurally and functionally characterized in vitro model of rabbit vocal fold epithelium.
abstract: In this paper, we describe a method for primary culture of a well differentiated electrically tight rabbit vocal fold epithelial cell multilayer and the measurement of transepithelial electrical resistance (TEER) for the evaluation of epithelial barrier function in vitro. Rabbit larynges were harvested and enzymatically treated to isolate vocal fold epithelial cells and to establish primary culture. Vocal fold epithelial cells were co-cultured with mitomycin C-treated feeder cells on collagen-coated plates. After 10-14 days in primary culture, cells were passaged and cultured until they achieved 70-90% confluence on collagen-coated plates. Epithelial cells were then passaged onto collagen-coated cell culture inserts using 4.5cm2membrane filters (1.0μm pore size) with 10% fetal bovine serum or 30μg/mL bovine pituitary extract to investigate the effects of growth-promoting additives on TEER. Additional experiments were performed to investigate optimal seeding density (1.1, 2.2, 4.4, or 8.9×105cells/cm2), the effect of co-culture with feeder cells, and the effect of passage number on epithelial barrier function. Characterization of in vitro cultures was performed using hematoxylin and eosin staining and immunostaining for vocal fold epithelial cell markers and tight junctions. Results revealed higher TEER in cells supplemented with fetal bovine serum compared to bovine pituitary extract. TEER was highest in cells passaged at a seeding density of 2.2×104cells/cm2, and TEER was higher in cells at passage two than passage three. Ultrastructural experiments revealed a well-differentiated epithelial cell multilayer, expressing the epithelial cell markers CK13, CK14 and the tight junction proteins occludin and ZO-1.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Mizuta
authors.affiliation: Department of Otolaryngology, Vanderbilt University Medical Center, 1215 21st Avenue South, Medical Center East, Nashville, TN 37232, USA.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kurita
authors.affiliation: Department of Hearing and Speech Sciences, Vanderbilt University School of Medicine, 1215 21st Avenue South, Medical Center East, Nashville, TN 37232, USA.
authors.forename: Emily E
authors.initials: EE
authors.lastname: Kimball
authors.affiliation: Department of Otolaryngology, Vanderbilt University Medical Center, and Hearing and Speech Sciences and Mechanical Engineering, Vanderbilt University School of Medicine, Medical Center East, 1215 21st Avenue South, Nashville, TN 37232, USA. Electronic address: bernard.rousseau@vanderbilt.edu.
authors.forename: Bernard
authors.initials: B
authors.lastname: Rousseau
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D008954:Models, Biological; D011817:Rabbits; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D014827:Vocal Cords / Q000166:cytology / Q000378:metabolism
keywords: Epithelial cells; In vitro; Primary culture; Transepithelial resistance; Vocal fold