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 4.5 cm2 membrane filters (1.0 μm pore size) with 10% fetal bovine serum or 30 μg/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 × 105 cells/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 × 104 cells/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.

Methodology for the establishment of primary porcine vocal fold epithelial cell cultures

“…Cells can also be incorporated into a three‐dimensional in vitro model of vocal fold mucosa. Previous success had been demonstrated incorporating human embryonic stem cell‐derived epithelial cells and rabbit vocal fold epithelial cells in in vitro vocal fold mucosa models. Porcine epithelial cell cultures will provide researchers with a valuable new in vitro tool to investigate vocal fold epithelium and mucus barriers.…”

“…However, our understanding of the role of epithelium and mucus in vocal fold health in the pathogenesis of voice disorders is hindered by a lack of methods for epithelial cell culture. There has been a limited number of reports describing laryngeal epithelial cells cultures from humans and rabitts . However, epithelial cells from different tissues and species often differ substantially in cell isolation techniques and culture requirements .…”

“…The vocal fold epithelium is composed of multiple layers of closely packed cells, tightly attached to the basement membrane. Consequently, we chose to utilize a dispase solution, which digests collagenous extracellular matrix in order to separate the epithelium from the underlying lamina propria prior to the isolation of individual cells . Such techniques are preferable because they limit the number of potentially contaminating cell lineages introduced to the culture.…”

“…Such techniques are preferable because they limit the number of potentially contaminating cell lineages introduced to the culture. Mizuta et al used a similar technique to isolate rabbit vocal fold epithelial cells. The choice of culture medium also differed between studies, mostly as related to the presence or absence of serum.…”

“…Animal models are frequently used to study vocal fold function in vivo and in vitro. There has been a report of successful culture and characterization of rabbit vocal fold epithelial cells . An ideal animal model of epithelium will demonstrate morphological and functional properties similar to those of humans.…”