Structurally and functionally characterized in vitro model of rabbit vocal fold
Authors Mizuta M, Kurita T, Kimball EE, Rousseau B
Submitted By Submitted Externally on 6/15/2017
Status Published
Journal Tissue & cell
Year 2017
Date Published 6/1/2017
Volume : Pages 49 : 427 - 434
PubMed Reference 28395914
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 4.5cm(2) membrane 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 8.9×10(5) cells/cm(2)), 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 2.2×10(4) cells/cm(2), 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.