Utility of cell viability assays for use with ex vivo vocal fold epithelial tissue

Abstract

Objectives/hypothesis: Ex vivo models are routinely used to investigate the barrier function of the vocal fold epithelium. However, there are limited reports on assays that can be used to investigate the effect of clinically relevant challenges on vocal fold epithelial tissue viability. Our objective was to determine the utility of two assays routinely used in cell culture-a cellular metabolic activity assay and a cell membrane integrity assay-to investigate the viability of ex vivo porcine vocal fold epithelium.

Study design: Prospective, ex vivo animal study.

Methods: Porcine vocal folds were exposed to acrolein, hydrochloric acid, or hydrogen peroxide challenge. An untreated, sham challenge was included as a control. Assays including metabolic activity, cell membrane integrity, and histology were used to determine whether challenges reduced epithelial viability as compared to sham.

Results: Cell membrane integrity and metabolic activity assays detected reductions in viability following hydrochloric acid and hydrogen peroxide challenges but not acrolein challenge as compared to sham. No challenge produced significant changes in epithelial appearance as evidenced by light microscopy.

Conclusions: Metabolic activity and cell membrane integrity assays are valuable tools that can be used to evaluate the viability of ex vivo vocal fold epithelial tissue following clinically relevant challenges. As viability is reduced, the ability of epithelial tissue to maintain its barrier function is compromised. Accurate assessment of viability may provide us clues into understanding mechanisms underlying vocal fold epithelial injury and disease.

Level of evidence: NA Laryngoscope, 125:E180-E185, 2015.

Keywords: Ex vivo model; viability assay; vocal fold epithelium.

Figure 1

Viability indices (abs/mg) of porcine vocal folds exposed to sham, acrolein, HCl, or H₂O₂ challenge or DMSO. Data are presented as mean and standard error of the mean. Viability indices significantly decreased following HCl and H₂O₂ challenges as compared to sham and acrolein. * p < 0.05

Figure 2

A) Representative fluorescent micrographs of the effect of sham (A), acrolein (B), HCl (C), and H₂O₂ (D) challenges on porcine vocal fold epithelial cell membrane integrity (Hoechst (blue), ethidium (red), 20×). Diffuse labeling of damaged cells (red) is observed in the HCl and H₂O₂, but not sham or acrolein challenged tissue. B) Epithelial cell damage rate (%) of porcine vocal folds exposed to sham, acrolein, HCl, or H₂O₂ challenge, Data are presented as mean and standard error of the mean. Damage rate significantly increased following HCl and H₂O₂ challenges as compared to sham and H₂O₂ challenge as compared to acrolein. * p < 0.05

Figure 3

Representative light micrographs of the effect of sham (A), acrolein (B), HCl (C), and H₂O₂ (D) challenges on vocal fold structure (H&E stain, 40×). No significant damage was observed following any challenge. Ep = stratified squamous epithelium, LP = lamina propria.