Human sinonasal explant system for testing cytotoxicity of intranasal agents

Abstract

Background: Intranasal agents play a critical role in the management of sinonasal disorders. There are ongoing efforts to develop new intranasal medications to combat sinonasal disease. Some intranasal agents, however, can have cytotoxic effects on human sinonasal tissue. In order to facilitate safe drug discovery, we developed a simple and reliable in vitro screening assay using human sinonasal explants to measure the cytotoxic profiles of intranasal agents.

Methods: We obtained sinonasal tissues from several regions of the nasal cavity from 12 patients undergoing endoscopic sinonasal surgery. These tissues were cultured on polytetrafluoroethylene membrane in serum-free growth medium. We determined the biochemical properties of these explants by measuring extracellular lactate dehydrogenase (LDH) levels and performing histological analyses over a period of 1 to 2 weeks. We then examined the cytotoxic profiles of 13 intranasal agents by measuring extracellular LDH levels using the human sinonasal explant system.

Results: Sinonasal explants exhibited a rapid reduction in extracellular LDH levels indicating stabilization in the culture environment within 2 days. Histological analysis showed maintenance of good cellular architecture for up to 2 weeks. The explants displayed intact epithelium and expressed βIII-tubulin and Ki-67. Of the 13 tested intranasal agents, 1% zinc sulfate (ZnSO(4) ), 5% ZnSO(4) , and Zicam application were cytotoxic.

Conclusion: Based on the unique biochemical properties of the human nasal explant culture system, we developed a simple and reliable in vitro screening assay to determine the cytotoxic profiles of various intranasal agents by examining extracellular LDH levels and histopathology.

Conflict of interest statement

Conflicts of interests: None

Copyright © 2011 American Rhinologic Society-American Academy of Otolaryngic Allergy, LLC.

Figures

Figure 1

Organotypic tissue culture setup for human nasal explants. A) Single well setup. B) 24-well setup. The nasal tissue (black arrowhead) is placed on a 0.4µm PTFE membrane (red arrowhead) with the epithelial side exposed to the air. Growth medium surrounds the membrane and provides the tissue with nutrients.

Figure 2

Human nasal explants in organotypic tissue culture system. A) Gross appearance of nasal explants from several different regions of the nasal cavity over 10 days in culture.

Figure 3

Hematoxylin and eosin staining of human nasal explants in culture. (A) Day 0 (B) Day 7 (C) Day 12. (D) In the absence of growth medium, significant degeneration of the epithelium and submucosal glands are also observed.

Figure 4

Lactate dehydrogenase (LDH) released by human nasal explants over 7 days in culture. LDH level is rapidly reduced and stabilized by day 2. LDH levels for each sample tissue is normalized to control growth medium.

Figure 5

Cultured human nasal explants (middle turbinate) express various cellular proteins at day 12. (A) Epithelial cells and neural bundles that traverse the submucosal layer express neural-specific β-tubulin. (B) Ki-67, a marker of cell proliferation, is visible in the basal layer.

Figure 6

Average LDH levels for each intranasal agent by day. The LDH levels are normalized to day 1. Saline and dH2O were used as controls. Four different concentrations of ZnSO4 (0.01%, 0.1%, 1%, and 5%) solutions were tested. Nasalcrom, Atrovent, Afrin, Astelin, Pantanase, Flonase, Omnaris, Rhinocort, Nasonex and Zicam were also examined. Asterisks indicate significant changes in LDH levels as compared to saline (*p<0.05; **p<0.01; ***p<0.001). The error bars indicate standard error of the mean.

Figure 7

Hematoxylin and eosin (H&E) stained human nasal explants following treatment with either saline, dH2O, 0.01% ZnSO4, 0.1% ZnSO4, 1% ZnSO4, 5% ZnSO4, Nasalcrom, Atrovent, Afrin, Astelin, Pantanase, Flonase, Omnaris, Rhinocort, Nasonex and Zicam. Note the significant cellular necrosis with epithelium sloughing in tissues treated with 1% ZnSO4, 5% ZnSO4 and Zicam. Black arrow indicates basal layer of the remaining epithelium.