Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro

Abstract

The purpose of this study was to compare cell growth characteristics, ciliated cell differentiation, and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures. Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods. Epithelial cell growth characteristics were observed by inverted phase contrast microscopy. Ciliated cell differentiation was detected by β-tubulin IV and ZO-1 immunocytochemistry. Basal and ATP-stimulated ciliary beat frequency (CBF) was measured using a high-speed digital microscopic imaging system. Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition, with both types of cultures comprising ciliated and non-ciliated epithelial cells. Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures. In both culture systems, the highest ciliated cell density appeared at 7th–10th culture day and declined with time, with the lifespan of ciliated cells ranging from 14 to 21 days. Overall, 10% of the cells in explant cultures and 20% of the cells in the dissociated tissue cultures were ciliated. These two cultures demonstrated similar ciliary beat frequency values at baseline (7.78±1.99 Hz and 7.91±2.52 Hz, respectively) and reacted equivalently following stimulation with 100 μM ATP. The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells, which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

Keywords

ciliated cells / ciliary beat frequency / dissociated tissue culture / explant culture / nasal epithelial cells

References

[1] Kojima T, Go M, Takano K, Kurose M, Ohkuni T, Koizumi J, Kamekura R, Ogasawara N, Masaki T, Fuchimoto J, Obata Ki, Hirakawa S, Nomura K, Keira T, Miyata R, Fujii N, Tsutsumi H, Himi T, Sawada N. Regulation of tight junctions in upper airway epithelium. Biomed Res Int 2013(2013): 947072

[2] Kato A, Schleimer RP. Beyond inflammation: airway epithelial cells are at the interface of innate and adaptive immunity. Curr Opin Immunol2007; 19(6): 711-720

[3] Devalia JL, Davies RJ. Airway epithelial cells and mediators of inflammation. Respir Med1993; 87(6): 405-408

[4] Gudis D, Zhao KQ, Cohen NA. Acquired cilia dysfunction in chronic rhinosinusitis. Am J Rhinol Allergy2012; 26(1): 1-6

[5] Antunes MB, Gudis DA, Cohen NA. Epithelium, cilia, and mucus: their importance in chronic rhinosinusitis. Immunol Allergy Clin North Am2009; 29(4): 631-643

[6] Gudis DA, Cohen NA. Cilia dysfunction. Otolaryngol Clin North Am2010; 43(3): 461-472, vii

[7] Comer DM, Elborn JS, Ennis M. Comparison of nasal and bronchial epithelial cells obtained from patients with COPD. PLoS ONE2012; 7(3): e32924

[8] Bukowy Z, Ziętkiewicz E, Witt M. In vitro culturing of ciliary respiratory cells—a model for studies of genetic diseases. J Appl Genet2011; 52(1): 39-51

[9] Dimova S, Brewster ME, Noppe M, Jorissen M, Augustijns P. The use of human nasal in vitro cell systems during drug discovery and development. Toxicol In Vitro2005; 19(1): 107-122

[10] Zhang L, Han D, Song X, Wang K, Wang H. Effect of oxymetazoline on healthy human nasal ciliary beat frequency measured with high-speed digital microscopy and mucociliary transport time. Ann Otol Rhinol Laryngol2008; 117(2): 127-133

[11] Nlend MC, Bookman RJ, Conner GE, Salathe M. Regulator of G-protein signaling protein 2 modulates purinergic calcium and ciliary beat frequency responses in airway epithelia. Am J Respir Cell Mol Biol2002; 27(4): 436-445

[12] Jiao J, Wang H, Lou W, Jin S, Fan E, Li Y, Han D, Zhang L. Regulation of ciliary beat frequency by the nitric oxide signaling pathway in mouse nasal and tracheal epithelial cells. Exp Cell Res2011; 317(17): 2548-2553

[13] Devalia JL, Sapsford RJ, Wells CW, Richman P, Davies RJ. Culture and comparison of human bronchial and nasal epithelial cells in vitro. Respir Med1990; 84(4): 303-312

[14] Rhee CS, Min YG, Lee CH, Kwon TY, Lee CH, Yi WJ, Park KS. Ciliary beat frequency in cultured human nasal epithelial cells. Ann Otol Rhinol Laryngol2001; 110(11): 1011-1016

[15] Dimova S, Vlaeminck V, Brewster ME, Noppe M, Jorissen M, Augustijns P. Stable ciliary activity in human nasal epithelial cells grown in a perfusion system. Int J Pharm2005; 292(1-2): 157-168

[16] Robertson A, Stannard W, Passant C, O’Callaghan C, Banerjee A. What effect does isoflurane have upon ciliary beat pattern: an in vivo study. Clin Otolaryngol Allied Sci2004; 29(2): 157-160

[17] Liu Z, Lu X, Wang H, You XJ, Gao QX, Cui YH. Group II subfamily secretory phospholipase A2 enzymes: expression in chronic rhinosinusitis with and without nasal polyps. Allergy2007; 62(9): 999-1006

[18] Agu RU, Jorissen M, Willems T, Augustijns P, Kinget R, Verbeke N. In-vitro nasal drug delivery studies: comparison of derivatised, fibrillar and polymerised collagen matrix-based human nasal primary culture systems for nasal drug delivery studies. J Pharm Pharmacol2001; 53(11): 1447-1456

[19] Werner U, Kissel T. Development of a human nasal epithelial cell culture model and its suitability for transport and metabolism studies under in vitro conditions. Pharm Res1995; 12(4): 565-571

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg