Summary
Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating tracheal epithelial cultures from Syrian golden hamsters and characterized the cultures for cell composition and function. Soon after initial plating, the epithelial cells reached a high transepithelial resistance and formed tight junctions. The cells differentiated into a heterogeneous, multicellular culture containing ciliated, secretory, and basal cells after culture at an air-liquid interface (ALI). The, secretory cell populations initially consisted of MUC5AC-positive goblet cells and MUC5AC/CCSP double-positive cells, but the makeup changed to predominantly Clara cell secretory protein (CCSP)-positive Clara cells after 14 d. The ciliated cell populations differentiated rapidly after ALI as judged by the appearance of β tubulin IV-positive cells. The cultures produced mucus, CCSP, and trypsin-like proteases and were capable of wound repair as judged by increased expression of matrilysin. Our method provides an efficient, high-yield protocol for producing differentiated hamster tracheal epithelial cells that can be used for a variety of in vitro studies including tracheal cell differentiation, airway disease mechanisms, and pathogen-host interactions.
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Ali, M. J.; Teh, C. Z.; Jennings, R.; Potter, C. W. Transmissibility of influenza viruses in hamsters. Arch. Virol. 72:187–197; 1982.
Bara, J.; Chastre, E.; Mahiou, J.; Singh, R. L.; Forgue-Lafitte, M. E.; Hollande, E.; Godeau, F. Gastric m1 mucin, an early oncofetal marker of colon carcinogenesis, is encoded by the MUC5AC gene. Int. J. Cancer 75:767–773; 1998.
Boers, J. E.; Ambergen, A. W.; Thunnissen, F. B. J. M. Number and proliferation of Clara cells in normal human airway epithelium. Am. J. Respir. Crit. Care Med. 159:1585–1591; 1999.
Buchholz, U. J.; Bukreyev, A.; Yang, L.; Lamirande, E. W.; Murphy, B. R.; Subbarao, K.; Collins, P. L. Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. Proc. Natl. Acad. Sci. USA 101:9804–9809; 2004.
Chua, K. B.; Bellini, W. J.; Rota, P. A., et al. Nipah virus: a recently emergent deadly paramyxovirus. Science 288:1432–1435; 2000.
Collier, A. M.; Peterson, L. P.; Baseman, J. B. Pathogenesis of infection with Bordetella pertussis in hamster tracheal organ culture. J. Infect. Dis. 136 (Suppl.):S196-S203; 1977.
Denker, B. M.; Nigam, S. K. Molecular structure and assembly of the tight junction. Am. J. Physiol. Renal Physiol. 274:F1-F9, 1998.
Dunsmore, S. E.; Saarialho-Kere, U. K.; Roby, J. D.; Wilson, C. L.; Matrisian, L. M.; Welgus, H. G.; Parks, W. C. Matrilysin expression and function in airway epithelium. J. Clin. Invest. 102:1321–1331; 1998.
Fisher, A. F.; Tesh, R. B.; Tonry, J.; Guzman, H.; Liu, D.; Xiao, S.-Y. Induction of severe disease in hamsters by two sandfly fever group viruses, Punta Toro and Gabek Forest (phlebovirus, bunyaviridae), similar to that caused by Rift Valley fever virus. Am. J. Trop. Med. Hyg. 69:269–276; 2003.
Goldman, W. E.; Baseman, J. B. Selective isolation and culture of a proliferating epithelial cell population from the hamster trachea. In Vitro 16:313–319; 1980.
Goldman, W. E.; Klapper, D. G.; Baseman, J. B. Detection, isolation, and analysis of a released Bordetella pertussis product toxic to cultured tracheal cells. Infect. Immun. 36:782–794; 1982.
Hayashi, T.; Ishii, A.; Nakai, S.; Hasegawa, K. Ultrastructure of goblet-cell metaplasia from Clara cell in the allergic asthmatic airway inflammation in a mouse model of asthma in vivo. Virchows Arch. 444: 66–73; 2004.
Hermans, C.; Bernard, A. Lung epithelium-specific proteins. Characteristics and potential applications as markers. Am. J. Respir. Crit. Care Med. 159:646–678; 1999.
Hooper, J. W.; Larsen, T.; Custer, D. M.; Schmaljohn, C. S. A lethal disease model for hantavirus pulmonary syndrome. Virology 289:6–14; 2001.
Kaartinen, L.; Nettesheim, P.; Adler, K. B.; Randell, S. H. Rat tracheal epithelial cell differentiation in vitro. In Vitro Cell. Dev. Biol. 29A:481–492; 1993.
Kalinichenko, V. V.; Gusarova, G. A.; Tan, Y.; Wang, I. C.; Major, M. L.; Wang, X.; Yoder, H. M.; Costa, R. H. Ubiquitous expression of the forkhead box m1b transgene accelerates proliferation of distinct pulmonary cell types following lung injury. J. Biol. Chem. 278:37888–37894; 2003.
Kalinichenko, V. V.; Lim, L.; Shin, B.; Costa, R. H. Differential expression of forkhead box transcription factors following butylated hydroxytoluene lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L695-L704; 2001.
Karp, P. H.; Moninger, T. O.; Weber, S. P; Nesselhauf, T. S.; Launspach, J. L.; Zabner, J.; Welsh, M. J. An in vitro model of differentiated human airway epithelia. Methods for establishing primary cultures. Methods Mol. Biol. 188:115–137; 2002.
Kelly, R. O.; Dekker, R. A.; Bluemink, J. G. Ligand-mediated osmium binding: its application in coating biological specimens for SEM. J. Ultrastruct. Res. 45: 254–258; 1973.
Kido, H.; Yokogoshi, Y.; Sakai, K.; Tashiro, M.; Kishino, Y.; Fukutomi, A.; Katunuma, N. Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of, the viral fusion glycoprotein. J. Biol. Chem. 267:13573–13579; 1992.
Kim, J. H.; Lee, S. Y.; Bak, S. M., et al. Effects of matrix metalloproteinase inhibitor on 1ps-induced goblet cell metaplasia. Am. J. Physiol. Lung Cell. Mol. Physiol. 287:L127-L133; 2004.
Ksiazek, T. G.; Erdman, D.; Goldsmith, C. S., et al. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348:1953–1966; 2003.
Lee, T. C.; Wu, R.; Brody, A. R.; Barrett, J. C.; Nettesheim, P. Growth and differentiation of hamster tracheal epithelial cells in culture. Exp. Lung Res. 6:27–45; 1984.
Li, Q.; Park, P. W.; Wilson, C. L.; Parks, W. C. Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell 111: 635–646; 2002.
Look, D. C.; Walter, M. J.; Williamson, M. R., et al. Effects of paramyxoviral infection on airway epithelial cell foxjl expression, ciliogenesis, and mucociliary function. Am. J. Pathol. 159:2055–2069; 2001.
Lopez, N.; Padula, P.; Rossi, C.; Lazaro, M. E.; Franze-Fernandez, M. T. Genetic identification of a new hamtavirus causing severe pulmonary syndrome in Argentina. Virology 220:223–226; 1996.
McCown, M.; Diamond, M. S.; Pekosz, A. The utility of siRNA transcripts produced by RNA polymerase I in down regulating viral gene expression and replication of negative- and positive-strand RNA viruses. Virology 313: 514–524; 2003.
McGuire, J. K.; Li, Q.; Parks, W.C. Matrilysin (matrix metalloproteinase-7) mediates E-cadherin ectodomain shedding in injured lung epithelium. Am. J. Pathol. 162: 1831–1843; 2003.
Melby, P. C.; Chandrasekar, B.; Zhao, W.; Coe, J. E The hamster as a model of human visceral leishmaniasis: progressive disease and impaired generation of nitric oxide in the face of a prominent th1-like cytokine response. J. Immunol. 166:1912–1920; 2001.
Milazzo, M. L.; Eyzaguirre, E. J.; Molina, C. P.; Fulhorst, C. F. Maporal viral infection in the Syrian golden hamster: a model of hantavirus pulmonary syndrome. J. Infect. Dis. 186:1390–1395; 2002.
Moller, P. C.; Partridge, L. R.; Cox, R.; Pellegrini, V.; Ritchie, D. C. An in vitro system for the study of tracheal epithelial cells. Tissue Cell 19:783–791; 1987.
Morrey, J. D.; Day, C. W.; Julander, J. G.; Olsen, A. L.; Sidwell, R. W.; Cheney, C. D.; Blatt, L. M. Modeling hamsters for evaluating West Nile virus therapies. Antiviral Res. 63:41–50; 2004.
Niles, R.; Kim, K. C.; Hyman, B.; Christensen, T.; Wasano, K.; Brody, J. Characterization of extended primary and secondary cultures of hamster tracheal epithelial cells. In Vitro Cell. Dev. Biol. 24:457–463; 1988.
Nordman, H.; Davies, J. R.; Lindell, G.; de Bolos, C.; Real, F.; Carlstedt, I. Gastric MUC5AC and MUC6 are large oligomeric mucins that differ in size, glycosylation and tissue distribution. Biochem. J. 364: 191–200; 2002.
Paessler, S.; Aguilar, P.; Anishchenko, M.; Wang, H. Q.; Aronson, J.; Campbell, G.; Cararra, A. S.; Weaver, S. C. The hamster as an animal model for eastern equine encephalitis—and its use in studies of virus entrance into the brain. J. Infect. Dis. 189:2072–2076; 2004.
Parks, W. C.; Lopez-Boado, Y. S.; Wilson, C. L. Matrilysin in epithelial repair and defense. Chest 120:36S-41S; 2001.
Parks, W. C.; Wilson, C. L.; Lopez-Boado, Y. S. Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat. Rev. Immunol. 4:617–629; 2004.
Paterson, R. G.; Lamb, R. A. The molecular biology of influenza viruses and paramyxoviruses. Molecular virology: a practical approach. Oxford, U.K.: Oxford University Press; 1993:35–73.
Reader, J. R.; Tepper, J. S.; Schelegle, E. S.; Aldrich, M. C.; Putney, L. F.; Pfeiffer, J. W.; Hyde, D. M. Pathogenesis of mucous cell metaplasia in a murine asthma model. Am. J. Pathol. 162: 2069–2078; 2003.
Robinson, T. W.; Dorio, R. J.; Kim, K. J. Formation of tight monolayers of guinea pig airway epithelial cells cultured in an air-interface: bioelectric properties. Biotechniques 15:468–473; 1993.
Shahzeidi, S.; Aujla, P. K.; Nickola, T. J.; Chen, Y.; Alimam, M. Z.; Rose, M. C. Temporal analysis of goblet cells and mucin gene expression in murine models of allergic asthma. Exp. Lung Res. 29:549–565; 2003.
Singh, G.; Katyal, S. L. Clara cells and Clara cell 10 kd protein (cc10). Am. J. Respir. Cell Mol. Biol. 17:141–143; 1997.
Sinn, P. L.; Williams, G.; Vongpunsawad, S.; Cattaneo, R.; McCray, P. B., Jr. Measles virus preferentially transduces the basolateral surface of well-differentiated human airway epithelia. J. Virol. 76:2403–2409; 2002.
Steinhauer, D. A. Role of hemagglutinin cleavage for the pathogenicity of influenza virus. Virology 258:1–20; 1999.
Takeda, M.; Pekosz, A.; Shuck, K.; Pinto, L. H.; Lamb, R. A. Influenza a virus M2 ion channel activity is essential for efficient replication in tissue culture. J. Virol. 76:1391–1399; 2002.
Tao, T.; Skiadopoulos, M. H.; Durbin, A. P.; Davoodi, F.; Collins, P. L.; Murphy, B.R. A live attenuated chimeric recombinant parainfluenza virus (PIV) encoding the internal proteins of PIV type 3 and the surface glycoproteins of PIV type 1 induces complete resistance to PIV1 challenge and partial resistance to PIV3 challenge. Vaccine 17:1100–1108; 1999.
Walters, R. W.; Freimuth, P.; Moninger, T. O.; Ganske, I.; Zabner, J.; Welsh, M. J. Adenovirus fiber disrupts car-mediated intercellular adhesion allowing virus escape. Cell 110:789–799; 2002.
Whitcutt, M. J.; Adler, K.B.; Wu, R. A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells. In Vitro Cell. Dev. Biol. 24:420–428; 1988.
Wielock, B.; Libert, C.; Wilson, C. Matrilysin (matrix metalloproteinase-7): a new promising drug target in cancer and inflammation?. Cytokine Growth Factor Rev. 15:111–115; 2004.
Wong, K. T.; Grosjean, I.; Brisson, C., et al. A golden hamster model for human acute Nipah virus infection. Am. J. Pathol. 163:2127–2137; 2003.
Wu, R. Growth and differentiation of tracheobronchial epithelial cells. In: Mcdonald, J. A., ed. Lung growth and development, vol. 100. New York: Marcel Dekker; 1997:211–241.
Wu, R.; Nolan, E.; Turner, C. Expression of tracheal differentiated functions in serum-free hormone-supplemented medium. J. Cell. Physiol. 125:167–181; 1985.
Wu, R.; Smith, D. Continuous multiplication of rabbit tracheal epithelial cells in a defined, hormone-supplemented medium. In Vitro 18:800–812; 1982.
Yamaya, M.; Finkbeiner, W. E.; Chun, S. Y.; Widdicombe, J. H. Differentiated structure and function of cultures from human tracheal epithelium. Am. J. Physiol. 262:L713-L724; 1992.
Yamaya, M.; Hosoda, H.; Suzuki, T.; Yamada, N.; Sasaki, H. Human airway epithelial cell culture. In: Wise, C., ed. Epithelial cell culture protocols. Totowa, NJ: Humana Press; 2002:7–16.
You, Y.; Richer, E. J.; Huang, T.; Brody, S. L. Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population. Am. J. Physiol. Lung Cell. Mol. Physiol. 283:L1315-L1321; 2002.
Zhang, L.; Peeples, M. E.; Boucher, R. C.; Collins, P. L.; Pickles, R. J. Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology. J. Virol. 76:5654–5666; 2002.
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Rowe, R.K., Brody, S.L. & Pekosz, A. Differentiated cultures of primary hamster tracheal airway epithelial cells. In Vitro Cell.Dev.Biol.-Animal 40, 303–311 (2004). https://doi.org/10.1290/0408056.1
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DOI: https://doi.org/10.1290/0408056.1