title: A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.
abstract: A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.
authors.affiliation: Department of Microbiology and Immunology, Program in Molecular Pathogenesis and Immunity, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Carterson
authors.forename: K
authors.initials: K
authors.lastname: Höner zu Bentrup
authors.forename: C M
authors.initials: CM
authors.lastname: Ott
authors.forename: M S
authors.initials: MS
authors.lastname: Clarke
authors.forename: D L
authors.initials: DL
authors.lastname: Pierson
authors.forename: C R
authors.initials: CR
authors.lastname: Vanderburg
authors.forename: K L
authors.initials: KL
authors.lastname: Buchanan
authors.forename: C A
authors.initials: CA
authors.lastname: Nickerson
authors.forename: M J
authors.initials: MJ
authors.lastname: Schurr
mesh_terms: D000941:Antigens / Q000378:metabolism; D000951:Antigens, Neoplasm; D015415:Biomarkers; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods; D024141:Collagen Type IV / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007378:Interleukins / Q000378:metabolism; D007797:Laminin / Q000378:metabolism; D008168:Lung / Q000378:metabolism / Q000382:microbiology*; D008954:Models, Biological; D055271:Mucin 5AC; D018396:Mucin-1; D009077:Mucins / Q000378:metabolism; D011552:Pseudomonas Infections; D011550:Pseudomonas aeruginosa; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000941:Antigens; D000951:Antigens, Neoplasm; D015415:Biomarkers; D024141:Collagen Type IV; D006023:Glycoproteins; D007378:Interleukins; D007797:Laminin; C060192:MUC1 protein, human; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D018396:Mucin-1; D009077:Mucins; D014409:Tumor Necrosis Factor-alpha