title: Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier.
abstract: OBJECTIVE
  The aim of this study was to compare the effect of liquid-covered culture (LCC) and air-interfaced culture (AIC) on Calu-3 cell layer morphology and permeability, thus assessing the fitness of these culture systems as models of airway epithelium barrier function.
  
  
  METHODS
  Cell layers were grown on 0.33 cm2 Transwell polyester cell culture supports. Cell layers grown using LCC and AIC were evaluated by using light and electron microscopy, transepithelial electrical resistance (TER), and permeability to the transepithelial flux of fluorescein sodium (flu-Na), and by varying molecular weight dextrans labeled with fluorescein isothiocyanate (FITC-dex). The tight junction protein, zona occludens protein-1 (ZO-1), was visualized by confocal microscopy and apical glycoprotein secretions were identified by using alcian blue.
  
  
  RESULTS
  Cells grown via AIC produced a more columnar epithelium with a more rugged apical topography and greater glycoprotein secretion compared to cells grown via LCC. Apical protrusions appearing to be cilia-like structures were observed on occasional cells using AIC, but typical airway ciliated cell phenotypes were not produced under either condition. Secretory granules were observed in cells cultured under both conditions. Cells cultured using LCC exhibited higher levels of ZO-1 protein than the AIC counterpart. The maximal TER of cells using LCC, 1,086 +/- 113 ohms cm2 at 11-16 days, was significantly greater than the TER of cells cultured using AIC, 306 +/- 53 ohms cm2 at 11-13 days. Apparent permeability (P(app)) values for the transport of flu-Na using LCC and AIC were 1.48 +/- 0.19x10(-7) and 3.36 +/- 0.47x10(-7) cm s(-1), respectively. Transport rates of flu-Na and FITC-dex were inversely proportional to molecular weight, and were significantly lower (p < 0.05) in cell layers grown using LCC than AIC. Renkin analysis fitted the data to single pore populations of radii 7.7 and 11.0 nm for LCC and AIC, respectively.
  
  
  CONCLUSIONS
  Distinct differences in morphology and permeability result when Calu-3 cells are grown using AIC or LCC. Cells cultured using AIC generate a model more morphologically representative of the airway epithelium than cells cultured using LCC.
authors.affiliation: Pharmaceutical Science Research Division, King's College London, Franklin Williams Building, London, SE1 9NH, UK.
authors.forename: Christopher I
authors.initials: CI
authors.lastname: Grainger
authors.forename: Leona L
authors.initials: LL
authors.lastname: Greenwell
authors.forename: David J
authors.initials: DJ
authors.lastname: Lockley
authors.forename: Gary P
authors.initials: GP
authors.lastname: Martin
authors.forename: Ben
authors.initials: B
authors.lastname: Forbes
mesh_terms: D001980:Bronchi / Q000378:metabolism* / Q000648:ultrastructure; D018929:Cell Culture Techniques; D002460:Cell Line; D002463:Cell Membrane Permeability; D048430:Cell Shape; D003911:Dextrans; D017097:Electric Impedance; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D006801:Humans; D008871:Microvilli; D020545:Respiratory Mucosa / Q000378:metabolism* / Q000648:ultrastructure; D019108:Tight Junctions / Q000378:metabolism
chemical_list: D003911:Dextrans; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate