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Culture of Calu-3 Cells at the Air Interface Provides a Representative Model of the Airway Epithelial Barrier

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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.


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.


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 Ω cm2 at 11–16 days, was significantly greater than the TER of cells cultured using AIC, 306 ± 53 Ω cm2 at 11–13 days. Apparent permeability (P app ) values for the transport of flu-Na using LCC and AIC were 1.48 ± 0.19×10−7 and 3.36 ± 0.47×10−7 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.


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.

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A :

surface area of filter insert (cm2)


air-interfaced culture

C :

initial concentration (μg/mL)

D :

diffusion coefficient (cm2/s)

F :

flux (rate of change in cumulative mass transported) (μg/s)


fluorescein isothiocyanate labeled dextran


fluorescein-sodium salt


liquid-covered culture

P app :

apparent permeability (cm/s)

r i :

molecular radius of solute (nm)

r p :

molecular radius of pore (nm)


transepithelial resistance (Ω cm2)


zona occludens protein-1

ɛ/L :

ratio of pore area to length


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The technical assistance of Tony Brain for electron microscopy and the advice of Toshinobu Seki and David Barlow regarding the Renkin analysis are gratefully acknowledged. This work was funded by a grant from the Safety and Environmental Assurance Centre, Unilever Colworth, UK.

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Correspondence to Ben Forbes.

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Grainger, C.I., Greenwell, L.L., Lockley, D.J. et al. Culture of Calu-3 Cells at the Air Interface Provides a Representative Model of the Airway Epithelial Barrier. Pharm Res 23, 1482–1490 (2006).

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