Purpose
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 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.
Conclusion
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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Abbreviations
- A :
-
surface area of filter insert (cm2)
- AIC:
-
air-interfaced culture
- C :
-
initial concentration (μg/mL)
- D :
-
diffusion coefficient (cm2/s)
- F :
-
flux (rate of change in cumulative mass transported) (μg/s)
- FITC-dex:
-
fluorescein isothiocyanate labeled dextran
- flu-Na:
-
fluorescein-sodium salt
- LCC:
-
liquid-covered culture
- P app :
-
apparent permeability (cm/s)
- r i :
-
molecular radius of solute (nm)
- r p :
-
molecular radius of pore (nm)
- TER:
-
transepithelial resistance (Ω cm2)
- ZO-1:
-
zona occludens protein-1
- ɛ/L :
-
ratio of pore area to length
References
G. Borchard (2002) ArticleTitleCalu-3 cells, a valid model for the airway epithelium? STP Pharma Sci. 12 205–211
C. Ehrhardt J. Fiegel S. Fuchs R. Abu-Dahab U. F. Schaefer J. Hanes C. M. Lehr (2002) ArticleTitleDrug absorption by the respiratory mucosa: cell culture models and particulate drug carriers J. Aerosol Med. 15 131–139 Occurrence Handle12184863 Occurrence Handle1:CAS:528:DC%2BD38Xmt1aisrg%3D Occurrence Handle10.1089/089426802320282257
B. I. Florea M. L. Cassara H. E. Junginger G. Borchard (2003) ArticleTitleDrug transport and metabolism characteristics of the human airway epithelial cell line Calu-3 J. Control. Release 87 131–138 Occurrence Handle12618029 Occurrence Handle1:CAS:528:DC%2BD3sXhsFOlsrc%3D Occurrence Handle10.1016/S0168-3659(02)00356-5
R. Q. Shen W. E. Finkbeiner J. J. Wine R. J. Mrsny J. H. Widdicombe (1994) ArticleTitleCalu-3—a human airway epithelial-cell line that shows C amp-dependent Cl- secretion Am. J. Phys. 266 L493–L501 Occurrence Handle1:CAS:528:DyaK2cXktlWqt7g%3D
K. A. Foster M. L. Avery M. Yazdanian K. L. Audus (2000) ArticleTitleCharacterization of the Calu-3 cell line as a tool to screen pulmonary drug delivery Int. J. Pharm. 208 1–11 Occurrence Handle11064206 Occurrence Handle1:CAS:528:DC%2BD3cXosVGjsbw%3D Occurrence Handle10.1016/S0378-5173(00)00452-X
B. Forbes C. Ehrhardt (2005) ArticleTitleHuman respiratory epithelial cell culture for drug delivery applications Eur. J. Pharm. Biopharm. 60 193–205 Occurrence Handle15939233 Occurrence Handle1:CAS:528:DC%2BD2MXltFCrsr8%3D Occurrence Handle10.1016/j.ejpb.2005.02.010
B. I. Florea M. Thanou H. E. Junginger G. Borchard (2006) ArticleTitleEnhancement of bronchial octreotide absorption by chitosan and N-trimethyl chitosan shows linear in vitro/in vivo correlation J. Control. Release 110 353–361 Occurrence Handle16269199 Occurrence Handle1:CAS:528:DC%2BD2MXhtlert7nP Occurrence Handle10.1016/j.jconrel.2005.10.001
J. Geys L. Coenegrachts J. Vercammen Y. Engelborghs A. Nemmar B. Nemery P. H. Hoet (2006) ArticleTitle In vitro study of the pulmonary translocation of nanoparticles: a preliminary study Toxicol. Lett. 160 218–226 Occurrence Handle16137845 Occurrence Handle1:CAS:528:DC%2BD2MXht1arsrfI Occurrence Handle10.1016/j.toxlet.2005.07.005
L. A. Sachs W. E. Finkbeiner J. H. Widdicombe (2003) ArticleTitleEffects of media on differentiation of cultured human tracheal epithelium In Vitro Cell. Dev. Biol., Anim. 39 56–62 Occurrence Handle1:CAS:528:DC%2BD3sXmvFygsLs%3D Occurrence Handle10.1290/1543-706X(2003)039<0056:EOMODO>2.0.CO;2
J. H. Widdicombe L. A. Sachs W. E. Finkbeiner (2003) ArticleTitleEffects of growth surface on differentiation of cultures of human tracheal epithelium In Vitro Cell. Dev. Biol., Anim. 39 51–55 Occurrence Handle1:STN:280:DC%2BD3szmtVGmuw%3D%3D Occurrence Handle10.1290/1543-706X(2003)039<0051:EOGSOD>2.0.CO;2
P. M. Dejong M. A. J. A. Vansterkenburg J. A. Kempenaar J. H. Dijkman M. Ponec (1993) ArticleTitleSerial culturing of human bronchial epithelial-cells derived from biopsies In Vitro Cell. Dev. Biol., Anim. 29A 379–387 Occurrence Handle1:STN:280:ByyA3c%2FntFw%3D
L. G. Johnson K. G. Dickman K. L. Moore L. J. Mandel R. C. Boucher (1993) ArticleTitleEnhanced Na+ transport in an air–liquid interface culture system Am. J. Physiol 264 L560–L565 Occurrence Handle8333549 Occurrence Handle1:CAS:528:DyaK3sXlsFGgtbs%3D
M. Yamaya W. E. Finkbeiner S. Y. Chun J. H. Widdicombe (1992) ArticleTitleDifferentiated structure and function of cultures from human tracheal epithelium Am. J. Physiol 262 L713–L724 Occurrence Handle1616056 Occurrence Handle1:CAS:528:DyaK38Xmt12ms74%3D
P. K. Jeffery (1983) ArticleTitleMorphologic features of airway surface epithelial-cells and glands Am. Rev. Respir. Dis. 128 S14–S20 Occurrence Handle6881701 Occurrence Handle1:STN:280:BiyB1c7gtVI%3D
P. K. Jeffery D. Gaillard S. Moret (1992) ArticleTitleHuman airway secretory cells during development and in mature airway epithelium Eur. Respir. J. 5 93–104 Occurrence Handle1577157 Occurrence Handle1:STN:280:By2B2cblslY%3D
A. V. Rogers A. Dewar B. Corrin P. K. Jeffery (1993) ArticleTitleIdentification of serous-like cells in the surface epithelium of human bronchioles Eur. Respir. J. 6 498–504 Occurrence Handle8491299 Occurrence Handle1:STN:280:ByyB2MbivFY%3D
M. E. Krouse J. F. Talbott M. M. Lee N. S. Joo J. J. Wine (2004) ArticleTitleAcid and base secretion in the Calu-3 model of human serous cells Am. J. Physiol. Lung. Cell. Mol. Physiol. 287 L1274–L1283 Occurrence Handle15310554 Occurrence Handle1:CAS:528:DC%2BD2cXhtFShsb7M Occurrence Handle10.1152/ajplung.00036.2004
P. B. R. Babu A. Chidekel L. Utidjian T. H. Shaffer (2004) ArticleTitleRegulation of apical surface fluid and protein secretion in human airway epithelial cell line Calu-3 Biochem. Biophys. Res. Commun. 319 1132–1137 Occurrence Handle1:CAS:528:DC%2BD2cXkvVCltbg%3D Occurrence Handle10.1016/j.bbrc.2004.05.101
M. Singh M. Krouse S. Moon J. J. Wine (1997) ArticleTitleMost basal I-sc in Calu-3 human airway cells is bicarbonate-dependent Cl- secretion Am. J. Physiol. Lung. Cell. Mol. Physiol. 16 L690–L698
I. Pezron R. Mitra D. Pal A. K. Mitra (2002) ArticleTitleInsulin aggregation and asymmetric transport across human bronchial epithelial cell monolayers (Calu-3) J. Pharm. Sci. 91 1135–1146 Occurrence Handle11948552 Occurrence Handle1:CAS:528:DC%2BD38XjtFKhu7k%3D Occurrence Handle10.1002/jps.10114
J. Patel D. Pal V. Vangala M. Gandhi A. K. Mitra (2002) ArticleTitleTransport of HIV-protease inhibitors across 1 alpha,25di-hydroxy vitamin D-3-treated Calu-3 cell monolayers: modulation of P-glycoprotein activity Pharm. Res. 19 1696–1703 Occurrence Handle12458676 Occurrence Handle1:CAS:528:DC%2BD38XotV2hu70%3D Occurrence Handle10.1023/A:1020761514471
D. Cooney M. Kazantseva A. J. Hickey (2004) ArticleTitleDevelopment of a size-dependent aerosol deposition model utilising human airway epithelial cells for evaluating aerosol drug delivery ATLA, Altern. Lab. Anim. 32 581–590 Occurrence Handle1:CAS:528:DC%2BD2MXivFGns70%3D
J. Fiegel C. Ehrhardt U. F. Schaefer C. M. Lehr J. Hanes (2003) ArticleTitleLarge porous particle impingement on lung epithelial cell monolayers—toward improved particle characterization in the lung Pharm. Res. 20 788–796 Occurrence Handle12751635 Occurrence Handle1:CAS:528:DC%2BD3sXjtFKgsLo%3D Occurrence Handle10.1023/A:1023441804464
E. M. Renkin (1954) ArticleTitleFiltration, diffusion, and molecular sieving through porous cellulose membranes J. Gen. Physiol. 38 225–243 Occurrence Handle13211998 Occurrence Handle1:STN:280:DyaG2M%2FitlKltw%3D%3D
T. Seki J. Mochida M. Okamoto O. Hosoya K. Juni K. Morimoto (2003) ArticleTitleMeasurement of diffusion coefficients of parabens and steroids in water and 1-octanol Chem. Pharm. Bull. 51 734–736 Occurrence Handle12808257 Occurrence Handle1:CAS:528:DC%2BD3sXls1KntLw%3D Occurrence Handle10.1248/cpb.51.734
O. Hosoya S. Chono Y. Saso K. Juni K. Morimoto T. Seki (2004) ArticleTitleDetermination of diffusion coefficients of peptides and prediction of permeability through a porous membrane J. Pharm. Pharmacol. 56 1501–1507 Occurrence Handle15563756 Occurrence Handle1:CAS:528:DC%2BD2MXhtl2q Occurrence Handle10.1211/0022357044878
M. Knowles G. Murray J. Shallal F. Askin V. Ranga J. Gatzy R. Boucher (1984) ArticleTitleBioelectric properties and ion flow across excised human bronchi J. Appl. Physiol. 56 868–877 Occurrence Handle6144659 Occurrence Handle1:CAS:528:DyaL2cXhvV2gurs%3D
J. T. Berger J. A. Voynow K. W. Peters M. C. Rose (1999) ArticleTitleRespiratory carcinoma cell lines—MUC genes and glycoconjugates Am. J. Respir. Cell Mol. Biol. 20 500–510 Occurrence Handle10030849 Occurrence Handle1:CAS:528:DyaK1MXitVWisL8%3D
N. R. Mathias J. Timoszyk P. I. Stetsko J. R. Megill R. L. Smith D. A. Wall (2002) ArticleTitlePermeability characteristics of Calu-3 human bronchial epithelial cells: in vitro–in vivo correlation to predict lung absorption in rats J. Drug Target. 10 31–40 Occurrence Handle1:CAS:528:DC%2BD38Xis1Oht78%3D Occurrence Handle10.1080/10611860290007504
P. M. Dejong M. A. J. A. Vansterkenburg S. C. Hesseling J. A. Kempenaar A. A. Mulder A. M. Mommaas J. H. Dijkman M. Ponec (1994) ArticleTitleCiliogenesis in human bronchial epithelial-cells cultured at the air–liquid interface Am. J. Respir. Cell Mol. Biol. 10 271–277 Occurrence Handle1:STN:280:ByuC287ps1Q%3D
W. E. Finkbeiner S. D. Carrier C. E. Teresi (1993) ArticleTitleReverse transcription-polymerase chain-reaction (RT-PCR) phenotypic analysis of cell-cultures of human tracheal epithelium, tracheobronchial glands, and lung carcinomas Am. J. Respir. Cell Mol. Biol. 9 547–556 Occurrence Handle7692897 Occurrence Handle1:CAS:528:DyaK2cXitFegu7g%3D
M. Yamaya W. E. Finkbeiner J. H. Widdicombe (1991) ArticleTitleIon-transport by cultures of human tracheobronchial submucosal glands Am. J. Physiol. 261 L485–L490 Occurrence Handle1767867 Occurrence Handle1:CAS:528:DyaK38XotV2qsg%3D%3D
B. Meyrick J. M. Sturgess L. Reid (1969) ArticleTitleA reconstruction of duct system and secretory tubules of human bronchial submucosal gland Thorax 24 729–736 Occurrence Handle5350723 Occurrence Handle1:STN:280:DyaE3c%2Fjt1ejsA%3D%3D Occurrence Handle10.1136/thx.24.6.729
H. Wan H. L. Winton C. Soeller G. A. Stewart P. J. Thompson D. C. Gruenert M. B. Cannell D. R. Garrod C. Robinson (2000) ArticleTitleTight junction properties of the immortalized human bronchial epithelial cell lines Calu-3 and 16HBE14o- Eur. Respir. J. 15 1058–1068 Occurrence Handle10885425 Occurrence Handle1:STN:280:DC%2BD3M%2FitV2jsQ%3D%3D Occurrence Handle10.1034/j.1399-3003.2000.01514.x
C. Ehrhardt C. Kneuer J. Fiegel J. Hanes U. F. Schaefer K. J. Kim C. M. Lehr (2002) ArticleTitleInfluence of apical fluid volume on the development of functional intercellular junctions in the human epithelial cell line 16HBE14o-: implications for the use of this cell line as an in vitro model for bronchial drug absorption studies Cell Tissue. Res. 308 391–400 Occurrence Handle12107432 Occurrence Handle1:CAS:528:DC%2BD38Xks1Srs78%3D Occurrence Handle10.1007/s00441-002-0548-5
S. Loman J. Radl H. M. Jansen T. A. Out R. Lutter (1997) ArticleTitleVectorial transcytosis of dimeric IgA by the Calu-3 human lung epithelial cell line: upregulation by IFN-gamma Am. J. Physiol. Lung Cell. Mol. Physiol. 16 L951–L958
C. Meaney B. I. Florea G. Borchard H. E. Junginger (1999) ArticleTitleCharacterisation of a human submucosal gland cell line (Calu-3) as an in vitro model of the airway epithelium Proc. Int. Symp. Control. Rel. Bioact. Mater. 26 198–199
Acknowledgments
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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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). https://doi.org/10.1007/s11095-006-0255-0
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DOI: https://doi.org/10.1007/s11095-006-0255-0