Pharmaceutical Research

, Volume 30, Issue 3, pp 781–791 | Cite as

Drug Delivery Characteristics of the Progenitor Bronchial Epithelial Cell Line VA10

  • Berglind Eva Benediktsdóttir
  • Ari Jón Arason
  • Skarphédinn Halldórsson
  • Thórarinn Gudjónsson
  • Már Másson
  • Ólafur Baldursson
Research Paper



To determine the integrity and permeability properties of the immortalized human VA10 bronchial epithelial cell line for its suitability as an in vitro drug permeation model.


Cells were grown under liquid-covered culture (LCC) or air-liquid interface (ALI) culture, characterized using electron microscopy and immunostaining. Integrity was measured using transepithelial electrical resistance (TER) and permeability of fluorescein sodium (Flu-Na). General permeability was established with dextrans and model drugs and P-glycoprotein (P-gp) function determined with bidirectional flux of rhodamine-123.


ALI culture resulted in 2–3 cell layers with differentiation towards ciliated cells but LCC showed undifferentiated morphology. VA10 cells formed TJ, with higher TER in LCC than ALI (∼2500 vs. ∼1200 Ω*cm2) and Flu-Na permeability ∼1–2 × 10−7 cm/s. ALI cultured cells expressed P-gp and distinguished between compounds depending on lipophilicity and size, consistent with previous data from Calu-3 and 16HBE14o-cell lines.


ALI cultured cell layers capture the in vivo-like phenotype of bronchial epithelium and form functional cell barrier capable of discriminating between compounds depending on physiochemical properties. The VA10 cell line is an important alternative to previously published cell lines and a relevant model to study airway drug delivery in vitro.


air-liquid interface culture airway permeability differentiation drug delivery human bronchial epithelial cells 





surface area (cm2)




air-liquid interface


basolateral to apical


bovine serum albumin


ciliary beating frequency


cystic fibrosis transmembrane conductance regulator


fluorescein isothiocyanate labeled dextran


fluorescein sodium


Hanks balanced salt solution


human papilloma virus-16


liquid-covered culture


normal human bronchial epithelial


apparent permeability (cm/s)


phosphate buffered saline




retinoblastoma tumor suppressor protein


rhodamine 123


scanning electron microscopy


transepithelial electrical resistance (Ω*cm2)


tight junction



Financial support from the Eimskip Fund of University of Iceland, the University of Iceland Research Fund, the Landspitali University Hospital Science Fund and the Bergthóru and Thorsteins Scheving Thorsteinssonar Fund is gratefully acknowledged. We thank Professor Magnus Karl Magnusson for critical discussion and good advice, Sigrún Kristjánsdóttir at the Pathology Department of Landspitali University Hospital for her contribution to the paraffin prepared samples and Bergthóra S. Snorradóttir at the University of Iceland for help with the HPLC.

Supplementary material

Supplementary Video

Video of beating cilia of differentiated VA10 cells cultured at ALI for 14 days was taken with Leica DMI3000 inverted microscopy, 40x objective and DIC filter, focus directed at apical surface of the cell layer. Before imaging, the surface of the cells was immersed in PBS. The ciliary beating can clearly be seen on individual cells and patches of ciliated cells that cover 10-15% of the surface area. (WMV 1794 kb)


  1. 1.
    Sakagami M. In vivo, in vitro and ex vivo models to assess pulmonary absorption and disposition of inhaled therapeutics for systemic delivery. Adv Drug Delivery Rev. 2006;58(9–10):1030–60.CrossRefGoogle Scholar
  2. 2.
    Guidance for industry: Waiver of in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage forms based on a biopharmaceutics classification system. August 2000, CDER/FDA.Google Scholar
  3. 3.
    Note for guidance on the investigation of bioavailability and bioequivalence. December 2000, EMEA.Google Scholar
  4. 4.
    Forbes B, Ehrhardt C. Human respiratory epithelial cell culture for drug delivery applications. Eur J Pharm Biopharm. 2005;60(2):193–205.PubMedCrossRefGoogle Scholar
  5. 5.
    Sporty JL, Horalkova L, Ehrhardt C. In vitro cell culture models for the assessment of pulmonary drug disposition. Expert Opin Drug Metab Toxicol. 2008;4(4):333–45.PubMedCrossRefGoogle Scholar
  6. 6.
    Shen BQ, Finkbeiner WE, Wine JJ, Mrsny RJ, Widdicombe JH. Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl- secretion. Am J Physiol Lung Cell Mol Physiol. 1994;266(5):L493–501.Google Scholar
  7. 7.
    Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, et al. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol. 1994;10(1):38–47.PubMedGoogle Scholar
  8. 8.
    Wan H, Winton H, Soeller C, Stewart G, Thompson P, Gruenert D, et al. Tight junction properties of the immortalized human bronchial epithelial cell lines Calu-3 and 16HBE14o. Eur Respir J. 2000;15(6):1058–68.PubMedCrossRefGoogle Scholar
  9. 9.
    Manford F, Tronde A, Jeppsson A-B, Patel N, Johansson F, Forbes B. Drug permeability in 16HBE14o- airway cell layers correlates with absorption from the isolated perfused rat lung. Eur J Pharm Sci. 2005;26(5):414–20.PubMedCrossRefGoogle Scholar
  10. 10.
    Mathias NR, Timoszyk J, Stetsko PI, Megill JR, Smith RL, Wall DA. Permeability characteristics of Calu-3 human bronchial epithelial cells: In vitro-in vivo correlation to predict lung absorption in rats. J Drug Target. 2002;10(1):31–40.CrossRefGoogle Scholar
  11. 11.
    Fogh J, Fogh JM, Orfeo T. 127 Cultured human tumor-cell lines producing tumors in nude mice. J Natl Cancer Inst. 1977;59(1):221–6.PubMedGoogle Scholar
  12. 12.
    Florea BI, Cassara ML, Junginger HE, Borchard G. Drug transport and metabolism characteristics of the human airway epithelial cell line Calu-3. J Control Release. 2003;87(1–3):131–8.PubMedCrossRefGoogle Scholar
  13. 13.
    Fiegel J, Ehrhardt C, Schaefer UF, Lehr C-M, Hanes J. Large porous particle impingement on lung epithelial cell monolayers—Toward improved particle characterization in the lung. Pharm Res. 2003;20(5):788–96.PubMedCrossRefGoogle Scholar
  14. 14.
    Stentebjerg-Andersen A, Notlevsen IV, Brodin B, Nielsen CU. Calu-3 cells grown under AIC and LCC conditions: Implications for dipeptide uptake and transepithelial transport of substances. Eur J Pharm Biopharm. 2011;78(1):19–26.PubMedCrossRefGoogle Scholar
  15. 15.
    Grainger C, Greenwell L, Lockley D, Martin G, Forbes B. Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier. Pharm Res. 2006;23(7):1482–90.PubMedCrossRefGoogle Scholar
  16. 16.
    Haghi M, Young PM, Traini D, Jaiswal R, Gong J, Bebawy M. Time- and passage-dependent characteristics of a Calu-3 respiratory epithelial cell model. Drug Dev Ind Pharm. 2010;36(10):1207–14.PubMedCrossRefGoogle Scholar
  17. 17.
    Finkbeiner WE, Carrier SD, Teresi CE. Reverse 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. 1993;9(5):547–56.PubMedGoogle Scholar
  18. 18.
    Ehrhardt C, Forbes B, Kim K-J. In vitro models of the tracheo-bronchial epithelium. In: Ehrhardt C, Kim K-J, editors. Drug absorption studies: in situ, in vitro and in silico models. New York: Springer US; 2008. p. 235–57.CrossRefGoogle Scholar
  19. 19.
    Ehrhardt C, Kneuer C, Fiegel J, Hanes J, Schaefer U, Kim K-J, et al. Influence 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. 2002;308(3):391–400.PubMedCrossRefGoogle Scholar
  20. 20.
    Pohl C, Hermanns MI, Uboldi C, Bock M, Fuchs S, Dei-Anang J, et al. Barrier functions and paracellular integrity in human cell culture models of the proximal respiratory unit. Eur J Pharm Biopharm. 2009;72(2):339–49.PubMedCrossRefGoogle Scholar
  21. 21.
    Halldorsson S, Asgrimsson V, Axelsson I, Gudmundsson GH, Steinarsdottir M, Baldursson O, et al. Differentiation potential of a basal epithelial cell line established from human bronchial explant. In Vitro Cell Dev Biol Anim. 2007;43(8–9):283–9.PubMedCrossRefGoogle Scholar
  22. 22.
    Franzdottir S, Axelsson I, Arason A, Baldursson O, Gudjonsson T, Magnusson M. Airway branching morphogenesis in three dimensional culture. Respir Res. 2010;11(1):162.PubMedCrossRefGoogle Scholar
  23. 23.
    Gudjonsson T, Villadsen R, Nielsen HL, Rønnov-Jessen L, Bissell MJ, Petersen OW. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties. Genes Dev. 2002;16(6):693–706.PubMedCrossRefGoogle Scholar
  24. 24.
    Gudjonsson T, Villadsen R, Rønnov-Jessen L, Petersen OW. Immortalization protocols used in cell culture models of human breast morphogenesis. Cell Mol Life Sci. 2004;61(19):2523–34.PubMedCrossRefGoogle Scholar
  25. 25.
    Zabner J, Karp P, Seiler M, Phillips SL, Mitchell CJ, Saavedra M, et al. Development of cystic fibrosis and noncystic fibrosis airway cell lines. Am J Physiol Lung Cell Mol Physiol. 2003;284(5):L844–54.PubMedGoogle Scholar
  26. 26.
    Forbes B, Shah A, Martin GP, Lansley AB. The human bronchial epithelial cell line 16HBE14o-as a model system of the airways for studying drug transport. Int J Pharm. 2003;257(1–2):161–7.PubMedCrossRefGoogle Scholar
  27. 27.
    Dorscheid D, Conforti A, Hamann K, Rabe K, White S. Characterization of cell surface lectin-binding patterns of human airway epithelium. Histochem J. 1999;31(3):145–51.PubMedCrossRefGoogle Scholar
  28. 28.
    Rojas A, Gonzalez I, Figueroa H. Cell line cross-contamination in biomedical research: a call to prevent unawareness. Acta Pharmacol Sin. 2008;29(7):877–80.PubMedCrossRefGoogle Scholar
  29. 29.
    Nardone R. Eradication of cross-contaminated cell lines: a call for action. Cell Biol Toxicol. 2007;23(6):367–72.PubMedCrossRefGoogle Scholar
  30. 30.
    Otton A. Cell culture forensics of Calu-3: a human lung epithelial cell line. Ethn Dis. 2009;19(2):S78–9.Google Scholar
  31. 31.
    Boers JE, Ambergen AW, Thunnissen FBJM. Number and proliferation of basal and parabasal cells in normal human airway epithelium. Am J Respir Crit Care Med. 1998;157(6):2000–6.PubMedGoogle Scholar
  32. 32.
    Rock JR, Onaitis MW, Rawlins EL, Lu Y, Clark CP, Xue Y, et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci. 2009;106(31):12771–5.PubMedCrossRefGoogle Scholar
  33. 33.
    Dvorak A, Tilley AE, Shaykhiev R, Wang R, Crystal RG. Do airway epithelium air–liquid cultures represent the in vivo airway epithelium transcriptome? Am J Respir Cell Mol Biol. 2011;44(4):465–73.PubMedCrossRefGoogle Scholar
  34. 34.
    Halldorsson S, Gudjonsson T, Gottfredsson M, Singh PK, Gudmundsson GH, Baldursson O. Azithromycin maintains airway epithelial integrity during pseudomonas aeruginosa infection. Am J Respir Cell Mol Biol. 2010;42(1):62–8.PubMedCrossRefGoogle Scholar
  35. 35.
    Mathias NR, Kim K-J, Robison TW, Lee VHL. Development and characterization of rabbit tracheal epithelial cell monolayer models for drug transport studies. Pharm Res. 1995;12(10):1499–505.PubMedCrossRefGoogle Scholar
  36. 36.
    Yamaya M, Finkbeiner WE, Chun SY, Widdicombe JH. Differentiated structure and function of cultures from human tracheal epithelium. Am J Physiol Lung Cell Mol Physiol. 1992;262(6):L713–24.Google Scholar
  37. 37.
    Clary-Meinesz C, Mouroux J, Huitorel P, Cosson J, Schoevaert D, Blaive B. Ciliary beat frequency in human bronchi and bronchioles. CHEST J. 1997;111(3):692–7.CrossRefGoogle Scholar
  38. 38.
    Clary-Meinesz C, Mouroux J, Cosson J, Huitorel P, Blaive B. Influence of external pH on ciliary beat frequency in human bronchi and bronchioles. Eur Respir J. 1998;11(2):330–3.PubMedCrossRefGoogle Scholar
  39. 39.
    Rutland J, Griffin WM, Cole PJ. Human ciliary beat frequency in epithelium from intrathoracic and extrathoracic airways. Am Rev Respir Dis. 1982;125(1):100–5.PubMedGoogle Scholar
  40. 40.
    Asgrimsson V, Gudjonsson T, Gudmundsson GH, Baldursson O. Novel effects of azithromycin on tight junction proteins in human airway epithelia. Antimicrob Agents Chemother. 2006;50(5):1805–12.PubMedCrossRefGoogle Scholar
  41. 41.
    Kim K-J. Bioelectrical characterization of cultured epithelial cell (mono)layers and excised tissues. In: Lehr CM, editor. Cell culture models of biological barriers. CRC Press; 2002. p. 41–51.Google Scholar
  42. 42.
    Ehrhardt C, Fiegel J, Fuchs S, Abu-Dahab R, Schaefer UF, Hanes J, et al. Drug absorption by the respiratory mucosa: cell culture models and particulate drug carriers. J Aerosol Med. 2002;15(2):131–9.PubMedCrossRefGoogle Scholar
  43. 43.
    Lin H, Li H, Cho H-J, Bian S, Roh H-J, Lee M-K, et al. Air-liquid interface (ALI) culture of human bronchial epithelial cell monolayers as an in vitro model for airway drug transport studies. J Pharm Sci. 2007;96(2):341–50.PubMedCrossRefGoogle Scholar
  44. 44.
    Foster KA, Avery ML, Yazdanian M, Audus KL. Characterization of the Calu-3 cell line as a tool to screen pulmonary drug delivery. Int J Pharm. 2000;208(1–2):1–11.PubMedCrossRefGoogle Scholar
  45. 45.
    Tronde A, Norden B, Jeppsson AB, Brunmark P, Nilsson E, Lennernas H, et al. Drug absorption from the isolated perfused rat lung-correlations with drug physicochemical properties and epithelial permeability. J Drug Target. 2003;11(1):61–74.PubMedCrossRefGoogle Scholar
  46. 46.
    Conradi RA, Burton PS, Borchardt RT. Physico-chemical and biological factors that influence a drug’s cellular permeability by passive diffusion. In: Pliška V, Testa B, van de Waterbeemd H, editors. Lipophilicity in drug action and toxicology. Weinheim: Wiley-VCH Verlag GmbH; 2008. p. 233–52.Google Scholar
  47. 47.
    Clark DE. Rapid calculation of polar molecular surface area and its application to the prediction of transport phenomena. 1. Prediction of intestinal absorption. J Pharm Sci. 1999;88(8):807–14.PubMedCrossRefGoogle Scholar
  48. 48.
    Balimane PV, Han YH, Chong SH. Current industrial practices of assessing permeability and P-glycoprotein interaction. AAPS J. 2006;8(1):E1–E13.PubMedCrossRefGoogle Scholar
  49. 49.
    Hamilton KO, Topp E, Makagiansar I, Siahaan T, Yazdanian M, Audus KL. Multidrug resistance-associated protein-1 functional activity in Calu-3 cells. J Pharmacol Exp Ther. 2001;298(3):1199–205.PubMedGoogle Scholar
  50. 50.
    Ehrhardt C, Kneuer C, Laue M, Schaefer UF, Kim K-J, Lehr C-M. 16HBE14o-human bronchial epithelial cell layers express P-glycoprotein, lung resistance-related protein, and caveolin-1. Pharm Res. 2003;20(4):545–51.PubMedCrossRefGoogle Scholar
  51. 51.
    Lechapt-Zalcman E, Hurbain I, Lacave R, Commo F, Urban T, Antoine M, et al. MDR1-Pgp 170 expression in human bronchus. Eur Respir J. 1997;10(8):1837–43.PubMedCrossRefGoogle Scholar
  52. 52.
    Madlova M, Bosquillon C, Asker D, Dolezal P, Forbes B. In-vitro respiratory drug absorption models possess nominal functional P-glycoprotein activity. J Pharm Pharmacol. 2009;61(3):293–301.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Berglind Eva Benediktsdóttir
    • 1
  • Ari Jón Arason
    • 2
    • 3
  • Skarphédinn Halldórsson
    • 2
    • 4
  • Thórarinn Gudjónsson
    • 2
    • 3
  • Már Másson
    • 1
  • Ólafur Baldursson
    • 5
  1. 1.Faculty of Pharmaceutical Sciences, School of Health SciencesUniversity of IcelandReykjavikIceland
  2. 2.Stem Cell Research Unit, Biomedical Center School of Health SciencesUniversity of IcelandReykjavikIceland
  3. 3.Department of Laboratory HematologyLandspitali - The National University Hospital of IcelandReykjavikIceland
  4. 4.Department of Biology, School of Engineering and Natural SciencesCenter for Systems Biology, University of IcelandReykjavikIceland
  5. 5.Department of Pulmonary MedicineLandspitali - The National University Hospital of IcelandReykjavikIceland

Personalised recommendations