Drug Delivery Characteristics of the Progenitor Bronchial Epithelial Cell Line VA10

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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.

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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

Papp :

apparent permeability (cm/s)


phosphate buffered saline




retinoblastoma tumor suppressor protein


rhodamine 123


scanning electron microscopy


transepithelial electrical resistance (Ω*cm2)


tight junction


  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.

  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.

  3. 3.

    Note for guidance on the investigation of bioavailability and bioequivalence. December 2000, EMEA.

  4. 4.

    Forbes B, Ehrhardt C. Human respiratory epithelial cell culture for drug delivery applications. Eur J Pharm Biopharm. 2005;60(2):193–205.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  29. 29.

    Nardone R. Eradication of cross-contaminated cell lines: a call for action. Cell Biol Toxicol. 2007;23(6):367–72.

  30. 30.

    Otton A. Cell culture forensics of Calu-3: a human lung epithelial cell line. Ethn Dis. 2009;19(2):S78–9.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

  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.

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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.

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Correspondence to Ólafur Baldursson.

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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)

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)

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Benediktsdóttir, B.E., Arason, A.J., Halldórsson, S. et al. Drug Delivery Characteristics of the Progenitor Bronchial Epithelial Cell Line VA10. Pharm Res 30, 781–791 (2013) doi:10.1007/s11095-012-0919-x

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