Time- and passage-dependent characteristics of a Calu-3 respiratory epithelial cell model

Drug Dev Ind Pharm. 2010 Oct;36(10):1207-14. doi: 10.3109/03639041003695113.


Background: Although standard protocols for the study of drug delivery in the upper airways using the sub-bronchial epithelial cell line Calu-3 model, particularly that of the air-liquid interface configuration, are readily available, the model remains un-validated with respect to culture conditions, barrier integrity, mucous secretion, and transporter function. With respect to the latter, the significance of functional P-glycoprotein (P-gp) activity in Calu-3 cells has recently been questioned, despite previous reports demonstrating a significant contribution by the same transporter in limiting drug uptake across the pulmonary epithelium. Therefore, the aim of this study was the standardization of this model as a tool for drug discovery.

Methods: Calu-3 cells were grown using air-interfaced condition (AIC) on polyester cell culture supports. Monolayers were evaluated for transepithelial electrical resistance (TEER), permeability to the paracellular marker fluorescein sodium (flu-Na), surface P-gp expression, and functionality. Mucous secretion was also identified by alcian blue staining.

Results: TEER and permeability values obtained for Calu-3 monolayers were shown to plateau between day 5 and day 21 in culture with values reaching 474 +/- 44 omega cm(2) and 2.33 +/- 0.36 x 10(-7) cm/s, respectively, irrespective of the passage number examined. 32.7 +/- 1.49% of Calu-3 cells cultured under these conditions detected positive for cell surface P-gp expression from day 7 onwards. Functional cell surface expression was established by rhodamine 123 drug extrusion assays.

Conclusion: This study establishes a clear dependence on culture time and passage number for optimal barrier integrity, mucous secretion, and cell-surface P-gp expression and function in Calu-3 cells. Furthermore it provides initial guidelines for the optimization of this model for high throughput screening applications.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / analysis
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • Biological Transport
  • Bronchi / cytology
  • Bronchi / metabolism
  • Cell Line*
  • Cell Membrane / metabolism
  • Cell Membrane Permeability
  • Drug Discovery / methods*
  • Electric Impedance
  • Epithelial Cells* / metabolism
  • Humans
  • Models, Biological
  • Pharmaceutical Preparations / metabolism
  • Respiratory Mucosa* / cytology
  • Respiratory Mucosa* / metabolism
  • Rhodamine 123 / metabolism
  • Time Factors


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Pharmaceutical Preparations
  • Rhodamine 123