title: Time- and passage-dependent characteristics of a Calu-3 respiratory epithelial cell model.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Faculty of Pharmacy, University of Sydney, NSW, Australia.
authors.forename: Mehra
authors.initials: M
authors.lastname: Haghi
authors.forename: Paul M
authors.initials: PM
authors.lastname: Young
authors.forename: Daniela
authors.initials: D
authors.lastname: Traini
authors.forename: Ritu
authors.initials: R
authors.lastname: Jaiswal
authors.forename: Joyce
authors.initials: J
authors.lastname: Gong
authors.forename: Mary
authors.initials: M
authors.lastname: Bebawy
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000378:metabolism; D001692:Biological Transport; D001980:Bronchi / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D055808:Drug Discovery / Q000379:methods*; D017097:Electric Impedance; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D004364:Pharmaceutical Preparations / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D020112:Rhodamine 123 / Q000378:metabolism; D013997:Time Factors
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004364:Pharmaceutical Preparations; D020112:Rhodamine 123