title: The characterization of the human cell line Calu-3 under different culture conditions and its use as an optimized in vitro model to investigate bronchial epithelial function.
abstract: In this study we have investigated the effects of different cell culture conditions on the Calu-3 epithelial cell model. Calu-3 cells were cultured in media A-MEM at the air-liquid (A-L) or liquid-liquid (L-L) interface for one or three wks (weeks). Different cryomethods were tested and the cell line was characterized using histochemistry, immunofluorescence, transmission and scanning electron microscopy, transepithelial resistance (TEER) measurements, permeability studies, and gene profiling of 84 drug transporters. Cell culture was successful in A-MEM with only 2.5% FBS. Cell proliferation and viability depended on the cryopreservation method. All Calu-3 models expressed CK7, occludin, and E-cadherin. The A-L interface resulted in a more biomimetic native bronchial epithelium displaying pseudostratified columnar epithelium with more microvilli and secretory vesicles than at the L-L interface, where the epithelium was cuboidal, but exhibited higher TEER values and lower dextran permeabilities. Longer time in culture significantly decreased dextran permeability and increased the expression of specific drug transporters. Drug transporter expression was also notably influenced by the culture interface, where the A-L interface yielded a higher expression of drug transporter genes than the L-L interface. Since cell culture interface and time in culture affect Calu-3 cell differentiation, barrier integrity, permeability properties, and drug transporter expression, culture conditions need to be considered and standardized when using the Calu-3 cell line as an in vitro model for aerosol drug delivery and screening of bronchial drug candidates.
authors.affiliation: Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. Electronic address: mateja.erdani@mf.uni-lj.si.
authors.forename: Mateja Erdani
authors.initials: ME
authors.lastname: Kreft
authors.affiliation: Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. Electronic address: urska.dragin@mf.uni-lj.si.
authors.forename: Urška Dragin
authors.initials: UD
authors.lastname: Jerman
authors.affiliation: Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. Electronic address: eva.lasic@mf.uni-lj.si.
authors.forename: Eva
authors.initials: E
authors.lastname: Lasič
authors.affiliation: Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. Electronic address: neli.hevir-kene@mf.uni-lj.si.
authors.forename: Neli
authors.initials: N
authors.lastname: Hevir-Kene
authors.affiliation: Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. Electronic address: tea.lanisnik-rizner@mf.uni-lj.si.
authors.forename: Tea Lanišnik
authors.initials: TL
authors.lastname: Rižner
authors.affiliation: Lek Pharmaceuticals, d.d., Sandoz Development Center Slovenia, Verovškova 57, 1526 Ljubljana, Slovenia. Electronic address: luka.peternel@sandoz.com.
authors.forename: Luka
authors.initials: L
authors.lastname: Peternel
authors.affiliation: Lek Pharmaceuticals, d.d., Sandoz Development Center Slovenia, Verovškova 57, 1526 Ljubljana, Slovenia. Electronic address: katja.kristan@sandoz.com.
authors.forename: Katja
authors.initials: K
authors.lastname: Kristan
mesh_terms: D000388:Air; D001980:Bronchi / Q000166:cytology*; D018929:Cell Culture Techniques; D002460:Cell Line / Q000378:metabolism* / Q000648:ultrastructure; D015925:Cryopreservation; D003911:Dextrans / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D010539:Permeability; D059467:Transcriptome
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D016650:Fluorescein-5-isothiocyanate
keywords: Bronchial epithelium; Calu-3 cell line; Drug transporters; Microscopy; Permeability; TEER