Culture of Calu-3 cells at the air interface provides a representative model of the airway epithelial barrier

被引:301
作者
Grainger, Christopher I.
Greenwell, Leona L.
Lockley, David J.
Martin, Gary P.
Forbes, Ben
机构
[1] Kings Coll, Pharmaceut Sci Res Div, London SE1 9NH, England
[2] Unilever Res Labs Colworth, Safety & Environm Assurance Ctr, Sharnbrook MK44 1LQ, Beds, England
关键词
drug delivery; permeability; respiratory cell culture; toxicology;
D O I
10.1007/s11095-006-0255-0
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Purpose. The aim of this study was to compare the effect of liquid-covered culture (LCC) and air-interfaced culture (AIC) on Calu-3 cell layer morphology and permeability, thus assessing the fitness of these culture systems as models of airway epithelium barrier function. Methods. Cell layers were grown on 0.33 cm(2) Transwell polyester cell culture supports. Cell layers grown using LCC and AIC were evaluated by using light and electron microscopy, transepithelial electrical resistance (TER), and permeability to the transepithelial flux of fluorescein sodium (flu-Na), and by varying molecular weight dextrans labeled with fluorescein isothiocyanate (FITC-dex). The tight junction protein, zona occludens protein-1 (ZO-1), was visualized by confocal microscopy and apical glycoprotein secretions were identified by using alcian blue. Results. Cells grown via AIC produced a more columnar epithelium with a more rugged apical topography and greater glycoprotein secretion compared to cells grown via LCC. Apical protrusions appearing to be cilia-like structures were observed on occasional cells using AIC, but typical airway ciliated cell phenotypes were not produced under either condition. Secretory granules were observed in cells cultured under both conditions. Cells cultured using LCC exhibited higher levels of ZO-1 protein than the AIC counterpart. The maximal TER of cells using LCC, 1,086 +/- 113 Omega cm(2) at 11-16 days, was significantly greater than the TER of cells cultured using AIC, 306 +/- 53 Omega cm(2) at 11-13 days. Apparent permeability (P-app) values for the transport of flu-Na using LCC and AIC were 1.48 +/- 0.19x10(-7) and 3.36 +/- 0.47x10(-7) cm s(-1), respectively. Transport rates of flu-Na and FITC-dex were inversely proportional to molecular weight, and were significantly lower (p < 0.05) in cell layers grown using LCC than AIC. Renkin analysis fitted the data to single pore populations of radii 7.7 and 11.0 nm for LCC and AIC, respectively. Conclusion. Distinct differences in morphology and permeability result when Calu-3 cells are grown using AIC or LCC. Cells cultured using AIC generate a model more morphologically representative of the airway epithelium than cells cultured using LCC.
引用
收藏
页码:1482 / 1490
页数:9
相关论文
共 37 条
[1]   Regulation of apical surface fluid and protein secretion in human airway epithelial cell line Calu-3 [J].
Babu, PBR ;
Chidekel, A ;
Utidjian, L ;
Shaffer, TH .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2004, 319 (04) :1132-1137
[2]   Respiratory carcinoma cell lines -: MUC genes and glycoconjugates [J].
Berger, JT ;
Voynow, JA ;
Peters, KW ;
Rose, MC .
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, 1999, 20 (03) :500-510
[3]  
Borchard G, 2002, STP PHARMA SCI, V12, P205
[4]   Development of a size-dependent aerosol deposition model utilising human airway epithelial cells for evaluating aerosol drug delivery [J].
Cooney, D ;
Kazantseva, M ;
Hickey, AJ .
ATLA-ALTERNATIVES TO LABORATORY ANIMALS, 2004, 32 (06) :581-590
[5]   SERIAL CULTURING OF HUMAN BRONCHIAL EPITHELIAL-CELLS DERIVED FROM BIOPSIES [J].
DEJONG, PM ;
VANSTERKENBURG, MAJA ;
KEMPENAAR, JA ;
DIJKMAN, JH ;
PONEC, M .
IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL, 1993, 29A (05) :379-387
[6]   CILIOGENESIS IN HUMAN BRONCHIAL EPITHELIAL-CELLS CULTURED AT THE AIR-LIQUID INTERFACE [J].
DEJONG, PM ;
VANSTERKENBURG, MAJA ;
HESSELING, SC ;
KEMPENAAR, JA ;
MULDER, AA ;
MOMMAAS, AM ;
DIJKMAN, JH ;
PONEC, M .
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, 1994, 10 (03) :271-277
[7]   Drug absorption by the respiratory mucosa: Cell culture models and particulate drug carriers [J].
Ehrhardt, C ;
Fiegel, J ;
Fuchs, S ;
Abu-Dahab, R ;
Schaefer, UF ;
Hanes, J ;
Lehr, CM .
JOURNAL OF AEROSOL MEDICINE-DEPOSITION CLEARANCE AND EFFECTS IN THE LUNG, 2002, 15 (02) :131-139
[8]   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 [J].
Ehrhardt, C ;
Kneuer, C ;
Fiegel, J ;
Hanes, J ;
Schaefer, UF ;
Kim, KJ ;
Lehr, CM .
CELL AND TISSUE RESEARCH, 2002, 308 (03) :391-400
[9]   Large porous particle impingement on lung epithelial cell monolayers - Toward improved particle characterization in the lung [J].
Fiegel, J ;
Ehrhardt, C ;
Schaefer, UF ;
Lehr, CM ;
Hanes, J .
PHARMACEUTICAL RESEARCH, 2003, 20 (05) :788-796
[10]   REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION (RT-PCR) PHENOTYPIC ANALYSIS OF CELL-CULTURES OF HUMAN TRACHEAL EPITHELIUM, TRACHEOBRONCHIAL GLANDS, AND LUNG CARCINOMAS [J].
FINKBEINER, WE ;
CARRIER, SD ;
TERESI, CE .
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, 1993, 9 (05) :547-556