Article
Reference
Approaches to study differentiation and repair of human airway epithelial cells
CRESPIN, Sophie, et al.
Abstract
One of the main functions of the airway mucosa is to maintain a mechanical barrier at the air-surface interface and to protect the respiratory tract from external injuries. Differentiation of human airway epithelial cells (hAECs) to polarized airway mucosa can be reproduced in vitro by culturing the cells on microporous membrane at the air-liquid interface. Here, we describe approaches to study differentiation as well as repair of the hAECs by using a commercially available airway cell culture model called MucilAir™.
CRESPIN, Sophie, et al . Approaches to study differentiation and repair of human airway epithelial cells. Methods in Molecular Biology , 2011, vol. 742, p. 173-85
DOI : 10.1007/978-1-61779-120-8_10 PMID : 21547732
Available at:
http://archive-ouverte.unige.ch/unige:21534
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Approaches to Study Differentiation and Repair of Human Airway Epithelial Cells
Sophie Crespin, Marc Bacchetta, Song Huang, Tecla Dudez, Ludovic Wiszniewski, and Marc Chanson
Abstract
One of the main functions of the airway mucosa is to maintain a mechanical barrier at the air–surface interface and to protect the respiratory tract from external injuries. Differentiation of human airway epithelial cells (hAECs) to polarized airway mucosa can be reproduced in vitro by culturing the cells on microporous membrane at the air–liquid interface. Here, we describe approaches to study differentia- tion as well as repair of the hAECs by using a commercially available airway cell culture model called MucilAirTM.
Key words: Human airway epithelial cell culture, MucilAirTM, differentiation, wounding techniques, repair, cystic fibrosis.
1. Introduction
The respiratory epithelium plays a fundamental role as a line of defense against pathogens. Among other lung diseases, cystic fibrosis (CF) has been associated with a damaged airway mucosa consequently to chronic lung inflammation and with an abnor- mal repair (1,2). Various models were developed by researchers aiming to study the behavior of the respiratory epithelium and its repair.
Animal models appear to be useful for CF research (see related chapter in this book). Focusing on lung functions and
M.D. Amaral, K. Kunzelmann (eds.), Cystic Fibrosis, Methods in Molecular Biology 742, DOI 10.1007/978-1-61779-120-8_10, © Springer Science+Business Media, LLC 2011
173
repair, several strategies have already been described in the litera- ture using these models (for a review, see (3)). In vivo approaches consist in the inhalation of gases or the intratracheal instillation of drugs. Ex vivo airway epithelial xenograft models have also been developed. In this case, immunocompromised mice were used to receive grafts subcutaneously. Using this approach, Hajj et al. (4) studied the regeneration of human CF airway epithelium and reported a delayed and abnormal re-differentiation. Nonethe- less, these animal-based models exhibit some troubles. First of all, the study of mouse lung regeneration in the context of CF brings into question that mice do not exhibit a CF phenotype in the control conditions (without injury). So, even if we may draw conclusions about repair of the normal epithelium, the extrapola- tion to the CF epithelium based on CFTR knockout mice might be biased. Second, in the xenograft model, grafts are transplanted heterotypically, leading to a complete change of the normal tissue environment.
Closer to the native human healthy or pathologic airway epithelium, several groups developed in vitro cultures using hAECs obtained from surgical resection or brushing. Epithelial cells from the airway epithelium are isolated by explant culture or enzymatic dissociation (for review, see (5)). A key concept is the switch from submerged cultures to an air–liquid interface allow- ing the development of differentiated airway epithelia (6,7). To this end, inserts with porous membranes in tissue culture wells were used (8). On the same basis of air–liquid interface culture, at least two kinds of models were developed. One is based on the plating of isolated hAECs on collagen IV-coated inserts (9).
The culture is usually viable for about 1 month after the begin- ning of the culture (4). The other model consists in a first step of hAEC proliferation followed by a second step of differentia- tion (10,11). These models present the advantage to amplifying a rare material, especially for CF tissues. The MucilAirTMsystem presents the characteristics of the human airway epithelium with the presence of basal cells, goblet cells, and ciliated cells organized in a pseudostratified epithelium. An advantage of this system is the possibility to keep the airway epithelium differentiated for up to 6–9 months enabling to monitor epithelial repair, a process that lasts days to weeks depending on the extent of the lesion.
The aim of this chapter is to first describe the characteris- tics of a well-differentiated airway epithelium maintained in pri- mary culture. Thus, different protocols concerning the states of differentiation are given: epithelial morphology and expression of specific markers (Ki67, β-tubulin, and connexins). Second, a strategy mimicking an injury in vitro is described. Finally, the last part of this chapter concerns methods to monitor epithelial repair, including measurement of the kinetics of wound closure, cell pro- liferation, and migration.
2. Materials
2.1. hAEC Cultures and Media 2.1.1. hAEC Cultures
Differentiation and repair of hAECs were evaluated on the commercially available in vitro airway epithelium cell model MucilAirTM(Epithelix, Plan-les-Ouates, Switzerland). The respi- ratory epithelium is reconstituted from primary hAECs freshly isolated from nasal polyps or from tracheal/bronchial biop- sies, according to methods that are extensively described in this book. Briefly, hAECs are seeded onto 33-mm2 Costar Transwell inserts (Costar, ref. number 3470) with transparent microporous membranes (0.4-μm pore). Two days after seeding, hAECs are switched to an air–liquid interface for at least 45 days. This leads to the differentiation of hAECs to a mucociliated pseu- dostratified airway epithelium that is maintained in a homeo- static state for months (11). The transparent microporous mem- brane allows direct observation of the cells under a conventional inverted microscope. Moreover, the polyethylene membranes are more resistant than polycarbonate membranes and can withstand mechanical forces.
2.1.2. hAEC Media Typically, the medium used to maintain hAEC cultures at the air–
liquid interface is a 3:1 mix of DMEM with GlutaMax (Invitro- gen, ref. number 31966-021) and F12 (Invitrogen, ref. number 21765-029), supplemented with penicillin–streptomycin (15,000 units and 30 μg/ml, respectively; GIBCO, ref. number 15140- 148) and amphotericin B (Amimed, ref. number 4-05F00-H).
Other commercially available “ready-to-use” media can also be purchased from Epithelix (Plan-les-Ouates, Switzerland), Clonet- ics Corp. (San Diego, CA), or PromoCell GmbH (Heidelberg, Germany).
2.2. Other Reagents and Solutions
1. Buffered NaCl solution: Isotonic saline solution contain- ing 0.9% NaCl supplemented with 10 mM HEPES and 1.25 mM CaCl2.
2. Dulbecco’s phosphate buffered saline (DPBS) with calcium and magnesium (GIBCO, ref. number 14040).
3. PFA solution: Make 4% paraformaldehyde solution freshly from powder (Sigma, ref. number P6148) in DPBS. Adjust pH at 7.2.
4. Triton X-100 solution: Make 0.3% Triton X-100 (Sigma, ref.
number T8787) solution freshly in DPBS.
5. 0.5 M NH4Cl solution in DPBS.
6. BSA solution: Make 2% bovine serum albumin solution in DPBS freshly.
7. Lucifer yellow 4% solution: Prepare the Lucifer yellow solu- tion (Sigma, ref. number L0259) in 150 mM LiCl buffered to pH 7.2 with 10 mM HEPES. Keep the solution at 4◦C in dark and spin down before use to remove any possible aggregates.
2.3. Materials for Wounding
1. A conventional airbrush (Triplex, Gabbert).
2. A source of compressed air.
3. Pressure regulator (0.1 and 0.5 bar).
4. Flexible pipe allowing the connection between the pressure regulator and the airbrush.
2.4. Other Equipments
1. Inverted microscope equipped with fluorescein filters and UV illumination, standing on an anti-vibration table. A micromanipulator is also required to position thin-tip micro- electrodes.
2. Borosilicate glass capillaries with an internal filament (for example, Kwik-FilTM 1B120F-4 from World Precision Instruments, Inc.) and a microelectrode puller (typically, we use a vertical Narishige PC-10, Tokyo, Japan).
3. Module allowing the measurement of the transepithelial electrical resistance (for example, EVOMX from World Pre- cision Instruments, Inc.).
3. Methods
3.1. Approaches to Study Differentiation of hAEC
hAECs grown on filters lose their differentiated features. Differ- entiation can be triggered by exposing the apical surface of hAECs to air (7). Below, we describe approaches to monitor the differ- entiation of hAECs to a full polarized airway epithelium.
3.1.1. Morphology of the Differentiating Airway Epithelium
The differentiation of hAEC can be evaluated by paraffin- embedded sections of the cultures, although the procedure on Transwell inserts is delicate:
1. Fix the hAEC culture with the 4% PFA solution for at least 1 h.
2. Carefully remove the microporous membrane from the insert using a scalpel blade. Avoid tearing/bending of the membrane.
3. Follow a usual protocol of dehydration (succession of baths of ethanol from 70 to 100%, xylol) and embedding in paraffin. Embed the membrane vertically in paraffin. We
use an automatic embedding machine for treating histo- logical examination (Tissue Processor; Leica Microsystems, TP1020).
4. Make 5-μm-thick sections with a microtome and mount them on charged slides (as SuperfrostR slides). See Note 1 for tips and tricks.
5. Rehydrate the samples (succession of baths of xylol, ethanol from 100 to 70%, distilled water) before staining (hemalun–
eosin, periodic acid Schiff coloration, Alcian blue, etc.).
The morphology of hAECs grown at the air–liquid interface for increasing the amount of time is illustrated in Fig.10.1(top panels). With time, hAECs become taller and cilia appear at the apical surface. The presence of basal and mucous cells is also observed. The typical morphology of the respiratory epithelium is maintained on the long term.
Fig. 10.1. Long-term differentiation of MucilAirTMhAEC cultures. Paraformaldehyde-fixed, paraffin-embedded (PFPE) sections of MucilAirTMcultures. hAECs organized as a monolayer during the first week of culture. At this time, a high proliferation rate (Ki-67 staining) is associated with an undifferentiated state (few cells stained forβ-tubulin) and a high level of Cx26 expression. After 7 weeks, the hAEC culture exhibits a pseudostratified ciliated epithelium with goblet cells (arrowhead). This differentiation is correlated with a low proliferation rate, a huge amount ofβ-tubulin staining, and the absence of Cx26 expression. The latter profile is maintained over months (as shown as an example at 14 weeks). Bar, 25μm.
3.1.2.
Immunohistochemical Detection of Markers of hAEC Differentiation
Typically, differentiation is associated with inhibition of hAEC proliferation and occurrence of neociliogenesis. Several markers of hAEC differentiation can be detected by immunohistochem- istry. To this end, immunofluorescence is performed for Ki-67 (a nuclear marker of cell proliferation) andβ-tubulin (a component of ciliae). We also use connexin26 (Cx26, a gap junction protein) and Cx43 as key markers of differentiated hAECs (12):
1. Fix the hAEC culture with the PFA solution for 15 min.
Wash with DPBS.
2. Permeabilize hAECs with the Triton X-100 solution for 15 min. Wash with DPBS.
3. To avoid non-specific staining, incubate the culture first in the NH4Cl solution for 15 min followed by the BSA solu- tion for 30 min.
4. Primary and secondary antibodies are incubated for at least 90 min at room temperature.
5. After careful washing, cut the microporous membrane out of the insert using a scalpel blade and mount between slide and coverslip with a photobleaching-preventing mounting medium (VectashieldR, Clinisciences, ref. number H-1000;
Aquamount, Thermo Scientific, ref. number 14-390-5). See Note 2for tips and tricks.
Examples of immunostaining for Ki-67,β-tubulin, and Cx26 at different times of culture are shown in Fig.10.1. At early time after the air–liquid interface has been established, hAEC cultures exhibit mostly a monolayer appearance, high proliferation rate, and absence of ciliae. With longer time at the air–liquid inter- face, proliferation ceased while differentiation is evidenced by the absence of Ki-67 detection and numerous cilia covering 90% of the epithelial surface after 45 days of culture. Of interest is the marked decrease in the expression of the gap junction proteins Cx26 and Cx43 with differentiation of hAECs (12), as illustrated in Fig.10.1for Cx26.
3.1.3. Monitoring Gap Junctional Intercellular Communication
The loss of Cx43 and Cx26 with time at the air–liquid interface allows evaluating hAEC differentiation by monitoring gap junc- tional intercellular communication. In human, 20 different genes coding for connexin have been found. These connexins are asso- ciated with specific pattern of tissue expression, and depending on connexin composition, gap junction channels exhibit differ- ent permeability to molecules and dyes. We describe below an approach to microinject the fluorescent dye Lucifer yellow in hAEC cultures:
1. Cut the top of the Transwell insert half a centimeter above the cell culture. See Note 3 for tips and tricks.
2. Place the insert on a drop of culture medium onto a glass slide and move the preparation to the stage of the inverted microscope equipped for fluorescein detection.
3. Pull a microelectrode (typically 20–50 M). Bend the pipette over a thin flame (away for the tip) to an angle of about 10–20◦. This will help to position the electrode onto the cell surface within the narrow space provided by the insert.
4. Fill the pipette tip with the Lucifer yellow solution. See Note 4for tips and tricks.
5. Connect the microelectrode to the micromanipulator and bring the tip in close contact to the cell surface. The flu- orescence of Lucifer yellow helps to locate the microelec- trode. The cell impalement should be as gentle as possible to maintain cell viability; usually one brief finger tap on the anti-vibration table is sufficient.
6. Allow the dye to diffuse out of the electrode into the cells for 3 min and then rapidly remove the pipette. Dye coupling can be evaluated immediately by counting the number of flu- orescent cells. Use a new electrode for each microinjection.
An extensive cell-to-cell diffusion of the tracer will be indica- tive for a not yet differentiated airway epithelium. Ciliated cells within a well-polarized and differentiated airway epithelium do not communicate in terms of Lucifer yellow diffusion. This does not mean that ciliated cells are devoid of gap junctions; in fact, they express Cx30, another gap junction protein which is not permeable to Lucifer yellow (12).
3.1.4. Cytokine and Mucin Production
The production of IL-8 and mucin is changing with hAEC dif- ferentiation. Il-8 was measured using an ELISA kit (CLB, Ams- terdam, The Netherlands or BD OptEIATM, BD Biosciences, UK) in basal medium that was collected every 2 days. Typically, a well-polarized and differentiated airway epithelium produces 5–15 ng/ml of IL-8 per day.
Quantification of mucin production can be evaluated with the enzyme-linked lectin assay (ELLA). At time 0, the apical surface is rinsed with the buffered NaCl solution and cultures are returned to the incubator for various amount of time. At appropriate time points, the accumulated mucus at the apical surface is recovered with 200 μl of buffered NaCl. Glycoproteins in the mucus are captured by Helix pomatia lectin (HPA–lectin) and then revealed by HPA–horseradish peroxidase lectin conjugate (HPA–HRP).
The amount of mucus secreted on the epithelial surface is calcu- lated by dividing the obtained values with the number of days of accumulation (ng/ml/day). Mucins are not present in the baso- lateral compartment.
3.2. Approaches to Study Repair of hAECs
Following injury, hAECs migrate and proliferate to cover the wound. Covered areas exhibit hAECs that stop proliferating but re-differentiate into a pseudostratified airway epithelium (13). All these processes take place simultaneously with progression of the wound closure. Below, we describe approaches to wound the air- way epithelium and to monitor its repair.
3.2.1. Wounding Several approaches may be considered for wounding a
MucilAirTM culture (see Notes 5–7 for alternative wounding techniques). In the specific context of in vitro hAEC cultures on Transwell inserts, these approaches are limited by the fragility of the microporous membrane and the lack of accessibility to the cell surface.
We report below the method we are using to make repro- ducible, regular, and circular wounds. Targeted cells are locally removed from the insert without damaging adjacent cells on the membrane by using an airbrush linked to a pressure regulator.
As shown in Fig. 10.2, the diameter of the airbrush fits in the
Fig. 10.2. Procedure for wounding well-differentiated airway epithelia.aClassic air- brush use for making a wound in hAEC cultures (top). The airbrush and the Transwell insert exhibit nearly similar diameters (middle), allowing standardization of the wound by keeping a distance of 4 mm between the airbrush nozzle and the microporous mem- brane (bottom).bTypical view of a wound (bottom) made with the airbrush under a 0.5 bar pressure maintained for 1 s. A non-wounded hAEC culture of at least 6 weeks old is shown for comparison (top).
Transwell inserts (Fig. 10.2a). As these inserts are particularly calibrated, the same distance between the airbrush nozzle and the epithelial surface is kept. Note that these parameters depend mainly on the companies where airbrush and inserts are from:
1. Rinse the apical surface of hAEC cultures with the buffered NaCl solution.
2. Every pieces of the airbrush should be carefully cleaned. Also manage a clean area under sterile conditions (typically a cul- ture hood) for wounding.
3. To avoid drying of the apical surface while wounding, the reservoir of the airbrush is filled with the buffered NaCl solu- tion.
4. Adjust the air pressure between 0.1 and 0.5 bar.
5. Introduce the airbrush head into the insert until the end (about 4 mm).
6. Apply a brief pulse of air for 1–2 s. This parameter is highly dependent on the equipment and should be adjusted by each user. Depending on the pressure and the number of pushes on the airbrush, the size of the wound varies between 2 and 15 mm2.
7. Remove debris and detached cells by carefully washing the surface with the buffered NaCl solution and return the cul- tures to the incubator to allow cell repair.
Images of the circular wound that is obtained following this procedure are shown in Fig.10.2b.
3.2.2. Monitoring Wound Closure: Kinetics
One of the most convenient, reliable, and non-destructive meth- ods to monitor wound closure is the measurement of transep- ithelial electrical resistance (TEER). The TEER of wounded cul- tures should be compared with that of empty Transwell inserts with culture medium in both basal and apical compartments.
With repair and re-establishment of junctional complexes, TEER sharply increases to about 400–500 cm2, which is typical for in vitro human airway epithelia (see Discussion in reference 11).
The kinetics of recovery obviously depends on the wound size.
Another approach to monitor the kinetics of wound closure is by image analysis. To this end, we use an automated inverted microscope (DMIRE2; LEICA) equipped with a DMSTC XY stage and a digital camera (Ds-5Mc; Nikon) connected to a per- sonal computer. At regular intervals, the surface area of each insert is scanned. Typically, 35 images using a 5×objective are needed to complete the scanning of one insert. Reconstitution of the culture surface is performed by the analysis of pictures with the Image Pro Plus 6.0 software (Media Cybernetics). Alternatively, the ImageJ software (National Institutes of Health, Bethesda) can also be used (http://rsb.info.nih.gov/ij/). Typical images of progression of hAEC repair are shown in Fig. 10.3a, b also
Fig. 10.3. Monitoring wound closure.aThe wound closure is monitored by the acqui- sition of images taken at different times using an automated inverted microscope equipped with an automatized XY stage and a digital camera. The area covered within a determined period of time allows estimating the rate of epithelial repair.bView of the morphology of the repairing airway epithelium, as investigated using PFPE sections. Bar, 50μm.
shows a paraffin section of an airway culture 96 h after wounding.
Note the change in the height of the epithelium characterized by non-ciliated migrating/proliferating hAECs. To quantify wound closure, the wound area X is measured at different times (T1, T2, etc.). The wound area X is considered as a circle, allowing the determination of its radius R:R=√
(X/π). The distance covered by the migrating/proliferating hAECs between time intervals is then given by the difference between R1and R2, with R1and R2 being the radii determined at times T1and T2, respectively.
3.2.3. Monitoring Wound Closure: Cell Behavior
It is also possible to monitor wound healing by live imaging. The approach is limited by many factors, including time (it takes days for the epithelium to repair), cell focus (change from a tall dif- ferentiated epithelium to a migrating cell monolayer), mainte- nance of pH of the culture medium, and humidity of the cell environment. However, live imaging can be used within shorter time frames (several hours) at higher resolution to monitor cell behavior at edges of the wound. Thus, depending on the time after wounding, it is in principle possible to observe cell migra- tion, division, and/or differentiation in specific and restricted areas of the repairing airway epithelium. Typically, one needs an inverted microscope equipped with a close chamber enabling control of temperature, CO2, and humidity. The microscope is
also equipped for time-lapse imaging. For good (but not optimal) resolution of cell behavior cultured on Transwell inserts, refer to Note 8for tips and tricks.
3.2.4. Criteria of Repaired Airway Epithelium
As expected from a well-differentiated and polarized airway epithelium, the very same criteria hold for a fully repaired epithe- lium after injury, including the following:
1. A tall pseudostratified mucociliated morphology.
2. Positive and massive immunostaining for β-tubulin but no detection of Ki-67, Cx26, and Cx43.
3. Lack of Lucifer yellow-mediated gap junctional intercellular communication.
4. Low basal production of IL-8 but sustained apical secretion of mucins.
4. Notes
1. SuperfrostR slides are positively charged allowing a better adhesion of samples and avoiding the use of fixation or glue.
Alternatively, regular slides may be used after a poly-lysine treatment.
2. To avoid pressing the airway mucosa during the mounting of the samples after immunostaining, an alternative is to use small strips of tape on each edge of the Superfrost slide to create a small chamber where the sample is placed. Then, cover the sample with a coverslip.
3. To easily cut off the top part of inserts, we used a thin inox wire connected to a 6.3-A, 2–8-V DC generator (typically generators used for microscope bulbs). We designed a system whereby the positioning of the wire height could be changed to adapt for various plastic wares (Transwell inserts, Petri dishes, etc.).
4. To easily backfill microelectrodes with the Lucifer yellow solution, we recommend the MicroFilTMneedle from World Precision Instruments, Inc. (Microfil MF34G).
5. Mechanical wound may be performed by using the tip of a pipette (14,15). This technique is particularly simple when performed on a plastic dish but becomes more challenging on membrane inserts. The main inconvenience remains the risk to damage the microporous membrane and to tear the tall airway epithelium apart.
6. Mechanical wound may also be performed follow- ing the methodology reported by Vermeer et al. (16). A
home-designed wounding device performs a reproducible ring-shaped wound when lowered onto cells and turned 360◦C.
7. Chemical wounding consists of the deposit of a small vol- ume of 1 M sodium hydroxide and its rapid neutralization with DPBS. Tournier et al. (17) reported that a drop of 1μl leads to a circular wound area of 30 mm2. Transposed to our model (insert area of 33 mm2), this technique would need to be adapted for smaller volumes.
8. To monitor the cell behavior by live imaging, we are using 35-mm Petri dishes with a glass bottom (12 mm, ref. num- ber 73911035; Milian) and for which a hole has been drilled through the covers. The diameter of the hole is slightly larger than that of the bottom of the insert. This simple trick allows holding the insert in proximity to the glass bottom, which is of help for imaging using a 40×objective. The dish is filled with culture medium, while 100μl is added to the wounded surface. This allows to bath cells with sufficient medium and to ensure humidity and gas exchange in the Petri dish.
Acknowledgments
This work was supported by the grants from the Swiss National Science Foundation and by Vaincre la Mucoviscidose.
References
1. Puchelle, E., and Zahm, J. M. (2006) Repair process of the airway epithelium, in (Lenfant, C., and Dekker, M., eds.), Airway Environ- ment: From Injury to Repair. Series Lung biology in health and diseases. Marcel Dekker, New York, NY, pp. 1576–1582.
2. Voynow, J. A., Fischer, B. M., Roberts, B.
C., and Proia, A. D. (2005) Basal-like cells constitute the proliferating cell population in cystic fibrosis airways. Am J Respir Crit Care Med 172, 1013–1038.
3. Liu, X., Driskell, R. R., and Engelhardt, J. F.
(2006) Stem cells in the lung. Methods Enzy- mol 419, 285–321.
4. Hajj, R., Lesimple, P., Nawrocki-Raby, B., Birembaut, P., Puchelle, E., and Coraux, C. (2007) Human airway surface epithelial regeneration is delayed and abnormal in cys- tic fibrosis. J Pathol 211 3, 340–350.
5. Gruenert, D. C., Finkbeiner, W. E., and Wid- dicombe, J. H. (1995) Culture and transfor-
mation of human airway epithelial cells. Am J Physiol 268 3 Pt 1, L347–360.
6. Yamaya, M., Finkbeiner, W. E., Chun, S. Y., and Widdicombe, J. H. (1992) Differenti- ated structure and function of cultures from human tracheal epithelium. Am J Physiol 262, L713–L724.
7. de Jong, P. M., van Sterkenburg, M. A., Hes- seling, S. C., Kempenaar, J. A., Mulder, A.
A., Mommaas, A. M., et al. (1994) Ciliogen- esis in human bronchial epithelial cells cul- tured at the air–liquid interface. Am J Respir Cell Mol Biol 24, 224–234.
8. Johnson, L. G., Dickman, K. G., Moore, K. L., Mandel, L. J., and Boucher, R. C.
(1993) Enhanced Na+ transport in an air–
liquid interface culture system. Am J Physiol 264, L560–L565.
9. Karp, P. H., Moninger, T. O., Weber, S.
P., Nesselhauf, T. S., Launspach, J. L., Zab- ner, J., et al. (2002) An in vitro model of
differentiated human airway epithelia. Meth- ods for establishing primary cultures. Methods Mol Biol 188, 115–137.
10. Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R., and Randell, S. H. (2005) Well-Differentiated Human Airway Epithe- lial Cell Cultures. Human Cell Culture Pro- tocols, 2nd ed, Methods Mol. Med., vol 107.
Springer, New York, NY, pp. 183–206.
11. Wiszniewski, L., Jornot, L., Dudez, T., Pagano, A., Rochat, T., Lacroix, J. S., et al.
(2006) Long-term cultures of polarized air- way epithelial cells from patients with cystic fibrosis. Am J Respir Cell Mol Biol 34, 39–48.
12. Wiszniewski, L., Sanz, J., Scerri, I., Gas- parotto, E., Dudez, T., Lacroix, J. S., et al.
(2007) Functional expression of connexin30 and connexin31 in the polarized human airway epithelium. Differentiation 75, 382–392.
13. Puchelle, E., Zahm, J. M., Tournier, J. M., and Coraux, C. (2006) Airway epithelial repair, regeneration, and remodeling after injury in chronic obstructive pulmonary dis- ease. Proc Am Thorac Soc 3, 726–733.
14. Planus, E., Galiacy, S., Matthay, M., Laurent, V., Galvrilovic, J., Murphy, G., et al. (1999) Role of collagenase in mediating in vitro alve- olar epithelial wound repair. J Cell Sci 112, 243–252.
15. Lechapt-Zalcman, E., Prulière-Escabasse, V., Advenier, D., Galiacy, S., Charrière-Bertrand, C., Coste, A., et al. (2006) Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new path- way for epithelial wound repair?. Am J Physiol Lung Cell Mol Physiol 290, L1277–L1282.
16. Vermeer, P. D., Einwalter, L. A., Moninger, T. O., Rokhlina, T., Kern, J. A., Zabner, J., et al. (2003) Segregation of receptor and lig- and regulates activation of epithelial growth factor receptor. Nature 422, 322–326.
17. Tournier, J. M., Maouche, K., Coraux, C., Zahm, J. M., Cloëz-Tayarani, I., Nawrocki- Raby, B., Bonnomet, A., et al. (2006) alpha3alpha5beta2-Nicotinic acetylcholine receptor contributes to the wound repair of the respiratory epithelium by modulating intracellular calcium in migrating cells. Am J Pathol 168, 55–68.
