1 Medical Research Council Human Genetics Unit, Western General Hospital, and 3 Medical Genetics Section, Molecular Medicine Centre, Department of Medical Sciences, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, United Kingdom; and 2 University of North Carolina Cystic Fibrosis Center, Chapel Hill, North Carolina 27599
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ABSTRACT |
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The goal of this study was to develop a
primary culture model of differentiated murine tracheal epithelium.
When grown on semipermeable membranes at an air interface, dissociated
murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances (~12
k · cm2) that remained viable for up to 80 days.
Immunohistochemistry and light and electron microscopy demonstrated
that the cells were epithelial in nature (cytokeratin positive,
vimentin and
-smooth muscle actin negative) and differentiated to
form ciliated and secretory cells from day 8 after seeding
onward. With RT-PCR, expression of the cystic fibrosis transmembrane
conductance regulator (Cftr) and murine
-defensin
(Defb) genes was detected (Defb-1 was
constitutively expressed, whereas Defb-2 expression was
induced by exposure to lipopolysaccharide). Finally, Ussing chamber
experiments demonstrated an electrophysiological profile compatible
with functional amiloride-sensitive sodium channels and cAMP-stimulated
CFTR chloride channels. These data indicate that primary cultures of
murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the
establishment of primary cultures of airway epithelium from transgenic
mouse models of human diseases.
cystic fibrosis; cystic fibrosis transmembrane conductance regulator; defensins; airway surface liquid; airway epithelium
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INTRODUCTION |
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THE DEVELOPMENT OF ROBUST TECHNIQUES for the primary culture of airway epithelial cells from a variety of species, including human (32), rat (4, 5), dog (14), guinea pig (1), rabbit (17), and hamster (31), has provided invaluable in vitro models for the study of airway physiology in health and disease. However, the development of successful techniques for the culture of mouse airway epithelial cells has remained elusive. The most successful published techniques resulted in the establishment of a relatively undifferentiated epithelium with few, if any, ciliated cells (6, 16). Although transgenic technology in mice has enabled the development of murine models for human pulmonary diseases and knockout mice with prominent pulmonary phenotypes (15, 19, 21, 30), there has been a frustrating inability to establish an adequate system for the primary culture of airway cells from these transgenic models.
Recent studies (20, 28, 33) have utilized primary cultures of human airway epithelium to investigate the pathogenesis of lung disease in cystic fibrosis (CF). This approach has permitted investigation of the characteristics of the airway surface liquid (ASL) that lines the conducting airways. Such studies have suggested that alterations in the ionic composition of the ASL, with consequent impairment of its salt-sensitive antimicrobial properties (28) or its dehydration, with resultant failure of mucociliary clearance (20), may play a key role in the development of the chronic infection and inflammation that characterize this disease (reviewed in Ref. 7).
Despite the prominent role that mouse models of CF have played in recent research into this disorder, studies to parallel and further develop these critical observations have been hampered by the absence of an acceptable primary culture model for murine airway epithelial cells.
To facilitate studies of tracheal epithelium from transgenic mouse models of human disease, we have developed a primary culture model of differentiated mouse tracheal epithelium. Here we describe the establishment of this model and investigate its characteristics using electron microscopy, histochemistry, immunohistochemistry, gene expression analysis, and electrophysiology.
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MATERIALS AND METHODS |
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Animals. Specific pathogen-free C57BL/6N mice of both sexes, age 5-7 wk, were used (Charles River UK, Margate, UK). All experimental work complied with the UK Animal (Scientific Procedures) Act 1986. The animals were killed by asphyxiation with CO2.
Isolation and culture of tracheal epithelial cells. On day 1, the mice were killed by asphyxiation with CO2 and doused with 70% ethanol, and the tracheae were excised and severed at the proximal surface of the thyroid cartilage and at the bifurcation of the bronchi. The thyroid glands and other adherent tissue were removed from the superolateral surfaces (while retaining the underlying proximal tracheae). The tracheae were cut open lengthwise, washed in phosphate-buffered saline (PBS), and transferred to collection medium (see Formulations) prewarmed to 37°C. Batches of eight tracheae were transferred to 20 ml of prewarmed dissociation medium containing Pronase and DNase (see Formulations) and incubated at 37°C for 60 min. To stop enzymatic digestion, 5 ml of sterile fetal calf serum were added. To dissociate epithelial cells, the tracheae were gently agitated (the tube containing the tracheae was carefully inverted 12 times, not shaken). The tracheal "husks" were removed from the suspension, placed in 10 ml of culture medium (see Formulations), and gently agitated 12 times as before to release more epithelial cells. The resultant cell suspensions were pooled and centrifuged at 1,000 rpm (120 g) for 5 min at room temperature (21-23°C). After the supernatants were removed, the pellets were resuspended and washed in 10 ml of culture medium, centrifuged at 1,000 rpm for 5 min, and resuspended in 5 ml of culture medium. This suspension was incubated at 37°C for 2 h in a 100-mm culture dish (Premaria, Becton Dickinson UK, Oxford, UK) to remove contaminating nonepithelial cells. After this incubation, the medium was removed with a fine-tip flexible pipette to collect the unattached cells, centrifuged at 1,000 rpm for 5 min, and resuspended in culture medium (200 µl for every two tracheae used). The dissociated cells from two tracheae (~4 × 105 cells) were seeded onto one tissue culture insert semipermeable support membrane (Costar Transwell clear, tissue culture-treated polyester membrane 24-well plate inserts, 0.4-µm pore; Corning Costar, High Wycombe, UK) in 200 µl of culture medium, with 600 µl outside the insert. To precoat these inserts with type VI acid-soluble human placental collagen (Sigma-Aldrich, Poole, UK), 100 µl of collagen solution (0.5 mg/ml of human placental collagen in distilled water with 0.2% glacial acetic acid) were added to the semipermeable membrane, air-dried overnight, and then washed twice with PBS before use. The cells were incubated at 37°C in 6% CO2 in a humidified incubator for 3 days. On day 4, the medium on the apical surface of the cultured cells was removed along with any nonadherent cells and debris, and the medium on the outside of the insert (bathing the basolateral surface) was replaced with 600 µl of Ultroser G (USG) medium (see Formulations). Once the cells had reached confluence, the apical surface of the insert appeared dry; this normally occurred from day 4 onward. The medium bathing the basolateral surface was replaced twice weekly. Primary cultures grown at an air-liquid interface remained viable for up to 80 days.
Formulations. Collection medium consisted of a 1:1 mix of Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL, Life Technologies, Paisley, UK) and Ham's F-12 medium (GIBCO BRL) containing penicillin (100 IU/ml)-streptomycin (100 µg/ml). Dissociation medium consisted of calcium- and magnesium-free minimum essential medium (44 mM NaHCO3, 54 mM KCl, 110 mM NaCl, 0.9 mM NaH2PO4, 0.25 µM FeN3O9, 1 µM sodium pyruvate, and 42 µM phenol red, pH 7.5), 60 IU/ml of penicillin-60 µg/ml of streptomycin, 1.4 mg/ml of Pronase (Boehringer Mannheim UK, Lewes, UK), and 0.1 mg/ml of DNase (Sigma-Aldrich). Culture medium consisted of a 1:1 mixture of DMEM and Ham's F-12 medium containing 100 IU/ml of penicillin-100 µg/ml of streptomycin, 5% fetal calf serum, and 120 IU/l of insulin (Nova Human Actrapid, AAH Pharmaceuticals, Glasgow, UK). USG medium consisted of a 1:1 mixture of DMEM and Ham's F-12 medium containing 100 IU/ml of penicillin-100 µg/ml of streptomycin and 2% USG serum substitute (GIBCO BRL).
Electron microscopy. Scanning (SEM) and transmission (TEM) electron microscopy were performed on primary cultures of murine tracheal epithelium and tracheal specimens from 5-wk-old mice. For SEM, the specimens were washed with PBS, then fixed first in 2.5% glutaraldehyde in PBS and second in 1% osmium tetroxide in PBS. Subsequently, the specimens were dehydrated through an alcohol series, treated with hexamethyldisilazane, and allowed to air-dry overnight. Primary culture specimens (cut from the plastic insert) and tracheal specimens were mounted on stubs with carbon adhesive disks and silver DAG (Agar Scientific, Stansted, UK), sputter coated, and viewed in a Cambridge S250 scanning electron microscope. Images were recorded on Kodak Tmax 100 film. For TEM, the specimens were fixed as for SEM. After dehydration, the specimens were infiltrated with Spurr resin, and serial sections of 80-110 nm were cut with a Leica UCT Ultramicrotome. Sections were viewed on a Philips CM120 Biotwin. TEM plates were Kodak SO 163.
To quantitatively assess the different cell populations present in the primary cultures of murine tracheal epithelium, morphometric analyses were performed on SEM images of primary cultures on days 4, 8, 14, and 28 after seeding and on tracheal specimens from 5-wk-old mice. We counted the number of ciliated and nonciliated cells inHistochemistry. Primary cultures of murine tracheal epithelium were washed in PBS and fixed with 1:1 acetone-methanol for 5 min. A standard periodic acid-Schiff stain was performed to indicate the presence of goblet cells.
Fluorescence immunohistochemistry.
Primary cultures of murine tracheal epithelium and tracheal sections
from 5-wk-old mice were characterized with antibodies to different
cytokeratins, vimentin, and -smooth muscle actin. Similar methods
were used for both cultured epithelium and mouse tracheal sections. We
used the following antibodies: mouse monoclonal anti-human pan
cytokeratin (1:100 dilution; Sigma-Aldrich), mouse monoclonal
anti-human
-smooth muscle actin (1:400 dilution; Sigma-Aldrich), mouse monoclonal anti-human vimentin (1:40 dilution; Sigma-Aldrich), mouse monoclonal anti-proliferating cell nuclear antigen (1:2,000 dilution; Sigma-Aldrich), and rabbit anti-mouse cytokeratin 14 and
rabbit anti-mouse cytokeratin 18. The antibodies cytokeratin 14 and
cytokeratin 18 were raised in rabbits immunized with peptide sequences
present in the mouse homologs of human cytokeratins 14 and 18 (CGKWSTHEQVLRTKN-COOH and CGRWSETNDTRVLRH-COOH, respectively), conjugated to maleimide-activated ovalbumin or bovine serum
albumin (Pierce, Rockford, IL), and affinity purified on peptides
linked to maleimide-activated Sepharose (Pierce). Negative controls
were performed by omitting the primary antibodies. Tracheal specimens were fixed in 1:1 acetone-methanol, processed to paraffin wax blocks,
cut to 4-µm sections with a microtome, deparaffinized in xylene, and
hydrated through an alcohol series. Primary culture specimens were
washed in PBS, fixed in 1:1 acetone-methanol for 5 min, washed in PBS,
and cut from the culture insert. When a mouse primary antibody was
used, the specimens were preblocked with blocking solution [PBS with
2% normal animal serum (of the species in which the secondary antibody
was raised), 0.2% Tween, 20 mg/ml of bovine serum albumin, and 7%
glycerol] with 1% unlabeled sheep anti-mouse IgG [Scottish Antibody
Production Unit (SAPU), Carluke, UK] or 1% unlabeled sheep anti-mouse
immunoglobulins (SAPU) for 20 min and then washed with PBS. The
specimens were incubated with blocking solution for 20 min before
incubation for 1 h at room temperature with the primary antibody
diluted in blocking solution. After a wash with PBS, a 30-min
incubation was performed with the secondary antibody [FITC-labeled
sheep anti-mouse IgG (SAPU) or FITC-labeled donkey anti-rabbit (SAPU)] diluted 1:100 in blocking solution. After being washed in PBS, the
specimens were mounted in Vectashield (Vector Laboratories, Peterborough, UK) containing 4,6-diamidino-2-phenylindole nuclear stain.
Gene expression analysis.
The gene expression profile of primary cultures of murine tracheal
epithelium was investigated with reverse transcriptase-polymerase chain
reaction (RT-PCR). Total RNA was isolated with RNAzol B (Biogenesis,
Poole, UK) and cDNA synthesis was performed with Ready-To-Go You-Prime
First-Strand Beads (Amersham Pharmacia Biotech, St. Albans, UK) as
described by the manufacturers. The resultant cDNAs were used as
templates for intron-spanning PCRs with primers designed to detect the
murine CF transmembrane conductance regulator (Cftr) and
mouse -defensin-1 (Defb-1) and
-defensin-2
(Defb-2) genes, with parallel amplification of the murine
hypoxanthine phosphoribosyltransferase (Hprt) gene as an
internal control, all as previously described (8, 22, 23).
The amplified products were analyzed by electrophoresis on 2% agarose
gels with a
X147HaeIII marker, amplified plasmid-positive controls,
and negative controls, with either heat-inactivated RT or distilled water substituted for RNA during cDNA synthesis. Defb-2 PCR
products were not visible on a stained agarose gel after 34 cycles of
PCR but were detectable by hybridization with a Defb-2
internal oligonucleotide probe as previously described
(22). Primary cultures were analyzed after a 30-min
exposure to either 80 µg of Escherichia coli
lipopolysaccharide (LPS; serotype 026:B6; Sigma-Aldrich) in PBS or
~2 × 105 colony-forming units of Pseudomonas
aeruginosa (CF clinical strain J1385; generous gift from Prof.
J. R. W. Govan, University of Edinburgh, Edinburgh, UK). For
both LPS and P. aeruginosa, the vehicle was in PBS.
Untreated control epithelia were simultaneously exposed to PBS alone.
Electrophysiological studies. To monitor the transepithelial resistance (Rte) of cultured murine tracheal epithelium, we used an EVOM epithelial voltohmmeter (World Precision Instruments, Stevenage, UK) after the addition and equilibration of 200 µl of USG medium prewarmed to 37°C to the apical surface. Rte was measured on days 4, 8, 14, and 28 after seeding.
Primary cultures of murine tracheal epithelium were mounted in a modified Ussing chamber apparatus (Jim's Instruments Manufacturing, Iowa City, IA), and both the apical and basolateral surfaces were bathed with a physiological solution containing 140 mM NaCl, 5 mM KCl, 0.36 mM K2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES, 4.2 mM NaHCO3, and 5 mM D-glucose, titrated to pH 7.2 with Tris. Physiological solutions were maintained at 37°C and gassed with 95% CO2-5% O2. The voltage (ELEC/CV, ADInstruments, Hastings, UK) and current (Ag-AgCl) electrode pairs were connected to the apical and basolateral solutions by polyethylene tubing (PE-160; Garridor) filled with 3 M KCl-3% (wt/vol) Noble agar (Sigma-Aldrich). Transepithelial voltage was clamped at 0 mV with a Physiological Instruments VCCMC2 dual-channel voltage-current clamp (ADInstruments). Short-circuit current (Isc) was continuously recorded with a MacLab/4s recorder and Chart for Macintosh (version 3) data acquisition and analysis software (both supplied by ADInstruments); Rte was monitored with 10-mV voltage steps (duration 0.1 s, period 10 s) and calculated with Ohm's law. A positive Isc value represents a flow of positive charge from the luminal (apical) to the basolateral solution (anions moving in the opposite direction). To investigate the electrophysiological characteristics of primary cultures of murine tracheal epithelium, the following agents were sequentially added: 1) amiloride (10 µM) to the apical reservoir to inhibit the epithelial Na+ channel (ENaC), 2) cAMP agonists [10 µM forskolin, 100 µM 3-isobutyl-1-methylxanthine (IBMX), and 500 µM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)] to the apical reservoir to activate the CFTR ClStatistical analysis. Results are expressed as means ± SD; n is the number of observations. To compare sets of data, we used Student's t-test. Differences were considered significant when P < 0.05.
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RESULTS AND DISCUSSION |
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Primary cultures of murine tracheal epithelium were established on semipermeable support membranes, grown at an air-liquid interface, and characterized to assess differentiation.
The short enzymatic dissociation of pooled tracheae at 37°C as described in MATERIALS AND METHODS generated ~2 × 105 cells/trachea, with cell viability of ~95% as assessed by trypan blue dye exclusion. Isolated cells were seeded at high density on collagen-coated semipermeable membranes. After 72 h of submerged culture, the apical medium along with any nonadherent cells and debris was removed to create an air-liquid interface at the apical surface of the cells. This air-liquid interface was retained for as long as the cultures remained viable (up to 80 days). Phase-contrast light microscopy demonstrated that primary cultures of murine tracheal epithelium had a smooth "cobblestone" appearance. Consistent with this finding, high Rte values indicated confluence, with the formation of tight junctions from day 4 onward. During the second week of culture, the development of beating cilia was observed, with the gradual appearance of scattered foci of "cell stacking" noted over the next few weeks.
Further morphological studies were performed with SEM, TEM, histochemistry, and immunohistochemistry. Additional characterization was performed with RT-PCR to analyze gene expression and with Ussing chamber experiments to study the electrophysiological profile of the primary cultures.
Electron microscopy.
SEM was performed on primary cultures of murine tracheal epithelium on
days 4, 8, 14, and 28 (n 2 observations for each time point) and tracheal
specimens from 5-wk-old animals (n = 2 observations) of
the same strain used to isolate epithelial cells for primary culture
(Fig. 1).
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Histochemical characterization.
A periodic acid-Schiff stain was performed on days 4,
8, 14, and 28 (n 4 observations for each time point). This stain revealed occasional
positively stained, magenta-colored cells, suggesting the presence of
mucus-producing goblet cells (Fig. 4).
These cells were not always present and were variable in number, with
no pattern observed on the basis of culture age. The reason for this
irregularity is not clear. Given the scarcity of these cells in the
cultures, it is perhaps not surprising that they were not detected by
TEM. This minimal presence of goblet cells reflected their paucity in
the native tracheal epithelium of healthy mice (24).
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Immunohistochemical characterization.
To characterize the cell types that constituted the primary cultures of
murine tracheal epithelia, immunohistochemical analyses were performed
on days 4, 8, 14, and 28 (n 3 observations for each antibody at each time
point) with a panel of antibodies and compared with murine tracheal
sections (n
3 specimens for each antibody)
(Figs. 5 and
6). Data were quantified as described in MATERIALS AND METHODS.
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Gene expression.
Recent investigations of the pathogenesis of CF lung disease have
focused on the characteristics of the ASL and its role in mucociliary
clearance and innate lung defenses. The analysis of ASL in vivo has
proven to be extremely difficult technically and has produced
conflicting results (reviewed in Ref. 11). Consequently, key studies
(20, 28, 33) addressing the ionic composition and
antibacterial activity of the ASL have been performed in primary cultures of human airway epithelium. To maximize the opportunities presented by the availability of mouse models of CF, it is critical to
develop a primary culture model for murine airway epithelial cells.
Toward this goal, primary cultures of murine tracheal epithelium on
days 12-13 after seeding (n 3 observations for each condition) were characterized with RT-PCR.
We investigated the expression of Cftr and mouse
Defb-1 and Defb-2, mouse homologs of the human
-defensins, which have been implicated in the pathogenesis of CF
lung disease (10, 27). Because Defb-2
expression is upregulated in response to proinflammatory stimuli in
vivo (22), these studies were performed with both
untreated cultures and cultures exposed to E. coli LPS.
Parallel amplification of Hprt was performed as a control
(Fig. 7). Figure 7 demonstrates that the
expression pattern of these genes replicated the in vivo situation
(22, 23). Cftr and Defb-1 were both
constitutively expressed (n = 5 observations; Fig.
7a), whereas Defb2 was induced by exposure to
E. coli LPS (Fig. 7b). Defb-2
expression was not observed without prior stimulation
(n = 3 observations) but was observed in response to
LPS exposure (n = 4 observations). Consistent with the
in vivo results of Morrison et al. (22), the level of
expression of Defb-2 after induction was too low to be
observed on a stained agarose gel after 34 cycles of PCR. However, it
was detected by hybridization to an internal oligonucleotide probe.
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Electrophysiological characterization.
To assess the confluence of primary cultures of murine tracheal
epithelium, Rte was monitored on days
4, 8, 14, and 28. Table 1 shows that the maximum value of
Rte, 28.4 k · cm2, was
measured on day 4 immediately after the establishment of an
air-liquid interface. Subsequently, Rte values
decreased to ~12 k
· cm2 by day 14,
after which they remained stable at about this value (Table 1). These
values of Rte are markedly higher than those reported for cultures of airway epithelium from other species including
human [200-3,000
· cm2
(32)] and dog [200-400
· cm2 (14)]. They also greatly
exceed those reported for PD primary cultures of murine tracheal
epithelium [~400
· cm2 (6)].
Interestingly, we found that although primary cultures of murine
tracheal epithelium that failed to achieve elevated values of
Rte were able to maintain an air-liquid
interface, they did not develop ciliated epithelial cells.
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ACKNOWLEDGEMENTS |
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We thank Gillian Morrison, Paul Perry, Wendy Hannant, Prof. John Govan, Euan Slorach, Brendan Doe, Norman Davidson, and Sheila Webb for advice and assistance with this work and John Findlay at the Biosem Unit at the Institute of Cell and Molecular Biology (Edinburgh University, Edinburgh, UK) for electron microscopy. Special thanks go to Prof. David Porteous for all his advice and support and to Aurita Puga, Phil Karp, and Prof. Michael Welsh (University of Iowa, Iowa City, IA) for their kind hospitality and invaluable tuition and assistance in the initial stages of these studies.
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FOOTNOTES |
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This work was supported by the Medical Research Council (UK), Biotechnology and Biological Sciences Research Council, Cystic Fibrosis Trust (UK), and National Heart, Lung, and Blood Institute Grant HL-58345.
Address for reprint requests and other correspondence: J. R. Dorin, MRC Human Genetics Unit, Western General Hospital, Crewe Rd., Edinburgh EH4 2XU, UK (E-mail: julia.dorin{at}hgu.mrc.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 November 1999; accepted in final form 8 May 2000.
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