J-S Paquette et al. Human asthmatic bronchial equivalents
Abstract
The isolation of human bronchial epithelial (HBEC) and
fibroblastic cells (HBFC) from biopsies of asthmatic and
non-asthmatic volunteers provided unique cellular materi-
als to be used for the production of bioengineered bron-
chial equivalents (BE) in vitro. The HBEC are grown on a
mesenchymal layer seeded with HBFC and the BE can be
maintained for at least 15 days in culture. Under the BE
culture conditions established previously, HBEC undergo
differentiation into ciliated and goblet cells, within a
pseudostratified organization comparable to human bron-
chi. We published previously the results from histologic and
functional analyses of such BE produced exclusively using
non-asthmatic HBEC and HBFC. We report here the com-
parative analyses of BE produced with non-asthmatic and
asthmatic living HBEC and HBFC (naBE and aBE, respec-
tively). Our data indicated that all asthmatic HBEC
populations grown on a mesenchymal layer, containing non-
asthmatic HBFC, slowly reached a confluent state but then
detached from the matrix upon culture time. These BE ap-
pear to be very good models to study the mechanisms in-
volved in asthma in vitro.
Key words: human bronchial equivalents, asthma, bioen-
gineering.
Introduction
In our modern society, thousands of people suffer from
mild to severe asthma. This bronchial disorder is mostly
associated with mucosal inflammation and airway
hyperresponsiveness (Jeffery et al., 1989; Djukanovic et
al., 1990; Boulet et al., 1993). Histologic analyses of bron-
chial biopsies taken on asthmatic patients report an appar-
ent basement membrane thickening most likely caused by
sub-epithelial fibrosis. It is postulated that fibroblasts, in-
volved in collagen synthesis and remodelling, could be
responsible for the acute fibrosis in response to cytokines
secreted by inflammatory or epithelial cells (Brewster et
al., 1990; Roche, 1991; Gauldie et al., 1992). Another
major change in the structural properties of asthmatic bron-
chi is their partial or complete desquamation upon the evo-
lution of the disease (Jeffery et al., 1989). Up to now, the
alterations of the bronchial tissues of asthmatic subjects
remain poorly understood and the various hypotheses raised
on the putative mechanisms responsible for the mainte-
nance and the progression of these changes have to be as-
sessed.
Besides live animals (often rats and dogs) (DiCosmo
et al., 1995; Chung, 1996; Widdicombe, 1996; Shichinoke
et al., 1996), several research groups use animal bronchial
tissues (Opazo-Saez and Pare, 1994; Baeza-Squiban et al.,
1994; Davenport and Nettesheim, 1996) or cells grown
in monolayers (De Jong et al., 1994; Gray et al., 1996) as
experimental models, to study various aspects of asthma
in vitro. To overcome interindividual variations among
animals used as experimental models, the number of sub-
jects needed to perform each study has to be quite consid-
erable. Moreover, animal models are complex and it be-
comes sometimes difficult to control all physiologic pa-
rameters which may modulate the results of comparative
studies between non-asthmatic and asthmatic groups. In
addition, some limitations are associated with bronchial
cell monolayers, particularly because heterotypic cellular
interactions are difficult to reproduce under these culture
conditions.
Over the last decade, bioengineering has enlarged the
possibilities to develop tissue models (Langer and Vacanti,
1993) by combining the conventional cell culture ap-
proaches to a concept according to which most cells can
adopt specific three-dimensional orientation and organi-
zation in an extracellular matrix, in response to proper
stimuli induced mechanically in vitro. This concept has
been confirmed in various bioengineered tissues produced
in culture, notably in skin, blood vessels and ligaments
(Bellows et al., 1982; Bouvard et al., 1992; Lopez-Valle
et al., 1992; L’Heureux et al., 1993; Auger et al., 1995;
TISSUE-ENGINEERED HUMAN ASTHMATIC BRONCHIAL EQUIVALENTS
Jean-Sébastien Paquette, Véronique Moulin, Pierrot Tremblay, Vincent Bernier, Michel Boutet, Michel Laviolette,
François A. Auger, Louis-Philippe Boulet and Francine Goulet
Laboratoire de Génie Tissulaire/LOEX, Hôpital de l’Enfant-Jésus du CHA and
Unité de Recherche en Pneumologie, Hôpital Laval, Québec, Canada.
Materials Sciences and Applications, 2009, 1, 17-26

Human bronchial cell isolation
The human bronchial cells were isolated by a new enzymatic
technique and amplified in culture according to methods
developed in our laboratory (Goulet et al., 1996a). Briefly,
the processing was performed within 2-3 hours following
the bronchoscopy. Collagen being the major constituent of
bronchi matrix, collagenase was chosen to digest the col-
lagen matrix of the biopsies. The human bronchial biop-
sies were digested in 0.1% (0.2 U/ml) collagenase H
(Boehringer Mannheim, Montreal, Canada) prepared in
Dulbecco-Vogt modification of Eagles medium (DMEM)
culture medium containing 10mM CaCl2 without any sup-
plement. Tissues were digested overnight at 4°C because a
collagenase digestion performed overnight at 37°C would
reduce cell yield and viability. Homogenates were centri-
fuged for 10 min at 300 g and the cell pellets were
resuspended in DMEM supplemented with 10% fetal calf
serum (FCS).
All cells were plated in several 35-mm Petri dishes.
The cultures were monitored daily and the dishes contain-
ing epithelial cells were selected for addition of some le-
thally irradiated 3T3 feeder cells (like it is done for skin
epithelial cells, see Green et al., 1979). This technique al-
lows the selection of pure epithelial cell populations
(Goulet et al., 1996a). After 8-12 days in culture, bron-
chial epithelial cells had reached 85% confluency and were
ready to be stored and subcultured. The bronchial epithe-
lial cells were cultured according to the method established
for human keratinocytes (Germain et al., 1993), originally
described by Green et al. (1979), in a combination of
DMEM with Ham’s F12 in a 3:1 proportion (Gibco BRL,
Burlington, Canada), supplemented with 24.3 µg/ml ad-
enine (Sigma Chemicals, St-Louis, MO), 10 ng/ml human
epidermal growth factor (EGF, Austral Biological, San
Ramon, CA), 5 µg/ml bovine crystallized insulin, 5 µg/ml
human transferrin (Boehringer Mannheim, Laval, Canada),
2x10-9 M 3,3',5', triiodo-L-thyronin (Sigma), 0.4 mg/ml
hydrocortisone (Calbiochem, La Jolla, CA), 10-10 M chol-
era toxin (ICN Biochemicals, Montreal, Canada), 10% re-
constituted newborn calf serum (Immunocorp Sciences inc.,
Montreal, Canada) 100 IU/ml penicillin G and 25 µg/ml
gentamicin (Sigma). Bronchial fibroblasts were obtained
in the dishes which did not contain epithelial cells. They
were cultured in DMEM supplemented with 10% fetal calf
serum, 100 IU/ml penicillin G and 25 µg/ml gentamicin.
They also reached 85% confluency after a week. The cul-
ture media was changed three times a week. All cultures
were kept in an 8% CO2 atmosphere at 37°C.
Human bronchial cell viability
The viability of epithelial cells over 3-4 passages and of
fibroblasts over 8-9 passages, were comparable between
asthmatic and non-asthmatic cell populations (over 85%
viability). However, the yields of epithelial cells obtained
from asthmatic biopsies were about 60% lower in primary
culture, compared to non-asthmatic biopsies. That is ex-
pected, since the epithelia of the asthmatic biopsies are of-
ten poorer in cells and sometimes disorganized. Moreover,
considering that we isolated the cells from 6-10
microbiopsies (1-2 mm-diameter), we believe that our
Goulet et al., 2000; Chakir et al., 2001; Paquette et al.,
2003).
Thus, we have used several populations of the human
bronchial cells isolated in our laboratory (Goulet et al.,
1996a) from bronchial biopsies of asthmatic and non-asth-
matic subjects, in an attempt to produce three-dimensional
bronchial equivalents (BE) in vitro. For the first time, a
bilayered BE containing human bronchial epithelial (HBEC)
and fibroblastic cells (HBFC) isolated from asthmatic bi-
opsies was obtained and maintained in culture for at least
15 days. An asthmatic HBEC layer grown on a mesenchy-
mal counterpart seeded with asthmatic HBFC formed a BE
referred to as asthmatic BE (a/aBE). Similarly, the non-asth-
matic BE (na/naBE) was produced by combining non-asth-
matic HBEC and HBFC in their respective layers. The in-
teresting results from comparative analyses of these BEs
are described in the present report.
Materials and Methods
Subjects
Non-smoking asthmatic and normal subjects aged from 20
to 45 years, evaluated at the Laval Hospital asthma clinic,
were enrolled in the study. The study was approved by the
local Ethics Committee and subjects had given informed
written consent. The normal subjects had a PC20 value
(metacholine provocation, see below) over 16 mg/ml. All
asthmatic subjects had a diagnosis of asthma according to
the American Thoracic Society criteria (American Thoracic
Society, 1987). All were atopic with at least one positive
response to common allergens on allergy skin prick tests.
Their asthma required only an inhaled β2 agonist agent on
demand. None of the subjects reported a respiratory infec-
tion or an increase in asthma symptoms in the month pre-
ceding the study. They were not currently exposed to aller-
gens to which they were sensitized apart from house-dust.
Spirometry and response to inhaled methacholine were
measured according to standardized procedures using aero-
sols generated with a Wright’s nebulizer at tidal breathing
for periods of 2 minutes (output = 0.13 ml/min) (Juniper
et al., 1991). The provocative concentration of methacholine
inducing a 20% fall in FEV1, the PC20, was determined.
Skin prick tests were performed with a battery of common
airborne allergens. Atopy was considered to be present if
there was one or more positive response (> 3 mm wheal) to
the inhalant allergens, with a positive reaction to histamine
phosphate but not the diluent.
Bronchoscopy and bronchial biopsies
Before the bronchoscopy, a 200 µg dose of salbutamol was
given using a metered-dose inhaler. All subjects received
oxygen at 5 l / min by nasal catheter during bronchoscopy.
After local anaesthesia of the throat, larynx and bronchi with
2% and 4% xylocaine, the flexible bronchoscope (Olym-
pus OES 10 fiberscope, Olympus, Markham, Canada) was
introduced into the bronchial tree and ten bronchial biop-
sies were taken from the carinae of the right upper and mid-
dle lobes and the segmental bronchi of the upper and lower
lobes on both sides using conventional forceps. Vital signs,
electrocardiograph and oximetry were recorded through-
out the procedure.
18
J. S. Paquette et al.

method can be considered as successful (Goulet et al., 1996a).
Human bronchial cell markers
The respective morphological features of each cell type are
very different and were confirmed by specific
immunolabeling. Epithelial cells were immunolabeled with
anti-keratin antibodies and fibroblasts were labeled with anti-
vimentin antibodies (Goulet et al., 1996a).
Production of three-dimensional human bronchial
equivalents (BEs)
Step 1: Preparation of the mesenchymal layer of the
BEs. The BEs were produced according to the method pub-
lished by Auger et al. (1995), with a few modifications.
Briefly, a mixture of bovine Type I collagen (1.5 mg/ml) was
prepared by dissolving the powder overnight at 4°C in sterile
0.017 M acetic acid. A solution of 0.84 ml of DMEM 2.7X
containing 200 IU/ml penicillin G and 50 µg/ml of gen-
tamicin, pH 8.0, was mixed with a second solution contain-
ing 0.56 ml of FCS, 1.43 ml of the stock collagen solution,
30 µl of NaOH 0.7N and 0.15 ml of a HBFC suspension (1
X 106 cells/ml). This mixture was quickly distributed in a
bacteriological Petri dish (35-mm diameter) already contain-
ing the peripheral anchorage (sterile ring of Whatman pa-
per), to produce the mesenchymal layers of each BE. The
anchorage method, which prevents collagen lattice contrac-
tion by the cells, was used as described previously (L’Heureux
et al., 1993; Auger et al., 1995). Non-asthmatic (na) and asth-
matic (a) HBFC were seeded individually in different mes-
enchymal layers to produce naBE and aBE, respectively. The
mesenchymal layers of the BE were covered with 2 ml of
DMEM supplemented with 10% FCS, 100 IU/ml penicillin
G and 25 mg/ml gentamicin following collagen polymeriza-
tion and cultured in this medium until their epithelialization.
Step 2: Epithelialization of the BE under submerged
culture conditions. Four days later, the epithelialization was
performed by seeding HBEC (8x105 cells / BE) on the mes-
enchymal equivalents, maintained under submerged culture
conditions until a confluent epithelial cell layer was obtained.
Again, non-asthmatic and asthmatic HBEC were seeded on
the corresponding mesenchymal counterparts. In some groups
of BEs, HBEC from non-asthmatic source were seeded on
mesenchymal layers containing asthmatic HBFC and vice
versa. During the first 3 days after epithelia1ization, all BEs
were cultured in the medium used for the culture of HBEC
(see the section Human bronchial cell isolation). On the fourth
day after epithelialization, the BEs were cultured in serum-
free medium, supplemented with 24.3 mg/ml adenine, 10 ng/
ml human EGF, 5 mg/ml bovine crystallized insulin, 5 mg/
ml human transferrin, 2x10-9 M 3,3',5', triiodo-L-thyronin,
0.4 mg/ml hydrocortisone, 10-10 M cholera toxin, and 5x10-8
M retinoic acid (RA). The culture media were changed daily.
Step 3: Culture at the air-liquid interface: The BEs were
raised at the air-liquid interface as soon as a confluent layer
of HBEC had covered their mesenchymal layers (6-8 days
after epithelialization, depending on cell growth rates that
could slightly vary from one experiment to another). The BEs
were placed on Petri dishes containing an internal elevated
support (Falcon No 3037). They were cultured at the air-
liquid interface for equal number of days (a period varying
from 5 to 7 days, according to each experiment), in the
same media used under submerged conditions, but in the
absence of EGF and cholera toxin since these growth
factors enhance the production of gelatinases by the cells,
which degrade the collagen matrix of the mesenchymal
layer in culture at the air-liquid interface (Auger et al.,
1995). The culture media were changed daily. Each ex-
periment was done 3 times on at least 3 samples per group
of BEs tested.
Conditioned medium
The conditioned medium (CM) was obtained from con-
fluent non-asthmatic HBFC populations. The cells were
grown until they had reached 95% confluence in a 75
cm2 Falcon culture dish. During the conditioning period
of 7 hrs, the HBFC were kept in 12 ml of the same se-
rum-free medium, used to culture the BEs under sub-
merged conditions. Each sample of fresh CM was passed
through a 0.22 µm Millipore low-binding protein filter
in order to eliminate all cellular fragments before being
transferred on the various groups of BEs. Fresh CM was
produced and used daily, without any dilution.
Histological analysis
All BEs were fixed with Bouin’s solution and then em-
bedded in paraffin. The 4 µm thick sections were stained
using two different methods: the hematoxylin, phloxine
and saffron staining and the PAS staining. To eliminate
cross-reaction with endogenous glycogen in situ, some
BE sections were digested before PAS staining with a
solution of 0.5% maltase (Fisher) in phosphate-buffered
saline (PBS) for 30 min at 37°C and washed 10 min with
distilled water. At least 10 tissue sections were analyzed
for each BE tested. We observed the entire sections to
take representative pictures.
Electron microscopy
Some BEs were also processed and mounted for trans-
mission and scanning electron microscope analyses as pre-
viously described (Auger et al., 1993). Briefly, the sam-
ples were fixed with a solution of 2% glutaraldehyde in
0.1 M sodium cacodylate buffer, pH 7.5 for 24 hrs, post-
fixed with 1% osmium tetroxide, dehydrated with etha-
nol and coated with gold (Sputtercoater, Nanotech, Man-
chester, U.K.). Micrographs were made using Polaroid
Polapan 400. A JEOL JSM-35CF scanning electron mi-
croscope was used.
Gelatinase activity assay
The active gelatinases secreted in the BE culture media
were semi-quantitatively analyzed by zymography, accord-
ing to the method described previously (Auger et al.,
1995). Briefly, one sample of all BE culture supernatants
were collected daily, filtered and immediately frozen at -
70°C until use. At least 5 samples collected for each BE,
during culture at the air-liquid interface, were analyzed
by zymography. A constant volume (25 µl) of each sam-
ple diluted in equal volume of 2x sample buffer was re-
solved under non-reducing conditions by SDS-PAGE
(Laemmli, 1970). The gels were rinsed and equilibrated
19
J. S. Paquette et al.

Figure 1. Histological analyses of different bilayered BE cultured for 6 days at the air-liquid interface in serum-free
medium supplemented with RA. Masson trichrome staining of a BE produced with both non-asthmatic HBEC and
HBFC (A), non-asthmatic HBEC and asthmatic HBFC (B-E), both asthmatic HBEC and HBFC (F-H), and asth-
matic HBEC and non-asthmatic HBFC (I). The dashed line indicates the border of the mesenchymal layers in A, D-
E, H-I. Note the holes between the epithelial and the mesenchymal layers (arrows) and the cell debris under the
basement membrane (arrowheads) of the BEs produced with asthmatic HBEC (F-I). Scale bar 36µm.
20
J. S. Paquette et al.

in the proper buffer systems and put overnight at 37°C in
digestion buffer (50 mM Tris, pH 7.4, containing 10 mM
CaCl2 and 100 mM NaCl), under slow agitation. The gels
were fixed with a 30% methanol solution containing 10%
acetic acid and stained with 0.05% Coomassie blue pre-
pared in the fixative. The gels were photographed and
scanned.
Results
Histological analysis of BEs
Histological analyses were performed on 4 different groups
of BEs cultured for 6 days at the air-liquid interface. One
of the advantages of our bilayered BE is the possibility to
produce different types of bioengineered tissues, contain-
ing HBEC and HBFC isolated from the same biopsies or
from biopsies of different subjects (non-asthmatic or asth-
matic). Thus, to evaluate and compare the growth of non-
asthmatic and asthmatic HBEC on mesenchymal layers
containing asthmatic and non-asthmatic HBFC, the four
types of BEs were produced and analyzed.
The first group of BEs was produced using both non-
asthmatic HBEC and HBFC, na/naBEs. Masson trichrome
staining of a na/naBEs is shown in Figure lA. The second
group of BEs was produced by seeding non-asthmatic
HBEC on a mesenchymal layer containing asthmatic HBFC,
na/aBEs (Fig. 1, B-E). The third group of BEs was pro-
duced using both asthmatic HBEC and HBFC, a/aBEs (Fig.
1, F-H). In the fourth group of BEs, asthmatic HBEC were
grown on a mesenchymal layer seeded with non-asthmatic
HBFC, a/naBEs (Fig. 1I).
The thickness of the mesenchymal layers of the differ-
ent BE groups showed differences upon culture time. As
shown in figure 1, the mesenchymal layers of some a/aBEs
(Fig. 1, F-G) and na/aBEs (Fig. 1, B-C) were degraded more
slowly at the air-liquid interface, than the mesenchymal
counterpart of na/naBEs (Fig. lA), and depending on the
asthmatic HBFC population seeded in the different groups
of a/aBEs and na/aBEs.
The non-asthmatic HBEC reached a confluent state on
the mesenchymal layers populated with non-asthmatic and
asthmatic fibroblasts: na/naBEs (Fig. 1A) and na/aBEs (Fig.
1, B-E), respectively. However, these cells covered the
mesenchymal layer of aBEs one day later than those seeded
in naBEs (data not shown). Similarly, HBEC isolated from
asthmatic subjects grew more slowly (in 8 days under sub-
merged culture conditions) on all mesenchymal layers,
Figure 2. Macroscopic view of rhodanile red stained BEs (A and B). Non-asthmatic (A) and asthmatic (B) HBEC
were grown on a BE mesenchymal counterpart containing non-asthmatic HBFC in serum-free medium supple-
mented with RA for 8 days under submerged culture conditions. Photomicrograph taken under phase constrast
microscopy of BE sections after 18 days under the same culture conditions (C and D). Note the confluent layer of
non-asthmatic HBEC (C), compared to the compact colonies (arrowheads indicate their borders) of asthmatic HBEC
(D) slowly detaching from the mesenchymal counterpart. Scale bars: A-B, 0.5cm; C-D, 9 µm.
21
J. S. Paquette et al.

populated with asthmatic HBFC (Fig, l, F-H) and non-asth-
matic HBFC (fig 1I). However, after reaching a confluent
state on the various mesenchymal counterparts, the asth-
matic HBEC slowly started detaching in plates from the
matrix, one or two days later. Some holes remained in the
reconstructed epithelia thereafter, combined to the presence
of cell debris observed under the basement membrane of
all a/aBEs and a/naBEs (Fig. l, F-I). Compared to the main-
tenance of a confluent epithelial layer of non-asthmatic
HBEC on all BEs (Fig. 2A), the striking detachment of the
asthmatic HBEC could be seen macroscopically after
rhodanile red staining in the corresponding BEs (Fig. 2B).
These observations were confirmed under phase constrast
microscopy of BE sections, (Fig. 2, C-D). Indeed, the con-
fluent layer of non-asthmatic HBEC (Fig. 2C), was very
different from the compact colonies of asthmatic HBEC
Figure 3. Scanning electron microscopy analyses of cili-
ated HBEC in two different BE cultured for 7 days at the
air-liquid interface. Photomicrograph of a small patch of
asthmatic HBEC (a) remaining after the partial detach-
ment of the epithelial layers on a BE mesenchymal layer
seeded with asthmatic HBFC, shown at higher magnifica-
tion (b) and compared to non-asthmatic HBEC (c) grown
on a mesenchymal counterpart seeded with non-asthmatic
HBFC.
Figure 4. Scanning electron microscopy analyses of cili-
ated HBEC in two different BE cultured for 7 days at the
air-liquid interface. Photomicrograph of a non-asthmatic
HBEC (a) grown on a mesenchymal counterpart seeded with
non-asthmatic HBFC. Note the numerous and long cilia
on its apical pole (a). Photomicrographs of asthmatic HBEC
on a BE mesenchymal layer seeded with asthmatic HBFC
(b-c). Note the differences in the number and length of cilia
among different asthmatic cells at the same magnification.
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J. S. Paquette et al.

Figure 5. Macroscopic view of rhodanile red stained
BE. Non-asthmatic HBEC were grown on a BE mesen-
chymal counterpart containing non-asthmatic HBFC (1)
and two different asthmatic populations of HBFC (2-
3), cultured in serum-free medium supplemented with
RA (2-3) and conditioned medium (CM) for 7 days un-
der submerged conditions. Note the differences in the
density of non-asthmatic HBEC observed in response
to the HBFC isolated from non-asthmatic (1) and asth-
matic (2-3) tissues, seeded in the mesenchymal coun-
terparts of the different BE. Note the stimulatory ef-
fects of the addition of CM (conditioned by non-asth-
matic HBFC) on the growth of HBEC seeded on the
two mesenchymal layers containing the respective asth-
matic HBFC used to produce the BE 2 and 3. Scale
bars 0.5cm.
(Fig. 2D), that were slowly detaching from their mesen-
chymal counterpart.
Electron microscopy analyses of the various groups of
BEs
Scanning electron microscopy analyses confirmed the par-
tial detachment of the asthmatic HBEC from the mesen-
chymal layers of the various BEs (Fig. 3a). A small patch
of asthmatic HBEC contained ciliated asthmatic HBEC in
a/aBEs cultured for 7 days at the air-liquid interface (Fig.
3a-b). Similar observations were made on a/naBEs (data
not shown). All non-asthmatic HBEC grown in a naBE
under the same culture conditions showed a ciliated layer
of cells of more elongated morphology (Fig. 3c). Similar
observations were also made in na/aBEs (data not shown).
Transmission electron microscopy analyses showed colla-
gen fibers surrounded by bronchial fibroblasts in the mes-
enchymal layers of the various groups of BEs, but no dif-
ference was detected between their matrix ultrastructural
features (data not shown).
Ciliogenesis in BEs
Ciliogenesis of the HBEC isolated from non-asthmatic and
asthmatic tissues and grown within the different groups of
BEs, was assessed by scanning electron microscopy analy-
ses, after 7 days at the air-liquid interface. Ciliogenesis
occurred in all HBEC, independently from their tissue of
origin. However, longer and more numerous cilia were
observed on the apical pole of all non-asthmatic HBEC
(Fig. 4a). In contrast, sparsely distributed shorter cilia (Fig.
4b) were observed on some HBEC isolated from asthmatic
tissues, while in some others of the same population,
ciliogenesis was comparable to non-asthmatic cells (Fig.
4c). Thus, ciliogenesis occurred at various stages in the
asthmatic HBEC but was higher and more consistently
observed in non-asthmatic HBEC, independently of the
mesenchymal layers the cells were seeded on.
Conditioned medium
On the basis of several publications reporting the impor-
tant and beneficial effects of epithelium-mesenchyme in-
teractions through factors secreted and exchanged between
both types of tissues (Bouvard et al., 1992; Goulet et al.,
1996b; Paquette et al., 2003), the effects of non-asthmatic
HBFC was assessed on the growth of non-asthmatic HBEC
in na/naBEs and na/aBEs. The growth of the non-asthmatic
HBEC in the different BEs, cultured for 7 days under sub-
merged conditions, was assessed by rhodanile red staining.
Non-asthmatic HBEC grown on a naBE were used as the
positive control corresponding to the highest epithelial cell
density observed in absence of CM (Fig. 5, picture 1). Non-
asthmatic HBEC were also grown on two types of
mesenchymes containing different asthmatic populations
of HBFC (Fig. 5, pictures 2-3). As expected, the addition
of CM on na/naBEs did not improve the growth of the
HBEC (data not shown). However, non-asthmatic HBEC,
seeded on mesenchymal layers populated with asthmatic
HBFC (na/aBEs) and cultured in CM, grew much faster
(Fig. 5, pictures 2-3, CM) than the corresponding BEs,
cultured in the same medium but not conditioned by the
non-asthmatic HBFC (Fig. 5, pictures 2-3).
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J. S. Paquette et al.

Gelatinases secretion by human bronchial cells
Gelatinases A, zymogen of MMP-2 (proMMP-2, 72 kDa),
and activated MMP-2 (active MMP-2, 62.5 kDa) and B
(MMP-9, 92 kDa) were recently described as secretion prod-
ucts of HBFC and HBEC, respectively (Hoshino et al.,
1998). The gelatinases secreted by the BEs cultured at the
air-liquid interface were analyzed by zymography. At least
5 different samples were investigated for each BE tested
and we show in Fig. 6 a representative zymogram. Our re-
sults showed that the active form of gelatinase A (62.5 kDa)
was secreted by all HBFC in the BE supernatants (Fig. 6,
lanes 1-4), including the supernatant of a/aBEs cultured in
presence of CM (Fig.6, lane 4). The gelatinase B (92 kDa)
was also actively secreted by the various HBEC in all BE
supernatants (Fig, 6, lanes 1-4), but the zymograms showed
slightly lower activities of this enzyme in the supernatants
taken from a/aBEs (Fig. 6, lane 2-4), compared to na/naBEs
(Fig. 6, lane 1).
Interestingly, the sample that contained the lowest
amounts of gelatinase A (72 kDa), associated with fibroblast
secretion products, corresponded to the culture supernatant
resolved in lane 3 (Fig. 6), collected from the a/aBE. This
BE showed the thicker mesenchymal layer on histological
sections (Fig. 1F), compared to the other BEs cultured for
6 days at the air-liquid interface. This sample (Fig. 6, lane
3), was compared to the supernatant of another a/aBE, pro-
duced with the same cells, but cultured in presence of me-
dium conditioned by non-asthmatic HBFC (Fig. 6, lane 4).
The CM-stimulated-a/aBE (Fig. 6, lane 4), contained more
of the precursor form of the gelatinase A (72 kDa). It also
contained less of the active 62.5 kDa gelatinase than the
other BEs (Fig. 6, lanes 1-3). However, this a/aBE showed
a slightly thinner mesenchymal layer on histological analy-
ses than the na/naBEs (Fig. 1A). Thus, it is difficult to es-
tablish a correlation between the amounts of gelatinases
secreted in the culture supernatants of the various BEs and
the rate of degradation of their mesenchymal layers in cul-
ture. Other types of proteases may be involved in this proc-
ess.
Figure 6. Zymograms (top) and corresponding SDS-PAGE (bottom) showing the relative gelatinase activities analyzed
from supernatants of naBE (lane 1), of two different a/aBEs (lanes 2 and 3) and of an a/aBE cultured in the medium
conditioned by HBFC (lane 4). The arrowheads indicate the gelatinase A (MMP-2, 72 kDa). The arrows indicate the
gelatinase B (MMP-9, 92Kda) and the active form of gelatinase A (62.5 kDa).
24
J. S. Paquette et al.

Discussion
The production of bioengineered BEs with non-asthmatic
and asthmatic cells allowed the comparative analyses of
severa1 histologic and functional parameters in vitro. Our
results suggest that HBEC isolated from asthmatic sub-
jects can grow on a mesenchymal layer seeded with non-
asthmatic and asthmatic HBFC. However, if they grow bet-
ter on a non-asthmatic than on an asthmatic type of mesen-
chymal layer, they all started to detach from their mesen-
chymal support one to two days after reaching confluence.
Such process continued thereafter, under submerged cul-
ture conditions and subsequently, at the air-liquid interface.
In contrast, the non-asthmatic HBEC never detached from
any type of mesenchymal layer tested. When asthmatic
HBEC are grown in monolayers on plastic, they can be
maintained in culture for at least 3-4 passages without de-
taching from the culture dishes, in a medium supplemented
with serum. When grown in BEs, their detachment from
the various mesenchymal layers, correlates with the for-
mation of holes and the deposition of cell debris at the in-
terface epithelium-mesenchyme. These data suggest that
asthmatic HBEC fail to maintain stable points of anchor-
age with the matrix support, probably due to an absence or
a loss of membranous anchorage proteins or receptors. In
absence of serum that contains several growth factors and
fibronectin, such defect may be observed earlier. We may
believe that the behavior of the HBEC isolated from asth-
matic subjects seeded on BEs is comparable to the obser-
vations reported in asthmatic tissues in vivo when desqua-
mation occurs (Jeffery et al., 1989).
Ciliogenesis was observed, at least partially, in all
groups of BEs. However, several asthmatic HBEC did not
show cilia as numerous and/or as long as the non-asthmatic
HBEC. These data suggest that the asthmatic HBEC do
not reach the same level of differentiation within a given
population isolated from the same tissues. Such observa-
tions were also reported from histologic analyses of severe
asthmatic bronchi, whereas several non-ciliated cells were
detected at the surface of their epithelium (Jeffery et al.,
1989). Again, it was not surprising to observe this phe-
nomena in our BE in vitro.
The non-asthmatic HBEC grew more slowly within a
na/aBE and the histologic organization of the na/aBEs was
less compact than in the na/naBEs. Inversely, the asthmatic
HBEC grew faster on a mesenchymal layer containing non-
asthmatic HBFC than in a aBE. These data strongly sug-
gest that the HBEC respond to some factors secreted by the
HBFC up to a certain degree, and may as well release other
factors to the mesenchymal cells. It was previously demon-
strated in vitro that epithelial cells are an important regula-
tor of airway remodeling by means of paracrine control of
bronchial fibroblasts (Zhang et al., 1999). Other evidence
supporting the existence of epithelium-mesenchyme inter-
actions in the BEs is the positive effect of the CM col-
lected from non-asthmatic HBFC on the growth of non-
asthmatic HBEC on a mesenchymal layer seeded with asth-
matic HBFC. It seems that the CM contains cytokines that
could compensate a lack of factors secreted by the asth-
matic HBFC in the na/aBE.
The comparative analyses of the active gelatinases se-
creted in the BEs culture supernatants showed some dif-
ferences, but the 92 kDa and the 62.5 kDa gelatinases were
detected in all BE supernatants. The supernatant of the a/
aBE showing the thicker mesenchymal layer may be ex-
plained by a lower secretion of gelatinase A (72 kDa) or by
the synthesis of other collagen types, such as Type V
(Hoshino et al., 1998). The apparent sub-epithelial fibro-
sis associated with asthma is postulated to be caused by
fibroblasts. These cells are involved in collagen synthesis
and remodelling and their functional status may be modu-
lated by cytokines secreted by inflammatory or epithelial
cells (Brewster et al., 1990; Roche, 1991; Gauldie et al.,
1992). Thus, several hypotheses could be considered to
explain our results. The zymograms on gelatin, a substrate
rich in collagen Type I, do not reveal several other
collagenases and proteases that may be secreted in differ-
ent amounts by asthmatic and non-asthmatic cells present
in our different BEs. It would be interesting to assess the
effects of specific inhibitors of MMP-9 and MMP-2 on the
maintenance of the mesenchymal layers of the different
groups of BEs.
Taken together, we conclude that the tissue-engineered
human BE is a good model to investigate cellular mecha-
nisms involved in bronchial disorders like asthma. This
approach allows the analyses of several parameters such as
the bronchial cell growth and differentiation, their organi-
zation and their secretion products in vitro. Comparative
toxicological and pharmacological studies of various hu-
man bronchial cell populations can be achieved in vitro,
using BE as a precious alternative to animal use. Eventu-
ally, other cell types, such as immune or smooth muscle
cells could be added to the model to investigate more com-
plex interactions.
Acknowledgements
The authors are grateful to Dr Hilda López Valle, M.D.,
pathologist, for her critical analyses of the histological and
ultrastructural properties of the bronchial equivalents, Mr.
Aristide Pusterla for his contribution in electron microscopy
analyses, Mr. Claude Marin for photographic assistance,
Mrs Nathalie Tremblay for her technical assistance on this
project. Mr. Jean-Sébastien Paquette was recipient from the
NSERC fellowship and a honorary fellowship from Laval
University. Drs F. Goulet, F.A. Auger, and L.-P. Boulet were
recipients of Scolarships from the Fonds de la Recherche
en Santé du Québec.
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