Departments of 1 Clinical
Genetics and 3 Otolaryngology, In the present study, we describe a novel
three-dimensional airway epithelial explant preparation and demonstrate
its use for ion transport studies by electrophysiological technique.
Suspension cultures of sheets of epithelial cells released by protease
treatment from cystic fibrosis (CF) and non-CF nasal polyps developed
free-floating, monolayered epithelial spheres, with the apical,
ciliated cell membrane facing the bath and the basolateral cell
membrane pointing toward a fluid-filled lumen. Microelectrode
impalement of both non-CF and CF spheroids revealed lumen-positive
transepithelial electrical potential differences (PDs) that were
inhibited by amiloride, indicating that the spheroids were inflated due
to amiloride-sensitive Na+
absorption followed by water. Transformation to a
Cl
amiloride; adenosine 5'-triphosphate; adenosine
3',5'-cyclic monophosphate; glucocorticoids; cystic
fibrosis
CONVENTIONAL TWO-DIMENSIONAL cell cultures, grown on
glass, plastic, or permeable supports, have contributed significantly to the understanding of airway epithelial transport functions, including the abnormal ion transport properties of cells derived from
patients with cystic fibrosis (CF) (6, 14, 27). The nature of cellular
support, however, has been demonstrated to be crucial for the
qualitative and quantitative properties of epithelial cells in culture,
resulting in diverging expression of the original phenotype of cells in
vitro (6, 18, 19). We therefore set out to improve an
observed natural disposition of dispersed airway epithelial sheets to
develop matrix-independent, fluid-filled epithelial spheroids and to
test whether this preparation can be used as an experimental model for
studies of transport mechanisms in the upper airway epithelium. The
free-floating three-dimensional cell culture concept has been practiced
for some years, mainly with the use of solid spheres of cells such as
liver, heart, neuronal, and various tumor cells, on which a variety of
biochemical studies (7, 16, 22) have been performed. However,
cyst-forming epithelial cultures have also been described in thyroid,
endometrial, epididymal, and transformed renal cells (15). Except for
one study (23) in 1984, in which steady-state trans- and intracellular potential differences (PDs) of thyroid spheroids were measured, electrophysiological studies have not been performed in these naturally
produced experimental "chambers." This also applies to studies
(2, 9) on epithelial spheroids derived from human airway epithelia in
which ciliary activity and morphological properties have been
described. The present study is the first demonstration of the
electrophysiological experimental potential of free-floating airway
epithelial spheroids.
Cellular material. Nasal polyps were
resected from 17 normal subjects (7 women and 10 men) and 9 CF patients
(4 men and 5 women), of which 8 were Scanning electron microscopy. The
epithelial spheroids were transferred to 2% glutaraldehyde in 0.05 M
cacodylate buffer, pH 7.4. After fixation for 24 h at 5°C and a
short rinse in 0.15 M cacodylate buffer (pH 7.4), the specimens were
postfixed in 1%
OsO4 in 0.12 M
cacodylate buffer (pH 7.4) for 1 h, rinsed in distilled water, and
prepared for scanning electron microscopy examination by the
osmium-thiocarbohydrazide method, consisting of a
thorough 1-h rinse in distilled water, a 30-min treatment with a
saturated and filtered solution of thiocarbohydrazide in distilled
water, and a second continuous rinse in distilled water, and then the
specimens were returned to 1%
OsO4 for 30 min. The latter four
steps were repeated, and after a final rinse in distilled water, the
specimens were gradually dehydrated to 100% ethanol within 24 h,
transferred to 100% ethanol for 1 h, and critical point
dried (Balzers CPD 030, Belzer Union,
Liechtenstein) with CO2. Specimens were mounted on
stubs with adhesive carbon tabs and sputter coated with chromium (XE200
Xenosput, Edwards High Vacuum, Crawley, UK). Examination and
photography were carried out in a Philips FEG 30 scanning electron
microscope operated at 0.5-5 kV.
Transmission electron microscopy.
After fixation and postfixation as described in
Scanning electron
microscopy, the specimens were centrifuged
to form a pellet and embedded in agar. The samples were dehydrated in a
graded series of ethanols, transferred to propylene oxide, and embedded
in Epon. Sections were cut with a Leica Ultracut UCT
microtome, collected on one-hole copper grids with Formvar-supporting
membranes, stained with uranyl acetate and lead citrate, and examined
and photographed in a Philips EM 208 transmission electron microscope
operated at an accelerating voltage of 80 kV.
Electrophysiology. The transepithelial
PD was measured in epithelial spheroids transferred to 700 µl of
HEPES-buffered Ringer solution [containing (in mM) 140 Na+, 5 K+, 1.2 Ca2+, 1.2 Mg2+, 131.2 Cl Cell culture media were obtained from ICN Biochemicals (Costa Mesa,
CA). Diphenylamine-2-carboxylate (2-DPC) was a gift from Dr. R. Greger
(Albert-Ludwigs-University, Freiburg, Germany). All other chemicals
were purchased from Sigma.
Sheets of epithelial cells from CF and non-CF nasal polyps released by
protease treatment and cultured in a defined, serum-free medium formed
fluid-filled spheroids with diameters of 50-800 µm. From each
resected polyp (pea size), >100 spheroids were formed, ready to be
used for electrophysiological experiments within only a couple of days
after resection. It should be pointed out that the spheroids represent
explants rather than the result of a primary culture procedure.
Accordingly, no mitotic figures were observed in the epithelial cells
at any time.
The yield of cells in spheroids by the present method seems to be
rather high. Thus, for example, 100 spheroids with a
diameter of 300 µm represent an epithelial area of ~0.3
cm2, which corresponds to a
significant fraction of the surface epithelium of a nasal polyp
(1-1.5 cm2). Furthermore,
the number of experimental preparations resulting from a single polyp
is higher (>100) with the present method than with the traditional
primary culturing of cells in flat sheets. However, the number of cells
(e.g., for biochemical studies) obtained in spheroids is
of course smaller than after upformation of primary cultures.
The epithelial morphology of the spheroids, which was stable for
several weeks of continuous culture, resembled that of the native
tissue and collagen-supported cell culture (6, 10, 13). Thus all
spheroids consisted of a fully differentiated monolayered epithelium,
with the apical cell membrane containing stubby microvilli facing the
bath and the basolateral membranes pointing toward the central, clear,
fluid-filled lumen (Figs. 1 and
2). We were unable to distinguish
between CF spheroid (CFS) and non-CFS (NCFS)
morphology. A large part of the cells carried cilia
located exclusively on the apical side (Fig. 1), and ciliary activity
and associated spheroid movements were present during the entire
observation period.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
secretory state was
achieved by addition of ATP to the bath, leading to the development of
a diphenylamine-2-carboxylate-sensitive PD. A cAMP-induced increase in
PD was seen in non-CF spheroids only. In response to hydrocortisone
treatment, Na+ transport reflected
by amiloride-sensitive PD increased and more so in CF than in non-CF
spheres. We concluded that this preparation is a useful model for the
airway surface epithelium and is suitable for studies of transport
mechanisms and regulation.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
F508 homozygous
and 1 had the
F508/2790-1G-C combination. The
polyps were placed in a 10-ml test tube with ~8 ml of Dulbecco's
modified Eagle's medium (room temperature, pH 7.30-7.40)
containing 0.1% protease type XIV (Sigma, St. Louis, MO),
105 U/l of penicillin, 100 mg/l of
streptomycin, and 50 mg/l of gentamicin. Sheets of epithelial cells
were dislodged from the polyps by intermittent shaking. After 1-2
h, fetal bovine serum (10% vol/vol; GIBCO, Grand Island, NY) was added
to neutralize the protease. The solid parts of the polyps were isolated
and discarded, and the remaining epithelial suspension was washed twice
(5 min at 110 g) in Ham's F-12
culture medium containing 1% Ultroser-G serum substitute (IBF Biotechnics, Savage, MD) and antibiotics as above. The pellet was
resuspended in 5 ml of culture medium in a 50-ml tissue culture flask
and incubated at 37°C with 5%
CO2. The medium was changed every
3-4 days after gentle centrifugation (20 g for 2 min). From the third day on,
gentamicin was omitted from the medium. In a series of experiments,
hydrocortisone (HC; 5 × 10
6 M) was added 1-2
days before electrophysiological experiments.
, 1.6 HPO2
4, 0.4 H2PO
4, 10 glucose, and 10 HEPES, titrated to pH 7.35] at 37°C in a
thermostat-controlled chamber placed on the stage of an inverted
microscope (Leitz). Spheroids were kept in position
by applying gentle suction with a Ringer-filled holding pipette (Swemed
Lab, Billdal, Sweden) with an internal tip diameter of ~25 µm.
Spheroids were impaled by microelectrodes pulled from filamented
borosilicate glass tubes (OD 1 mm; Clark Electromedical, Reading, UK)
on a horizontal puller (P-87, Sutter Instruments,
Novato, CA) and backfilled with 0.3 M KCl (tip resistance 70-100
M
). The holding pipette and microelectrode were operated by
micromanipulators, and spheroid microelectrode impalement was provided
by a piezo translator (MPM-10, World Precision Instruments, Sarasota, FL). The microelectrode was
connected to a high-impedance electrometer (Duo 773, World Precision
Instruments), and the bath was grounded via a Ringer-agar bridge and an
Ag-AgCl electrode. Thus recorded transepithelial PDs
express spheroid lumen (serosal side) with respect to bath (mucosal
side). Data are presented as means ± SE. During microelectrode
impalements, the microscopic appearance of the spheroids was stored on
video and simultaneously displayed on a monitor.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
View larger version (141K):
[in a new window]
Fig. 1.
A: scanning electron
microscopy of an ~250-µm epithelial spheroid after 4 wk in culture
medium. B and
C: considerable number of epithelial
cells are covered with cilia (arrows), and remainder of cell membrane
is equipped with stubby microvilli (arrowhead). Magnifications and bar
lengths: ×750, 100 µm (A);
×9,000, 10 µm (B); and
×22,500, 5 µm (C).
View larger version (158K):
[in a new window]
Fig. 2.
Transmission electron microscopy survey of epithelial
cells. A: outer (apical) cell membrane
(O) faces culture medium (M) and is equipped with microvilli (solid
arrowheads). Inner (basolateral) cell membrane (I) faces fluid-filled
lumen (L); no basal membrane was observed. Epithelial cells are joined
with zonula occludens junctions toward
the culture medium (box), and lateral cell membranes are closely
opposed (arrows). Each epithelial cell contains a nucleus (N), numerous
mitochondria (Mi), endoplasmic reticulum studded with ribosomes (open
arrowheads), Golgi complexes (G), and lipid inclusions (Li).
Magnification, ×17,000; bar, 1 µm.
B: high magnification (×144,500)
of framed area in A displaying the
terminal bar region of 2 epithelial cells consisting of a zonula
occludens with 3 sealing strands (arrowheads). Bar, 0.1 µm.
The average size of individual spheroids appeared to be almost constant until final collapse. No differences in size were observed between the CFSs and NCFSs. The diameter of six isolated NCFSs, measured daily from the age of 2 wk until collapse 2-4 wk later, fluctuated 23-41% around the average diameter (225 µm). Such volume fluctuations might reflect the inflation of the spheroids due to an inward fluid transport, interrupted by periods of shrinkage caused by stretch-induced opening of the tight junctions (17). The presence of an active inward solute and water transport, resulting in an increased intraluminal pressure, was supported by an observed shrinkage after deliberate puncture of the spheroids, in which the diameter was reduced to ~75% of the original value. Most likely, this reflects an active inward transport of Na+ via apical membrane Na+ channels and basolateral membrane Na+-K+ pumps (25), giving rise to NaCl accumulation in the closed inner compartment followed by an inward flux of water due to osmotic coupling.
To test this hypothesis, we measured the transepithelial PD by
introducing microelectrodes into the lumen of the spheroids (Fig.
3). By this procedure, we found positive
intraluminal (basolateral) PDs ranging from 2.2 to 14.5 mV, with no
significant difference between CFSs and NCFSs. When 100 µM amiloride
was added to the bath (apical side), the PD of CFSs was abolished (from
6.1 ± 0.4 to 0.0 ± 0.1 mV; n = 29 spheroids), whereas the PD of NCFSs was reduced by
~60% (from 6.9 ± 0.5 to 2.5 ± 0.3 mV;
n = 35 spheroids). These results
confirm the notion that amiloride-sensitive
Na+ absorption is the main
determinant of airway epithelial PD (25). The difference in responses
to amiloride between NCFSs and CFSs is also well known (12). It
probably reflects that amiloride hyperpolarizes the apical cell
membrane, which in NCFSs induces a small
Cl secretion, whereas this
possibility is absent in CFSs because of the missing expression of the
CF transmembrane conductance regulator (CFTR) in the apical cell
membrane. The finding that control PD values were equal in NCFSs and
CFSs contrasts with a previous study (10) where the PD of CF airway
epithelia was higher than that of non-CF airway epithelia. In a few
studies (24, 28, 29), however, identical non-CF and CF PD values have
been reported. The explanation for these discrepancies is not clear but
may involve differences in epithelial resistance in response to
different experimental conditions or differences in hormonal
stimulation of transport mechanisms (see below). A detailed
quantitative analysis of transport properties in epithelia includes
measurements of epithelial resistance (to calculate equivalent short-circuit current) and cellular PDs with conventional and ion-selective microelectrodes (to calculate ionic driving forces). Such
measurements are under current development in the present spheroid
preparation. However, because natural upper airway epithelia are
low-resistance epithelia (20), it is unlikely that the presently observed drug-induced changes in PD can be explained merely by changes
in paracellular resistance; furthermore, the PD changes resemble those
observed in other preparations.
|
During formation of the spheroids in Ham's F-12 culture medium, this
medium is trapped in the interior (basolateral compartment). Regarding
ions of major importance (Na+,
Cl, and
K+), this medium has a
composition close to that of the Ringer solution used as the external
(apical) medium in the present study. Thus significant contributions of
diffusion potentials to the transepithelial PD resulting from
asymmetric ion concentrations are unlikely. This is further supported
by the observation that the transepithelial PD is zero when active
Na+ absorption is blocked by
amiloride and Cl
secretion
is absent (CFSs).
After the addition of amiloride, the residual PD of NCFSs was sensitive
to the addition of the
Cl-channel blocker 2-DPC
(250 µM) (5a), which reduced the residual PD by 60.0 ± 8.4%. In
contrast, the addition of 2-DPC to NCFSs before amiloride had no effect
on PD. This is in accordance with the hypothesis that 2-DPC-sensitive,
unstimulated apical membrane Cl
outflux in non-CF airway
epithelium is triggered by membrane hyperpolarization caused by
amiloride (26). The pathway for this 2-DPC-sensitive
Cl
outflux is obviously
related to CFTR because no effect of 2-DPC on PD (before or after
amiloride) was observed in the CFS. The Cl
-channel blocker DIDS
(400 µM) proved to be without any significant effect on PD before or
after amiloride addition in both CFSs and NCFSs.
In a series of experiments, amiloride-treated spheroids were exposed to
200 µM ATP to investigate the possible presence of an ATP-sensitive
Cl channel in the apical
cell membrane (4, 11). An abrupt rise in the basolateral side positive
PD followed by a decrease to a still increased steady level was
observed in both CFSs and NCFSs in response to ATP addition to the bath
(Fig. 4). However, in contrast to previous
reports (4, 11), the change in PD was not larger in CFSs than in NCFSs
(Fig. 4). The sustained level was insensitive to DIDS but
sensitive to 2-DPC in both NCFSs [change in PD (
PD) =
4.3 ± 0.3 mV; n = 11)] and CFSs (
PD =
0.8 ± 0.2 mV,
n = 14). Pretreatment with 2-DPC, but
not with DIDS, prevented a subsequent ATP response. In six experiments,
a brief negative deflection in PD preceded the usual initial positive
spike, indicating a short-lasting ATP-dependent activation of
conductances other than that carrying
Cl
, such as
K+ (5).
|
During individual micropuncture experiments lasting 20-30 min, we
were unable to detect fluid transport-dependent volume changes of the
spheroids from the video frames, primarily because the microscope
focusing was not continuously at the plane of the spheroid perimeter.
Furthermore, it was difficult to detect spheroid volume changes from
short-lasting diameter measurements if unstimulated fluid absorption in
this preparation was as low as in primary cultured human nasal
epithelium (8, 21) or in human bronchial xenografts (29), i.e.,
0.1-4
µl · cm2 · h
1.
Finally, the experimental protocol did not allow observations of
unperturbed spheroids for >10 min before inhibition of absorption by
amiloride and stimulation of secretion.
The dominating amiloride-sensitive
Na+ absorption in airway epithelia
is subject to long-term regulation, probably by steroid hormones (3).
To test this possibility, we measured the PD of NCFSs and CFSs after
addition of HC to the incubation medium 1-2 days before
electrophysiological measurements. The presence of HC resulted in
significantly larger basolateral side positive PDs than in the absence
of hormone (see above) and more so in CFSs [18.0 ± 2.2 mV (n = 19)
vs. 6.1 ± 0.4 mV
(n = 29)] than in NCFSs
[11.4 ± 0.8 mV (n = 26) vs. 6.9 ± 0.5 mV
(n = 35)]. These larger control
PD values doubtlessly represent increased levels of amiloride-sensitive
Na+ absorption because apical
amiloride (104 M) decreased
PD to the same level as in the absence of HC (see above). Thus, in the presence of HC, amiloride
decreased the PD in NCFSs from 11.4 ± 0.8 to 2.2 ± 0.3 mV
(n = 26) and in CFSs from 18.0 ± 2.2 to 0.0 ± 0.1 mV (n = 19).
Furthermore, the data suggest that the previously reported
upregulation of amiloride-sensitive Na+ channels in CF airway
epithelial cells lacking CFTR may be influenced by hormonal
stimulation, in this case by HC (3).
cAMP has been demonstrated to activate an apical
Cl conductance in
epithelial cells expressing CFTR (1). In normal airway epithelia, this
may result in cAMP-stimulated
Cl
secretion, whereas a
similar stimulation in CF epithelia is not possible because of the lack
of CFTR. In accordance with this notion, we observed a small but
significant increase in the PD of HC-stimulated and amiloride-treated
NCFSs (from 2.2 ± 0.3 to 3.1 ± 0.4 mV;
P < 0.001;
n = 26) 2-3 min after addition of
0.25 mM dibutryl cAMP and 0.25 mM IBMX to the apical
bath, whereas the PD of CFSs did not respond to this treatment (from
0.0 ± 0.1 to 0.1 ± 0.1 mV; not significant;
n = 19). The rather small increase in
PD of NCFSs may be related to the fact that the driving force for
Cl
across the apical cell
membrane is small even in the presence of amiloride (26). Subsequent
apical application of 200 µM ATP stimulated the basolateral side
positive PD slightly more in CFSs than in NCFSs
(P = 0.13). Thus, in CFSs, the maximal
PD increased from 0.1 ± 0.1 to 9.4 ± 1.6 mV (
PD = 9.3 ± 1.6 mV; P < 0.001; n = 19) and in NCFSs from 2.9 ± 0.4 to 8.7 ± 1.1 mV (
PD = 5.8 ± 1.0 mV;
P < 0.001;
n = 19).
This first study of functional characteristics of spheroid-shaped airway epithelial explants demonstrates that it is possible, from small amounts of primary material, to rapidly make large numbers of differentiated ready-to-use preparations of non-CF and CF airway epithelia with electrophysiological properties resembling those of the native tissue. The spheroid preparation may be suitable for application of several additional types of experimental techniques. The relative inaccessibility of the enclosed basolateral compartment may be a drawback. However, this medium may be accessible for applications or sampling of, e.g., agonists, tracers, and metabolites by use of high-pressure vacuum micropipettes and iontophoresis or by centrifugation of the spheroids out of suspension. Furthermore, the structural arrangement of the epithelium without underlying distracting supports may represent a unique possibility to reach the serosal cell membrane for impalements or patch clamping after opening of the spheroid. Thus this preparation is an experimental model useful for investigations of regulatory mechanisms of airway epithelial transport functions.
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ACKNOWLEDGEMENTS |
---|
We thank N. J. Brandt, J. Rostgaard, R. Sinding, and N. Rasmussen for help and encouragement during this study.
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FOOTNOTES |
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This work was supported by grants from the Danish Research Council of Health Sciences, the Novo-Nordisk Foundation, the Danish Heart Association, and the Velux Foundation.
Address for reprint requests: P. S. Pedersen, Dept. of Clinical Genetics (4061), Rigshospitalet, Blegdamsvej 9, DK 2100 Copenhagen, Denmark.
Received 23 September 1997; accepted in final form 18 September 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, M. P.,
D. P. Rich,
R. J. Gregory,
A. E. Smith,
and
M. J. Welsh.
Generation of cAMP-activated chloride currents by expression of CFTR.
Science
251:
679-682,
1991[Medline].
2.
Bridges, M. A.,
D. C. Walker,
R. A. Harris,
B. R. Wilson,
and
A. G. F. Davidson.
Cultured human nasal epithelial multicellular spheroids: polar cyst-like model tissues.
Biochem. Cell Biol.
69:
102-108,
1991[Medline].
3.
Champigny, G.,
N. Voilley,
E. Lingueglia,
V. Friend,
P. Barbry,
and
M. Lazdunski.
Regulation of expression of the lung amiloride-sensitive Na+ channel by steroid hormones.
EMBO J.
13:
2177-2181,
1994[Abstract].
4.
Clarke, L. L.,
and
R. C. Boucher.
Chloride secretory response to extracellular ATP in human normal and cystic fibrosis nasal epithelia.
Am. J. Physiol.
263 (Cell Physiol. 32):
C348-C356,
1992
5.
Clarke, L. L.,
T. Chinet,
and
R. C. Boucher.
Extracellular ATP stimulates K+ secretion across cultured human airway epithelium.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L1084-L1091,
1997
5a.
Di Stefano, A., M. Wittner, E. Schlatter, H. J. Lang,
H. Englert, and R. Greger. Diphenylamine-2-carboxylate, a blocker
of the Cl-conductive
pathway in Cl
-transporting
epithelia. Pflügers Arch. 405, Suppl. 1, 95-100, 1985.
6.
Gruenert, D. C.,
W. E. Finkbeiner,
and
J. H. Widdicombe.
Culture and transformation of human airway epithelial cells.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L347-L360,
1995
7.
Hoffman, R. M.
To do tissue culture in two or three dimensions? That is the question.
Stem Cells
11:
105-111,
1993[Abstract].
8.
Jiang, C.,
W. E. Finkbeiner,
J. H. Widdicombe,
P. B. McCray,
and
S. S. Miller.
Altered fluid transport across airway epithelium in cystic fibrosis.
Science
262:
424-427,
1993[Medline].
9.
Jorissen, M.,
and
A. Bessems.
Normal ciliary beat frequency after ciliogenesis in nasal epithelial cells cultured sequentially as monolayer and in suspension.
Acta Otolaryngol. (Stockh.)
115:
66-70,
1995[Medline].
10.
Knowles, M. R.,
J. L. Carson,
A. M. Collier,
J. T. Gatzy,
and
R. C. Boucher.
Measurements of nasal transepithelial electric potential differences in normal human subjects in vivo.
Am. Rev. Respir. Dis.
124:
484-490,
1981[Medline].
11.
Knowles, M. R.,
L. L. Clarke,
and
R. C. Boucher.
Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis.
N. Engl. J. Med.
325:
533-538,
1991[Abstract].
12.
Knowles, M.,
J. Gatzy,
and
R. Boucher.
Relative ion permeability of normal and cystic fibrosis nasal epithelium.
J. Clin. Invest.
71:
1410-1417,
1983[Medline].
13.
Larsen, P. L.,
and
M. Tos.
Nasal polyps: epithelium and goblet cell density.
Laryngoscope
99:
1274-1280,
1989[Medline].
14.
Lechner, L. F.,
A. Haugen,
H. Autrup,
I. A. McClendon,
B. F. Trump,
and
C. C. Harris.
Clonal growth of epithelial cells from normal adult human bronchus.
Cancer Res.
41:
2294-2304,
1981[Abstract].
15.
McAteer, J. A.,
G. S. Dougherty,
K. D. Gardner, Jr.,
and
A. P. Evan.
Polarized epithelial cysts in vitro: a review of cell and explant culture systems that exhibit epithelial cyst formation.
Scanning Microsc.
2:
1739-1763,
1988[Medline].
16.
Mueller-Klieser, W.
Multicellular spheroids.
J. Cancer Res. Clin. Oncol.
113:
101-122,
1987[Medline].
17.
Nitsch, L.,
and
S. H. Wollman.
Sudden volume changes of the lumen of inverted thyroid follicles in suspension cultures.
Exp. Cell Res.
162:
278-283,
1986[Medline].
18.
Opas, M.
Expression of the differentiated phenotype by epithelial cells in vitro is regulated by both biochemistry and mechanics of the substratum.
Dev. Biol.
131:
281-293,
1989[Medline].
19.
Robinson, C. B.,
and
R. Wu.
Mucin synthesis and secretion by cultured tracheal cells: effects of collagen gel substratum thickness.
In Vitro Cell. Dev. Biol. Anim.
29A:
469-477,
1993.
20.
Röpke, M.,
S. Carstens,
M. Holm,
and
O. Frederiksen.
Ion transport mechanisms in native rabbit nasal airway epithelium.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L637-L645,
1996
21.
Smith, J. J.,
P. H. Karp,
and
M. J. Welsh.
Defective fluid transport by cystic fibrosis airway epithelia.
J. Clin. Invest.
93:
1307-1311,
1994[Medline].
22.
Sutherland, R. M.
Cell and environment interactions in tumor microregions: the multicell spheroid model.
Science
240:
177-184,
1988[Medline].
23.
Takasu, N.,
Y. Handa,
Y. Shimizu,
and
T. Yamada.
Electrophysiological and morphological cell polarity and iodine metabolism in cultured porcine and human thyroid cells.
J. Endocrinol.
101:
189-197,
1984[Abstract].
24.
Verbeek, E.,
H. R. de Jonge,
J. Bijman,
J. Keulemans,
M. Sinaasappel,
A. W. M. van der Kamp,
and
B. J. Scholte.
Chloride transport in cultured nasal epithelium of cystic fibrosis patients.
Pflügers Arch.
415:
540-546,
1990[Medline].
25.
Welsh, M. J.
Electrolyte transport by airway epithelia.
Physiol. Rev.
67:
1143-1184,
1987
26.
Willumsen, N. J.,
C. W. Davis,
and
R. C. Boucher.
Intracellular Cl activity and cellular Cl
pathways in cultured human airway epithelium.
Am. J. Physiol.
256 (Cell Physiol. 25):
C1033-C1044,
1989
27.
Yankaskas, J. R.,
C. U. Cotton,
M. R. Knowles,
T. Gatzy,
and
R. C. Boucher.
Culture of human nasal epithelial cells on collagen matrix support.
Am. Rev. Respir. Dis.
132:
1281-1287,
1985[Medline].
28.
Yankaskas, J. R.,
J. T. Gatzy,
M. R. Knowles,
and
R. C. Boucher.
Persistance of abnormal chloride ion permeability in cystic fibrosis nasal epithelial cells in heterologous culture.
Lancet
27:
954-956,
1985.
29.
Zhang, Y.,
J. Yankaskas,
J. Wilson,
and
J. F. Engelhardt.
In vivo analysis of fluid transport in cystic fibrosis airway epithelia of bronchial xenografts.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1326-C1335,
1996