Murine nasal septa for respiratory epithelial air-liquid interface cultures.
ABSTRACT Air-liquid interface models using murine tracheal respiratory epithelium have revolutionized the in vitro study of pulmonary diseases. This model is often impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. We describe a simple technique to harvest the murine nasal septum and grow the epithelial cells in an air-liquid interface. The degree of ciliation of mouse trachea, nasal septum, and their respective cultured epithelium at an air-liquid interface were compared by scanning electron microscopy (SEM). Immunocytochemistry for type IV beta-tubulin and zona occludens-1 (Zo-1) are performed to determine differentiation and confluence, respectively. To rule out contamination with olfactory epithelium (OE), immunocytochemistry for olfactory marker protein (OMP) was performed. Transepithelial resistance and potential measurements were determined using a modified vertical Ussing chamber SEM reveals approximately 90% ciliated respiratory epithelium in the nasal septum as compared with 35% in the mouse trachea. The septal air-liquid interface culture demonstrates comparable ciliated respiratory epithelium to the nasal septum. Immunocytochemistry demonstrates an intact monolayer and diffuse differentiated ciliated epithelium. These cultures exhibit a transepithelial resistance and potential confirming a confluent monolayer with electrically active airway epitheliumn containing both a sodium-absorptive pathway and a chloride-secretory pathway. To increase the yield of respiratory epithelial cells harvested from mice, we have found the nasal septum is a superior source when compared with the trachea. The nasal septum increases the yield of respiratory epithelial cells up to 8-fold.
- SourceAvailable from: Akiva Cohen[Show abstract] [Hide abstract]
ABSTRACT: Traumatic brain injury (TBI) afflicts up to two million people annually in the United States and is the primary cause of death and disability in young adults and children. Previous TBI studies have focused predominantly on the morphological, biochemical and functional alterations of gray matter structures, such as the hippocampus. However, little attention has been given to the brain ventricular system, despite the fact that altered ventricular function is known to occur in brain pathologies. In the present study, we investigate anatomical and functional alterations to mouse ventricular cilia that result from mild traumatic brain injury. We demonstrate that TBI causes a dramatic decrease in cilia. Furthermore, using a particle tracking technique, we demonstrate that CSF flow is diminished, thus potentially negatively affecting waste and nutrient exchange. Interestingly, injury-induced ventricular system pathology resolves completely by thirty days after injury as ependymal cell ciliogenesis restores cilia density to uninjured levels in the affected lateral ventricle.Journal of neurotrauma 04/2014; · 4.25 Impact Factor
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ABSTRACT: Primary human airway epithelial cells cultured in an air-liquid interface (ALI) develop a well-differentiated epithelium. However, neither characterization of mucociliar differentiation overtime nor the inflammatory function of reconstituted nasal polyp (NP) epithelia have been described.PLoS ONE 06/2014; 9(6):e100537. · 3.53 Impact Factor
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ABSTRACT: We have previously demonstrated that Sinupret, an established treatment prescribed widely in Europe for respiratory ailments including rhinosinusitis, promotes transepithelial chloride (Cl-) secretion in vitro and in vivo. The present study was designed to evaluate other indicators of mucociliary clearance (MCC) including ciliary beat frequency (CBF) and airway surface liquid (ASL) depth, but also investigate the mechanisms that underlie activity of this bioflavonoid.PLoS ONE 08/2014; 9(8):e104090. · 3.53 Impact Factor
Vol. 43 ı No. 2 ı 2007 www.biotechniques.com ı BioTechniques ı 195
Short Technical Reports
The development of primary culture
models of transgenic mouse tracheal
epithelial cells has greatly facili-
tated the study of human respiratory
diseases. These primary culture models
are confluent, fully differentiated,
ciliated respiratory epithelium at an
air-liquid interface on a semipermeable
membrane that mimic many charac-
teristics of murine tracheal epithelial
cells in vivo. Prior to the development
of murine models, suboptimal methods
involving non-polarized and sometimes
poorly differentiated primary cultures
and immortalized cell lines were used
for respiratory epithelial cell research.
These models made logistical sense
because they often supplied copious
numbers of cells through expansion and
passage of the cells. While the murine
air-liquid interface models are ideal for
the development of differentiated respi-
ratory epithelial cells, expansion and
passage of these cells will ultimately
decrease the ability for these cells to
differentiate (1). Therefore, one of the
primary limitations of in vitro murine
models includes the large number of
mice required to obtain a significant
number of tracheal epithelial cells.
Typically, dissociation of respiratory
epithelium from two mouse tracheas
is required for the development of an
air-liquid interface on one transwell
membrane (6.5-mm diameter). This can
be cost-prohibitive when attempting
to develop in vitro airway models of
transgenic mice. An additional source
of respiratory epithelium will increase
the utility of these mice and, at the same
time, decrease expenses.
Our recent investigations have found
that the nasal septum is a superb source
of murine respiratory epithelium. Most
research involving murine nasal septa
has primarily focused on olfaction, so
the potential for respiratory cell culture
has largely been ignored. The two sides
of the murine nasal septum are covered
with mucosa, and the overall surface
area is much larger in comparison
to the mouse trachea. The olfactory
system of the mouse has four anatomi-
cally distinct chemosensory areas on
the nasal septum. (Figure 1) The main
olfactory epithelium (OE), the septal
organ of Masera (SO), and the vomero-
nasal organ (VNO) have bipolar sensory
neurons that reside in a pseudostratified
neuroepithelium. The MOE and SO are
part of the main olfactory system, which
primarily detects odorant molecules,
while the VNO detects pheromones
and is the major component of the
accessory olfactory system (2,3). The
fourth anatomically distinct chemo-
sensory area of the nasal septum is the
septal organ of Grüneberg (SOG) (4).
This chemosensory area is submucosal
in location and covered with respiratory
epithelium. Approximately 50% of the
total surface area of the septum is respi-
ratory epithelium. It has been presumed
that nasal respiratory epithelium is
very similar to tracheal respiratory
epithelium in structure and function.
Furthermore, sinonasal disorders
mimic or coexist with many respi-
ratory diseases, such as cystic fibrosis,
aspirin-sensitive asthma with polyps,
and allergic fungal sinusitis (the upper
airway correlates to allergic bronchopul-
monary aspergillosis) (5). Thus we have
focused our efforts on establishing air-
liquid interface cultures from the mouse
nasal septum and demonstrate that our
technique increases the yield of respi-
ratory epithelium 8-fold. Additionally,
our technique for removing the nasal
septum is simple and straightforward.
MATERIALS AND METHODS
Tissue Culture Technique
Harvest of mouse nasal septum.
Following euthanasia with a CO2 gas
chamber and cervical dislocation, the
mouse was placed on a Styrofoam®
dissection table in the prone position
and secured with several 18-gauge
needles. The skin at the nape of the
Murine nasal septa for respiratory epithelial
air-liquid interface cultures
Marcelo B. Antunes1, Bradford A. Woodworth1, Geeta Bhargave1, Guoxiang
Xiong1, Jorge L. Aguilar2, Adam J. Ratner2, James L. Kreindler3, Ronald C.
Rubenstein1, and Noam A. Cohen1
1University of Pennsylvania, Philadelphia, PA, 2Columbia University, New York, NY,
and 3University of Pittsburgh, Pittsburgh, PA, USA
BioTechniques 43:195-204 (August 2007)
Air-liquid interface models using murine tracheal respiratory epithelium have revolution-
ized the in vitro study of pulmonary diseases. This model is often impractical because of the
small number of respiratory epithelial cells that can be isolated from the mouse trachea. We
describe a simple technique to harvest the murine nasal septum and grow the epithelial cells
in an air-liquid interface. The degree of ciliation of mouse trachea, nasal septum, and their
respective cultured epithelium at an air-liquid interface were compared by scanning electron
microscopy (SEM). Immunocytochemistry for type IV β-tubulin and zona occludens-1 (Zo-1)
are performed to determine differentiation and confluence, respectively. To rule out contami-
nation with olfactory epithelium (OE), immunocytochemistry for olfactory marker protein
(OMP) was performed. Transepithelial resistance and potential measurements were deter-
mined using a modified vertical Ussing chamber. SEM reveals approximately 90% ciliated
respiratory epithelium in the nasal septum as compared with 35% in the mouse trachea. The
septal air-liquid interface culture demonstrates comparable ciliated respiratory epithelium
to the nasal septum. Immunocytochemistry demonstrates an intact monolayer and diffuse
differentiated ciliated epithelium. These cultures exhibit a transepithelial resistance and po-
tential confirming a confluent monolayer with electrically active airway epithelium contain-
ing both a sodium-absorptive pathway and a chloride-secretory pathway. To increase the
yield of respiratory epithelial cells harvested from mice, we have found the nasal septum is
a superior source when compared with the trachea. The nasal septum increases the yield of
respiratory epithelial cells up to 8-fold.
196 ı BioTechniques ı www.biotechniques.com
Vol. 43 ı No. 2 ı 2007
Short Technical Reports
neck was incised with fine dissecting
scissors, and the incision rotated
around the entire neck. This skin
was then dissected anteriorly and
completely removed, exposing the
bone over the entire skull and nose. The
skull was then sectioned in the coronal
plane posterior to the eyes (Figure 2A,
cut no. 1). The remnant of the brain
was completely removed, leaving
the anterior aspect of the skull base
exposed. The most anterior point of
the skull base on either side is directly
posterior to the mouse nasal cavities.
The remaining portion of the skull was
removed to the posterior aspect of the
nasal cavity (Figure 2A, cut no. 2).
The dorsum of the mouse nose
has two lines representing embryonic
fusion planes formed between the two
maxillas (laterally) and the ethmoid
bone (medially). Using this as a guide,
a scissor was inserted into the posterior
aspect of the nasal cavity, and the suture
line was incised separating one side of
the septum (medially) from the maxilla
(laterally) (Figure 2, A and B, cut no. 3).
The scissor was then turned inferiorly to
section the palate, and thus completely
separated the septum from the lateral
nasal wall. The procedure was then
repeated on the other side (Figure 2,
A and C, cut no. 4). The remaining
attachment at the nasal tip was severed
and the septum completely removed.
Coating semipermeable support
membranes. The tissue culture insert
semipermeable support membranes
(Costar® Transwell® clear 24-well
plate inserts, 0.4-μm pore; Corning
Life Sciences, Lowell, MA, USA) were
coated with 100 µL 50 µg/mL human
placental collagen (type VI; Rockland
Immunochemicals, Gilbertsville, PA,
USA) using a sterile technique 24–48 h
prior to tissue harvest. The inserts were
incubated in a 37°C biosafety incubator
over 24 h; the collagen solution was
removed, and the inserts were washed
twice with phosphate-buffered saline
(PBS) before use.
Culture of septal respiratory
epithelial cells. Mouse air-liquid
interface (ALI) cultures were adapted
from previously published methods
(6). After isolation of the mouse
septum, it was placed temporarily in a
50-mL conical tube containing either
PBS or a 1:1 mixture of Dulbecco’s
modified Eagle’s medium (DMEM),
Nutrient Mixture Ham’s F-12 medium
(Invitrogen, Carlsbad, CA, USA), 100
IU/mL penicillin, and 100 μg/mL strep-
tomycin if a long dissection is antici-
pated. Upon finishing the dissection of
all harvested mouse septa, they were
transferred into 20-mL volumes of
dissociation media containing minimal
essential medium (MEM; Invitrogen),
penicillin (60 IU/mL)-streptomycin (60
μg/mL), 1.4 mg/mL Pronase (Sigma-
Aldrich, St. Louis, MO, USA), and 0.1
μg/mL DNase (Roche Applied Science,
Indianapolis, IN, USA). We generally
place up to eight mouse septa per 20
mL dissociation media. This media was
prewarmed in a 5% CO2 chamber at
37°C for 1 h prior to use with the cap
loosely fitted to allow for diffusion of
CO2 into the media. The septa were
incubated in the dissociation media in
the 5% CO2 chamber at 37°C for 1 h.
To stop the enzymatic dissociation, 5
mL sterile 5% fetal bovine serum (FBS;
HyClone, Logan, UT, USA) were
added, followed by a further 2-min
incubation. The epithelial cells were
dissociated by gentle agitation of the
sinonasal tissue, achieved by 12 inver-
sions of the tube.
The tissue was removed from the
suspension and transferred to 10 mL
culture media consisting of a 1:1 mixture
Figure 1. The distribution of respiratory and olfactory epithelium (OE) on the murine nasal sep-
tum. The septum contains the main OE, the septal organ of Masera (SO), the vomeronasal organ (VNO),
and the septal organ of Grüneberg (SOG), which is submucosal and covered with respiratory epithelium.
Note that approximately 50% of the surface area is respiratory epithelium on both sides of the septum.
Figure 2. Harvesting the mouse septum. (A) Cut
no. 1: the skin is dissected and completely removed
from the skull and nose, and the skull sectioned in
the coronal plane. Cut no 2: next, the remainder
of the anterior skull is removed. The posterior as-
pect of the skull base has been removed and the
scissor inserted into the posterior nasal cavity. Cut
no. 3: the suture line is then incised bilaterally re-
vealing the nasal septum and the oral tongue be-
low (B) The scissor will separate the two maxillas
(laterally) and the ethmoid bone (medially) along
the distinct suture line. (C) Cut no. 4: the upper
palate and anterior nasal tip are removed next to
complete the dissection. The structure of the bony
septum is shown in panel A (left).
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Vol. 43 ı No. 2 ı 2007
Short Technical Reports
of DMEM and Nutrient Mixture Ham’s
F-12 medium containing penicillin (100
IU/mL)-streptomycin (100 μg/mL), 5%
heat-inactivated FBS, and 120 IU/mL
ITS™ universal culture supplement
(BD Biosciences, Bedford, MA, USA).
This tube was gently inverted 12 times,
as before, to release further epithelial
cells. The tissue was removed from
this suspension, and the two resultant
cell suspensions were pooled and
centrifuged at 120 × g for 5 min at room
temperature (21°–23°C). The super-
natant was removed, and the cell pellet
was gently resuspended and washed
in 10 mL culture media, centrifuged at
120× g for 5 min at room temperature
and then resuspended in 5 mL culture
media. This suspension was incubated
at 37°C for 2 h in 100 mm Primaria™
culture dishes (BD Biosciences)
to remove the nonepithelial cells.
Approximately one septum per Primaria
dish is needed to efficiently reduce
fibroblast contamination. Any visible
undigested tissue fragments were
removed, after which the cell suspension
containing the nonadherent cells was
collected with a fine-tip pipet. Next, the
suspension was centrifuged at 120× g
for 5 min at room temperature. The cell
pellet was gently resuspended in culture
media and mixed with a fine-tip pipet.
The cell yield was then counted using a
hemocytometer. The cell yield for one
trachea using this technique is approxi-
mately 2 × 105 cells. The cell yield for
one nasal septum is approximately 1.5
× 106 for one septum.
The dissociated cells were seeded at
a density of 4 × 105 cells per semiper-
meable support membrane in 200 μL
culture media, with 600 μL culture
media outside the insert in the basal
compartment. The cells were incubated
at 37°C in 5% CO2 in a humidified
incubator for 3 days. On day four, the
medium on the apical surface was
removed along with any nonadherent
cells and debris. The basal medium was
replaced with 600 μL differentiation
media consisting of a 1:1 mixture of
DMEM and Nutrient Mixture Ham’s
F-12 medium containing 100 IU/mL
penicillin, 100 μg/mL streptomycin,
and 2% NuSerum™ (BD Biosciences).
The basal medium was replaced twice
weekly. Primary cultures grown at
an air-liquid interface in this manner
reach maximal confluency at 1 week
on approximately 80%–90% of the
permeable support membrane plated
at the above concentration. Maximal
differentiation (generation of cilia),
however, is achieved at 3 weeks. Cilia
begin to appear between 1 and 2 weeks.
Depending on the contamination of
nonepithelial cells, generally 80% of
the surface will have differentiated cilia
by 2 weeks. One nasal septum yields
approximately four air-liquid interface
cultures. In general, we use 6- to 10-
week-old female Balb/c or C57 mice.
We have not found a difference in
yield or characteristics of the cultures
between these two strains of mice,
although we did not investigate the ideal
age for optimizing yield; waiting until
the mice have grown to adult size will
likely increase the number of epithelial
cells due to a larger septal area.
Scanning Electron Microscopy
SEM was performed on a mouse
nasal septum, trachea, and air-liquid
interface cultures at 17 days. The
formalin-fixed mouse trachea, septal
mucosa, and air-liquid interface
membranes were dehydrated in a
progression of increasing ethanol
concentrations, up to 100% ethanol.
The specimens were then critical-point
dried in CO2, mounted on scanning
electron microscope stubs, and sputter-
coated with gold palladium to a depth
of 12 nm. The surface of the trachea
and septum were then examined with
an AMR-1400 scanning electron micro-
scope at an accelerating voltage of 20
kV. Representative photomicrographs
were taken at various angles to effec-
tively display the specimen so that any
error in assessment is minimized due to
the tilt of the specimen or other artifact.
Photomicrographs were evaluated for
the percentage of ciliated epithelium in
the mouse trachea, nasal septum, and
air-liquid interface monolayer.
Localization by immunocytochem-
istry of cilia (type IV β-tubulin) and
tight junctions (Zo-1) was performed
on cultured monolayers in transwell
inserts to confirm differentiation and
confluence, respectively. Mouse anti-
human type IV β-tubulin monoclonal
antibodies and rabbit anti-human Zo-1
polyclonal antibodies were obtained
from Invitrogen. Negative controls
were performed in parallel without
a primary antibody incubation step.
Nonspecific staining was blocked
with 5% goat serum and 1% bovine
serum albumin (BSA). The cells were
permeabilized with 0.3% Triton® X-
100 and incubated in primary antibody
(type IV β-tubulin, 1:500; Zo-1, 1:100)
overnight at 4°C. After three washes
with PBS, the transwell insert was
incubated in fluorescein isothiocyanate
(FITC)-coupled goat anti-mouse
immunoglobulin G (IgG; 1:500) and
rhodamine-coupled goat anti-rabbit
IgG (1:500) at room temperature for
90 min. The membranes were washed
three times in 1× PBS and then cut from
the plastic support mold. They were
mounted with Gel Mount™ aqueous
mounting medium (Sigma-Aldrich)
on a glass slide. The slides were then
imaged on a Zeiss LSM510META
Because OE comprises approxi-
mately half of the nasal septum,
cultured epithelial monolayers were
incubated with OMP antibodies
(specific to OE) and type IV β-tubulin
(specific to respiratory cilia). Septal
olfactory mucosa was harvested and
fixed for positive control. Mouse
septal air-liquid interface cultures were
incubated in primary antibodies (type
IV β-tubulin, 1:500; OMP, 1:1000)
overnight at 4°C. After three washes
with PBS, the transwell insert was
incubated in FITC-coupled goat anti-
rabbit IgG (1:500) and rhodamine-
coupled goat anti-mouse IgG (1:500)
at room temperature for 90 min.
The remainder of the protocol was
performed in an identical fashion to our
previous methods, with the exception
that the slides were counterstained with
Solutions and chemicals. The bath
solution contained 120 mM NaCl, 25
mM NaHCO3, 3.3 mM KH2PO4, 0.8
mM K2HPO4, 1.2 mM MgCl2, 1.2 mM
CaCl2, and 10 mM glucose. The pH of
this solution is 7.3–7.4 when gassed
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with a mixture of 95% O2-5% CO2 at
37°C. Chemicals were obtained from
Sigma-Aldrich. Each chemical was
made as a 1000× stock and used at 1×
in the Ussing chamber. Amiloride was
made as an aqueous solution. Forskolin
and glybenclamide were dissolved in
dimethyl sulfoxide (DMSO).
Short circuit measurements.
Transwell inserts (Corning Life
Sciences) were mounted in a modified,
vertical Ussing chamber, and the
monolayers were continuously short-
circuited after fluid resistance compen-
sation using automatic voltage clamps
(VCC 600; Physiologic Instruments,
San Diego, CA, USA). Transwell
filters were mounted in bath solution
warmed to 37°C, and the solution was
continuously gas-lifted with a 95% O2-
5% CO2 mixture. The Ussing chambers
were placed on heated stage, but were
not mounted in a jacketed holder. The
short circuit current (ISC) was digitized
at one sample per second, and data
were stored on a computer hard drive
using Acquire & Analyze software
build 2.2 (Physiologic Instruments).
Transepithelial resistance (RT) was
measured every 60 s by passing a
2-mV, 2-s, bipolar pulse across the
monolayer and calculating RT by Ohm’s
law (V = IR). By convention, a positive
deflection in ISC is defined as the net
movement of a cation in the serosal to
RESULTS AND DISCUSSION
Mouse tracheal epithelium was
comparable to that reported in the liter-
ature with approximately 30%–35%
of the surface consisting of ciliated
respiratory epithelium imaged with
SEM (7) (Figure 3A). This percentage
is consistent with scanning electron
micrographs of cultured tracheal
epithelial cells at an air-liquid interface
and previously published reports (6)
(Figure 3B). On the other hand, the
mouse septum had approximately 90%
of the epithelial surface covered with
cilia. (Figure 3C) This was consistent
with the scanning electron micro-
graph of the air-liquid interface grown
from dissociated mouse nasal septal
epithelial cells at 17 days (Figure 3D).
Immunocytochemistry performed on
the epithelial air-liquid interface at 17
days demonstrates an intact monolayer
with tight junctions and a differentiated
cell composition with cilia (Figure
4). Tight junctions are cell-to-cell
adhesion structures in epithelial cells
that constitute the epithelial junctional
complex with adherens junctions and
desmosomes. Tight junctions seal
cells to create a primary barrier to the
diffusion of solutes and function as
a boundary between the apical and
basolateral membrane domains to
produce cellular polarization (8). Zo-1
is one of three major scaffold proteins
concentrated at the cytoplasmic
surfaces of the junctional complexes.
Type IV β-tubulin is one of the major
subtypes of β-tubulin in cilia and an
excellent marker. Demonstration of
Zo-1 and type IV β-tubulin by immuno-
cytochemistry indicates an intact
polarized monolayer with a differen-
tiated cell population.
Colocalization of type IV tubulin
and OMP was performed to determine
whether a significant proportion of
the air-liquid interface culture was
olfactory epithelial cells. OMP is a
specific marker for OE (Figure 5A).
No OMP was detected in the air-liquid
interface cultures by immunocyto-
chemistry (Figure 5B).
We studied three mature, visually
ciliated monolayers. At baseline, the
monolayers had a mean RT of 528 ±
42.4 Ω × cm2 and a mean ISC of -3.3 ±
1.46 μA/cm2. For two of three filters,
prior to short-circuiting the monolayer,
we studied the monolayers under open
circuit conditions. These filters had a
mean transepithelial potential of -1.9 ±
To study vectorial ion movement in
the nasal epithelial preparations, we
used three common pharmacological
manipulations. First, we applied
amiloride (10 μM) to mucosal surface.
This resulted in approximately 75%
inhibition of resting ISC, suggesting
that the baseline ISC in these cells is due
predominantly to sodium absorption.
In the continued presence of amiloride,
we then applied forskolin (2 μM) to
both the serosal and mucosal surfaces.
Forskolin resulted in increased ISC to a
mean of 7.4 ± 1.5 μA/cm2 (n = 3). The
forskolin-stimulated ISC was partially
Figure 3. Morphologic comparison of in situ and culture of mouse trachea and nasal septum.
(A) Scanning electron micrograph of a mouse trachea specimen. Only 35% ciliated respiratory epithelia
are present, while the remainder are non-ciliated and clara cells. (B) The morphology of a tracheal epithe-
lial air-liquid interface culture (fixed at 17 days) is very similar to native trachea. (C) This electron micro-
graph demonstrates the typical shag carpet of cilia present on the nasal septum. Approximately 90% of the
surface has cilia. (D) Another scanning electron micrograph of an intact epithelial air-liquid interface from
a mouse nasal septum demonstrates the high percentage of ciliated epithelial cells (17 days).
202 ı BioTechniques ı www.biotechniques.com
Vol. 43 ı No. 2 ı 2007
Short Technical Reports
inhibited by glybenclamide (mean
inhibition 23%). A representative
tracing is shown in Figure 6.
The established techniques for the
primary culture of airway epithelial
cells from transgenic mouse tracheas
have greatly facilitated the study of
pulmonary diseases. Numerous investi-
gators have described the maintenance
of airway epithelial cells in a differen-
tiated state in primary culture (6,9–11).
However, the total number of cells
that can be isolated from the mouse
trachea is very small. The tracheal
epithelium of the mouse has ciliated
cells that occur only in scattered
patches (7). In an attempt to increase
the yield of tracheal epithelial cells,
Kumar et al. (12) has recommended
expansion of the population through
serum-free growth media. However,
the expansion and passage of murine
respiratory epithelium has resulted in
limited differentiation even after just
one passage (1).
We have established a technique for
the development of air-liquid interface
cultures through the use of mouse nasal
septa. Our technique of harvesting the
nasal septum is simple and straight-
forward. This method increases the
yield of respiratory epithelia 8-fold
over tracheal cultures. The composition
of murine nasal epithelium compares
favorably to the nearly 90% ciliated
respiratory epithelium we see in the
developed air-liquid interface culture.
Septal air-liquid interface cultures
exhibit full differentiation with cilia
and confluence with tight junctions.
OE, which comprises approximately
half of the murine nasal septum, is
absent on the differentiated air-liquid
interface. The reasons for this are
currently unknown, but likely due to
the method of culture and absence of
neuronal growth factors.
The presence of a resting ISC
and of a resting electrical potential
in these preparations is consistent
with the presence of baseline net ion
transport. The fact that the ISC was
largely inhibited by amiloride suggests
that at baseline these preparations are
primarily sodium absorbing under
short-circuit conditions. Furthermore,
the presence of a forskolin-stimulated
current that is partially inhibited by
glybenclamide is strongly suggestive
that these preparations are capable of
secreting anions (most likely chloride)
via the cystic fibrosis transmembrane
Figure 4. Type IV β-tubulin (green) staining for cilia and zona occludens-1 (Zo-1; red). Staining for
tight junctions is demonstrated using confocal laser scanning microscopy (63×) following immunocy-
tochemistry of cultured epithelium on the air-liquid interface. This shows the green staining of the cilia
at the top of the cell (differentiation) and the red staining of the tight junctions (confluence) between the
cells of the monolayer.
Figure 5. Olfactory marker protein (OMP) and
type IV staining. (A and B) Immunofluorescence
[septal olfactory epithelium (OE; panel A)] and
immunofluorescence (septal air-liquid interface
culture; panel B) for both OMP (green) and
type IV β-tubulin (red). As expected, the septal
OE stains heavily for OMP, while the air-liquid
interface culture reveals staining only for type
IV β-tubulin. Blue staining for nuclei is present
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Vol. 43 ı No. 2 ı 2007
Short Technical Reports
The absolute value of our measured
ISC is less than that calculated by others
(13). This difference may be due to
differences in mouse strain or age, or
to differences in culture conditions.
Also, our experiments were performed
essentially at room temperature, which
may have further contributed to this
variation. Nevertheless, the overall
phenotype of ion transport in these
cells is consistent with an electrically
active, mucociliary airway epithelium
containing both a sodium-absorptive
pathway and a chloride-secretory
The increased yield and percentage
of ciliated respiratory epithelium
makes the murine nasal septa air-liquid
interface model ideal for the study of
sinonasal and pulmonary diseases and
ciliary physiology. Air-liquid interface
cultures grown from nasal septa exhibit
a transepithelial potential. Thus,
studying the growth and differentiation
of septal respiratory epithelium will
enable more physiologically relevant
analysis of sinonasal and pulmonary
diseases in vitro, and allow for the
evaluation of novel therapies.
M.B.A. and B.A.W. contributed
equally to this work. This work was sup-
ported by the Young Investigator Award
from the Sinus and Allergy Health
Partnership (N.A.C.), and NIH grant
nos. AI065450 (A.J.R.), K08HL081080
(J.L.K.), and R01 DK58046 (R.C.R.).
The authors declare no competing
1. You, Y., E.J. Richer, T. Huang, and S.L.
Brody. 2002. Growth and differentiation of
mouse tracheal epithelial cells: selection of a
proliferative population. Am. J. Physiol. Lung
Cell. Mol. Physiol. 283:L1315-L1321.
2. Doty, R.L. 1986. Odor-guided behavior in
mammals. Experientia 42:257-271.
3. Halpern, M. and A. Martinez-Marcos. 2003.
Structure and function of the vomeronasal sys-
tem: an update. Prog. Neurobiol. 70:245-318.
4. Storan, M.J. and B. Key. 2006. Septal organ
of Gruneberg is part of the olfactory system. J.
Comp. Neurol. 494:834-844.
5. Benninger, M.S., B.J. Ferguson, J.A.
Hadley, D.L. Hamilos, M. Jacobs, D.W.
Kennedy, D.C. Lanza, B.F. Marple, et al.
2003. Adult chronic rhinosinusitis: definitions,
diagnosis, epidemiology, and pathophysiolo-
gy. Otolaryngol. Head Neck Surg. 129:S1-32.
6. Davidson, D.J., F.M. Kilanowski, S.H.
Randell, D.N. Sheppard, and J.R. Dorin.
2000. A primary culture model of differentiat-
ed murine tracheal epithelium. Am. J. Physiol.
Lung Cell. Mol. Physiol. 279:L766-L778.
7. Pack, R.J., L.H. Al-Ugaily, G. Morris, and
J.G. Widdicombe. 1980. The distribution and
structure of cells in the tracheal epithelium of
the mouse. Cell Tissue Res. 208:65-84.
8. Gumbiner, B. 1987. Structure, biochemistry,
and assembly of epithelial tight junctions. Am.
J. Physiol. 253:C749-C758.
9. Davidson, D.J., M.A. Gray, F.M.
Kilanowski, R. Tarran, S.H. Randell, D.N.
Sheppard, B.E. Argent, and J.R. Dorin.
2004. Murine epithelial cells: isolation and
culture. J. Cyst. Fibros. Suppl 2:59-62.
10. Davidson, D.J. and M. Rolfe. 2001. Mouse
models of cystic fibrosis. Trends Genet. 17:
11. Lankford, S.M., M. Macchione, A.L. Crews,
S.A. McKane, N.J. Akley, and L.D. Martin.
2005. Modeling the airway epithelium in al-
lergic asthma: interleukin-13-induced effects
in differentiated murine tracheal epithelial
cells. In Vitro Cell. Dev. Biol. Anim. 41:217-
12. Kumar, R.K., S.E. Maronese, and R.
O’Grady. 1997. Serum-free culture of mouse
tracheal epithelial cells. Exp. Lung Res.
13. Grubb, B.R., R.N. Vick, and R.C. Boucher.
1994. Hyperabsorption of Na+ and raised
Ca(2+)-mediated Cl- secretion in nasal epi-
thelia of CF mice. Am. J. Physiol. 266:C1478-
14. Adler, K.B., P.W. Cheng, and K.C. Kim.
1990. Characterization of guinea pig tracheal
epithelial cells maintained in biphasic organo-
typic culture: cellular composition and bio-
chemical analysis of released glycoconjugates.
Am. J. Respir. Cell Mol. Biol. 2:145-154.
15. Gray, T.E., K. Guzman, C.W. Davis,
L.H. Abdullah, and P. Nettesheim. 1996.
Mucociliary differentiation of serially pas-
saged normal human tracheobronchial epi-
thelial cells. Am. J. Respir. Cell Mol. Biol.
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Figure 6. Representative short circiu current (ISC) tracing of polarized murine nasal epithelial
cells. Murine nasal epithelial cells grown on Transwell permeable supports were mounted in Ussing
chambers under short-circuit conditions and sequentially exposed to amiloride, forskolin, and glyben-
clamide. Note that baseline ISC is predominantly inhibited by amiloride and that forskolin-stimulated ISC
is partially blocked by glybenclamide. These data are consistent with the presence of both electrogenic
sodium absorption and anion secretion.
Received 9 December 2006; accepted
23 May 2007.
Address correspondence to Bradford A.
Woodworth, Department of Otorhino-
laryngology, 3400 Spruce Street, 5 Ravdin,
Philadelphia, PA 19104, USA. e-mail: