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Short Technical Reports Murine nasal septa for respiratory

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									                                                                                                   Short Technical Reports


Murine nasal septa for respiratory epithelial                                                      research involving murine nasal septa
                                                                                                   has primarily focused on olfaction, so
air-liquid interface cultures                                                                      the potential for respiratory cell culture
                                                                                                   has largely been ignored. The two sides
Marcelo B. Antunes1, Bradford A. Woodworth1, Geeta Bhargave1, Guoxiang                             of the murine nasal septum are covered
Xiong1, Jorge L. Aguilar2, Adam J. Ratner2, James L. Kreindler3, Ronald C.                         with mucosa, and the overall surface
Rubenstein1, and Noam A. Cohen1                                                                    area is much larger in comparison
1University
                                                                                                   to the mouse trachea. The olfactory
          of Pennsylvania, Philadelphia, PA, 2Columbia University, New York, NY,
                                                                                                   system of the mouse has four anatomi-
and 3University of Pittsburgh, Pittsburgh, PA, USA
                                                                                                   cally distinct chemosensory areas on
                                                                                                   the nasal septum. (Figure 1) The main
BioTechniques 43:195-204 (August 2007)                                                             olfactory epithelium (OE), the septal
doi 10.2144/000112493                                                                              organ of Masera (SO), and the vomero-
                                                                                                   nasal organ (VNO) have bipolar sensory
Air-liquid interface models using murine tracheal respiratory epithelium have revolution-          neurons that reside in a pseudostratified
ized the in vitro study of pulmonary diseases. This model is often impractical because of the      neuroepithelium. The MOE and SO are
small number of respiratory epithelial cells that can be isolated from the mouse trachea. We       part of the main olfactory system, which
describe a simple technique to harvest the murine nasal septum and grow the epithelial cells       primarily detects odorant molecules,
in an air-liquid interface. The degree of ciliation of mouse trachea, nasal septum, and their      while the VNO detects pheromones
respective cultured epithelium at an air-liquid interface were compared by scanning electron       and is the major component of the
microscopy (SEM). Immunocytochemistry for type IV β-tubulin and zona occludens-1 (Zo-1)            accessory olfactory system (2,3). The
are performed to determine differentiation and confluence, respectively. To rule out contami-      fourth anatomically distinct chemo-
nation with olfactory epithelium (OE), immunocytochemistry for olfactory marker protein            sensory area of the nasal septum is the
(OMP) was performed. Transepithelial resistance and potential measurements were deter-             septal organ of Grüneberg (SOG) (4).
mined using a modified vertical Ussing chamber. SEM reveals approximately 90% ciliated             This chemosensory area is submucosal
respiratory epithelium in the nasal septum as compared with 35% in the mouse trachea. The          in location and covered with respiratory
septal air-liquid interface culture demonstrates comparable ciliated respiratory epithelium        epithelium. Approximately 50% of the
to the nasal septum. Immunocytochemistry demonstrates an intact monolayer and diffuse              total surface area of the septum is respi-
differentiated ciliated epithelium. These cultures exhibit a transepithelial resistance and po-    ratory epithelium. It has been presumed
tential confirming a confluent monolayer with electrically active airway epithelium contain-       that nasal respiratory epithelium is
ing both a sodium-absorptive pathway and a chloride-secretory pathway. To increase the             very similar to tracheal respiratory
yield of respiratory epithelial cells harvested from mice, we have found the nasal septum is       epithelium in structure and function.
a superior source when compared with the trachea. The nasal septum increases the yield of          Furthermore, sinonasal disorders
respiratory epithelial cells up to 8-fold.                                                         mimic or coexist with many respi-
                                                                                                   ratory diseases, such as cystic fibrosis,
                                                                                                   aspirin-sensitive asthma with polyps,
INTRODUCTION                                      the development of differentiated respi-         and allergic fungal sinusitis (the upper
                                                  ratory epithelial cells, expansion and           airway correlates to allergic bronchopul-
    The development of primary culture            passage of these cells will ultimately           monary aspergillosis) (5). Thus we have
models of transgenic mouse tracheal               decrease the ability for these cells to          focused our efforts on establishing air-
epithelial cells has greatly facili-              differentiate (1). Therefore, one of the         liquid interface cultures from the mouse
tated the study of human respiratory              primary limitations of in vitro murine           nasal septum and demonstrate that our
diseases. These primary culture models            models includes the large number of              technique increases the yield of respi-
are confluent, fully differentiated,              mice required to obtain a significant            ratory epithelium 8-fold. Additionally,
ciliated respiratory epithelium at an             number of tracheal epithelial cells.             our technique for removing the nasal
air-liquid interface on a semipermeable           Typically, dissociation of respiratory           septum is simple and straightforward.
membrane that mimic many charac-                  epithelium from two mouse tracheas
teristics of murine tracheal epithelial           is required for the development of an
cells in vivo. Prior to the development           air-liquid interface on one transwell            MATERIALS AND METHODS
of murine models, suboptimal methods              membrane (6.5-mm diameter). This can
involving non-polarized and sometimes             be cost-prohibitive when attempting              Tissue Culture Technique
poorly differentiated primary cultures            to develop in vitro airway models of
and immortalized cell lines were used             transgenic mice. An additional source               Harvest of mouse nasal septum.
for respiratory epithelial cell research.         of respiratory epithelium will increase          Following euthanasia with a CO2 gas
These models made logistical sense                the utility of these mice and, at the same       chamber and cervical dislocation, the
because they often supplied copious               time, decrease expenses.                         mouse was placed on a Styrofoam®
numbers of cells through expansion and               Our recent investigations have found          dissection table in the prone position
passage of the cells. While the murine            that the nasal septum is a superb source         and secured with several 18-gauge
air-liquid interface models are ideal for         of murine respiratory epithelium. Most           needles. The skin at the nape of the
Vol. 43 ı No. 2 ı 2007                                                                            www.biotechniques.com ı BioTechniques ı 195
Short Technical Reports


                                                                                                         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
Figure 1. The distribution of respiratory and olfactory epithelium (OE) on the murine nasal sep-         sinonasal tissue, achieved by 12 inver-
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.    sions of the tube.
Note that approximately 50% of the surface area is respiratory epithelium on both sides of the septum.      The tissue was removed from the
                                                                                                         suspension and transferred to 10 mL
                                                                                                         culture media consisting of a 1:1 mixture
neck was incised with fine dissecting                   Coating semipermeable support
scissors, and the incision rotated                   membranes. The tissue culture insert
                                                                                                          A
around the entire neck. This skin                    semipermeable support membranes
was then dissected anteriorly and                    (Costar® Transwell® clear 24-well
completely removed, exposing the                     plate inserts, 0.4-μm pore; Corning
bone over the entire skull and nose. The             Life Sciences, Lowell, MA, USA) were
skull was then sectioned in the coronal              coated with 100 µL 50 µg/mL human
plane posterior to the eyes (Figure 2A,              placental collagen (type VI; Rockland
cut no. 1). The remnant of the brain                 Immunochemicals, Gilbertsville, PA,
was completely removed, leaving                      USA) using a sterile technique 24–48 h
the anterior aspect of the skull base                prior to tissue harvest. The inserts were
exposed. The most anterior point of                  incubated in a 37°C biosafety incubator
the skull base on either side is directly            over 24 h; the collagen solution was
posterior to the mouse nasal cavities.               removed, and the inserts were washed                 B
The remaining portion of the skull was               twice with phosphate-buffered saline
removed to the posterior aspect of the               (PBS) before use.
nasal cavity (Figure 2A, cut no. 2).                    Culture of septal respiratory
    The dorsum of the mouse nose                     epithelial cells. Mouse air-liquid
has two lines representing embryonic                 interface (ALI) cultures were adapted
fusion planes formed between the two                 from previously published methods
maxillas (laterally) and the ethmoid                 (6). After isolation of the mouse
bone (medially). Using this as a guide,              septum, it was placed temporarily in a
a scissor was inserted into the posterior            50-mL conical tube containing either
aspect of the nasal cavity, and the suture           PBS or a 1:1 mixture of Dulbecco’s
                                                                                                         Figure 2. Harvesting the mouse septum. (A) Cut
line was incised separating one side of              modified Eagle’s medium (DMEM),                     no. 1: the skin is dissected and completely removed
the septum (medially) from the maxilla               Nutrient Mixture Ham’s F-12 medium                  from the skull and nose, and the skull sectioned in
(laterally) (Figure 2, A and B, cut no. 3).          (Invitrogen, Carlsbad, CA, USA), 100                the coronal plane. Cut no 2: next, the remainder
The scissor was then turned inferiorly to            IU/mL penicillin, and 100 μg/mL strep-              of the anterior skull is removed. The posterior as-
                                                                                                         pect of the skull base has been removed and the
section the palate, and thus completely              tomycin if a long dissection is antici-             scissor inserted into the posterior nasal cavity. Cut
separated the septum from the lateral                pated. Upon finishing the dissection of             no. 3: the suture line is then incised bilaterally re-
nasal wall. The procedure was then                   all harvested mouse septa, they were                vealing the nasal septum and the oral tongue be-
repeated on the other side (Figure 2,                transferred into 20-mL volumes of                   low (B) The scissor will separate the two maxillas
A and C, cut no. 4). The remaining                   dissociation media containing minimal               (laterally) and the ethmoid bone (medially) along
                                                                                                         the distinct suture line. (C) Cut no. 4: the upper
attachment at the nasal tip was severed              essential medium (MEM; Invitrogen),                 palate and anterior nasal tip are removed next to
and the septum completely removed.                   penicillin (60 IU/mL)-streptomycin (60              complete the dissection. The structure of the bony
                                                     μg/mL), 1.4 mg/mL Pronase (Sigma-                   septum is shown in panel A (left).


196 ı BioTechniques ı www.biotechniques.com                                                                                           Vol. 43 ı No. 2 ı 2007
Short Technical Reports


of DMEM and Nutrient Mixture Ham’s            reach maximal confluency at 1 week            human type IV β-tubulin monoclonal
F-12 medium containing penicillin (100        on approximately 80%–90% of the               antibodies and rabbit anti-human Zo-1
IU/mL)-streptomycin (100 μg/mL), 5%           permeable support membrane plated             polyclonal antibodies were obtained
heat-inactivated FBS, and 120 IU/mL           at the above concentration. Maximal           from Invitrogen. Negative controls
ITS™ universal culture supplement             differentiation (generation of cilia),        were performed in parallel without
(BD Biosciences, Bedford, MA, USA).           however, is achieved at 3 weeks. Cilia        a primary antibody incubation step.
This tube was gently inverted 12 times,       begin to appear between 1 and 2 weeks.        Nonspecific staining was blocked
as before, to release further epithelial      Depending on the contamination of             with 5% goat serum and 1% bovine
cells. The tissue was removed from            nonepithelial cells, generally 80% of         serum albumin (BSA). The cells were
this suspension, and the two resultant        the surface will have differentiated cilia    permeabilized with 0.3% Triton® X-
cell suspensions were pooled and              by 2 weeks. One nasal septum yields           100 and incubated in primary antibody
centrifuged at 120 × g for 5 min at room      approximately four air-liquid interface       (type IV β-tubulin, 1:500; Zo-1, 1:100)
temperature (21°–23°C). The super-            cultures. In general, we use 6- to 10-        overnight at 4°C. After three washes
natant was removed, and the cell pellet       week-old female Balb/c or C57 mice.           with PBS, the transwell insert was
was gently resuspended and washed             We have not found a difference in             incubated in fluorescein isothiocyanate
in 10 mL culture media, centrifuged at        yield or characteristics of the cultures      (FITC)-coupled goat anti-mouse
120× g for 5 min at room temperature          between these two strains of mice,            immunoglobulin G (IgG; 1:500) and
and then resuspended in 5 mL culture          although we did not investigate the ideal     rhodamine-coupled goat anti-rabbit
media. This suspension was incubated          age for optimizing yield; waiting until       IgG (1:500) at room temperature for
at 37°C for 2 h in 100 mm Primaria™           the mice have grown to adult size will        90 min. The membranes were washed
culture dishes (BD Biosciences)               likely increase the number of epithelial      three times in 1× PBS and then cut from
to remove the nonepithelial cells.            cells due to a larger septal area.            the plastic support mold. They were
Approximately one septum per Primaria                                                       mounted with Gel Mount™ aqueous
dish is needed to efficiently reduce          Scanning Electron Microscopy                  mounting medium (Sigma-Aldrich)
fibroblast contamination. Any visible                                                       on a glass slide. The slides were then
undigested tissue fragments were                 SEM was performed on a mouse               imaged on a Zeiss LSM510META
removed, after which the cell suspension      nasal septum, trachea, and air-liquid         confocal microscope.
containing the nonadherent cells was          interface cultures at 17 days. The               Because OE comprises approxi-
collected with a fine-tip pipet. Next, the    formalin-fixed mouse trachea, septal          mately half of the nasal septum,
suspension was centrifuged at 120× g          mucosa, and air-liquid interface              cultured epithelial monolayers were
for 5 min at room temperature. The cell       membranes were dehydrated in a                incubated with OMP antibodies
pellet was gently resuspended in culture      progression of increasing ethanol             (specific to OE) and type IV β-tubulin
media and mixed with a fine-tip pipet.        concentrations, up to 100% ethanol.           (specific to respiratory cilia). Septal
The cell yield was then counted using a       The specimens were then critical-point        olfactory mucosa was harvested and
hemocytometer. The cell yield for one         dried in CO2, mounted on scanning             fixed for positive control. Mouse
trachea using this technique is approxi-      electron microscope stubs, and sputter-       septal air-liquid interface cultures were
mately 2 × 105 cells. The cell yield for      coated with gold palladium to a depth         incubated in primary antibodies (type
one nasal septum is approximately 1.5         of 12 nm. The surface of the trachea          IV β-tubulin, 1:500; OMP, 1:1000)
× 106 for one septum.                         and septum were then examined with            overnight at 4°C. After three washes
   The dissociated cells were seeded at       an AMR-1400 scanning electron micro-          with PBS, the transwell insert was
a density of 4 × 105 cells per semiper-       scope at an accelerating voltage of 20        incubated in FITC-coupled goat anti-
meable support membrane in 200 μL             kV. Representative photomicrographs           rabbit IgG (1:500) and rhodamine-
culture media, with 600 μL culture            were taken at various angles to effec-        coupled goat anti-mouse IgG (1:500)
media outside the insert in the basal         tively display the specimen so that any       at room temperature for 90 min.
compartment. The cells were incubated         error in assessment is minimized due to       The remainder of the protocol was
at 37°C in 5% CO2 in a humidified             the tilt of the specimen or other artifact.   performed in an identical fashion to our
incubator for 3 days. On day four, the        Photomicrographs were evaluated for           previous methods, with the exception
medium on the apical surface was              the percentage of ciliated epithelium in      that the slides were counterstained with
removed along with any nonadherent            the mouse trachea, nasal septum, and          4′,6-diamidino-2-phenylindole (DAPI)
cells and debris. The basal medium was        air-liquid interface monolayer.               nuclear stain.
replaced with 600 μL differentiation
media consisting of a 1:1 mixture of                                                        Electrophysiology
DMEM and Nutrient Mixture Ham’s               Immunocytochemistry
F-12 medium containing 100 IU/mL                  Localization by immunocytochem-              Solutions and chemicals. The bath
penicillin, 100 μg/mL streptomycin,           istry of cilia (type IV β-tubulin) and        solution contained 120 mM NaCl, 25
and 2% NuSerum™ (BD Biosciences).             tight junctions (Zo-1) was performed          mM NaHCO3, 3.3 mM KH2PO4, 0.8
The basal medium was replaced twice           on cultured monolayers in transwell           mM K2HPO4, 1.2 mM MgCl2, 1.2 mM
weekly. Primary cultures grown at             inserts to confirm differentiation and        CaCl2, and 10 mM glucose. The pH of
an air-liquid interface in this manner        confluence, respectively. Mouse anti-         this solution is 7.3–7.4 when gassed
198 ı BioTechniques ı www.biotechniques.com                                                                       Vol. 43 ı No. 2 ı 2007
Short Technical Reports


with a mixture of 95% O2-5% CO2 at                Immunocytochemistry performed on                       specific marker for OE (Figure 5A).
37°C. Chemicals were obtained from            the epithelial air-liquid interface at 17                  No OMP was detected in the air-liquid
Sigma-Aldrich. Each chemical was              days demonstrates an intact monolayer                      interface cultures by immunocyto-
made as a 1000× stock and used at 1×          with tight junctions and a differentiated                  chemistry (Figure 5B).
in the Ussing chamber. Amiloride was          cell composition with cilia (Figure                            We studied three mature, visually
made as an aqueous solution. Forskolin        4). Tight junctions are cell-to-cell                       confluent, and morphologically
and glybenclamide were dissolved in           adhesion structures in epithelial cells                    ciliated monolayers. At baseline, the
dimethyl sulfoxide (DMSO).                    that constitute the epithelial junctional                  monolayers had a mean RT of 528 ±
    Short circuit measurements.               complex with adherens junctions and                        42.4 Ω × cm2 and a mean ISC of -3.3 ±
Transwell inserts (Corning Life               desmosomes. Tight junctions seal                           1.46 μA/cm2. For two of three filters,
Sciences) were mounted in a modified,         cells to create a primary barrier to the                   prior to short-circuiting the monolayer,
vertical Ussing chamber, and the              diffusion of solutes and function as                       we studied the monolayers under open
monolayers were continuously short-           a boundary between the apical and                          circuit conditions. These filters had a
circuited after fluid resistance compen-      basolateral membrane domains to                            mean transepithelial potential of -1.9 ±
sation using automatic voltage clamps         produce cellular polarization (8). Zo-1                    0.25 mV
(VCC 600; Physiologic Instruments,            is one of three major scaffold proteins                        To study vectorial ion movement in
San Diego, CA, USA). Transwell                concentrated at the cytoplasmic                            the nasal epithelial preparations, we
filters were mounted in bath solution         surfaces of the junctional complexes.                      used three common pharmacological
warmed to 37°C, and the solution was          Type IV β-tubulin is one of the major                      manipulations. First, we applied
continuously gas-lifted with a 95% O2-        subtypes of β-tubulin in cilia and an                      amiloride (10 μM) to mucosal surface.
5% CO2 mixture. The Ussing chambers           excellent marker. Demonstration of                         This resulted in approximately 75%
were placed on heated stage, but were         Zo-1 and type IV β-tubulin by immuno-                      inhibition of resting ISC, suggesting
not mounted in a jacketed holder. The         cytochemistry indicates an intact                          that the baseline ISC in these cells is due
short circuit current (ISC) was digitized     polarized monolayer with a differen-                       predominantly to sodium absorption.
at one sample per second, and data            tiated cell population.                                    In the continued presence of amiloride,
were stored on a computer hard drive              Colocalization of type IV tubulin                      we then applied forskolin (2 μM) to
using Acquire & Analyze software              and OMP was performed to determine                         both the serosal and mucosal surfaces.
build 2.2 (Physiologic Instruments).          whether a significant proportion of                        Forskolin resulted in increased ISC to a
Transepithelial resistance (RT) was           the air-liquid interface culture was                       mean of 7.4 ± 1.5 μA/cm2 (n = 3). The
measured every 60 s by passing a              olfactory epithelial cells. OMP is a                       forskolin-stimulated ISC was partially
2-mV, 2-s, bipolar pulse across the
monolayer and calculating RT by Ohm’s           A                                                          B
law (V = IR). By convention, a positive
deflection in ISC is defined as the net
movement of a cation in the serosal to
mucosal direction.


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
                                                C                                                          D
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        Figure 3. Morphologic comparison of in situ and culture of mouse trachea and nasal septum.
with the scanning electron micro-             (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-
graph of the air-liquid interface grown       lial air-liquid interface culture (fixed at 17 days) is very similar to native trachea. (C) This electron micro-
from dissociated mouse nasal septal           graph demonstrates the typical shag carpet of cilia present on the nasal septum. Approximately 90% of the
epithelial cells at 17 days (Figure 3D).      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).

200 ı BioTechniques ı www.biotechniques.com                                                                                          Vol. 43 ı No. 2 ı 2007
Short Technical Reports


                                                                                                                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


                                                                                                                   A




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.                                                                                            B
inhibited by glybenclamide (mean                         respiratory epithelium has resulted in
inhibition 23%). A representative                        limited differentiation even after just
tracing is shown in Figure 6.                            one passage (1).
    The established techniques for the                      We have established a technique for
primary culture of airway epithelial                     the development of air-liquid interface
cells from transgenic mouse tracheas                     cultures through the use of mouse nasal
have greatly facilitated the study of                    septa. Our technique of harvesting the
pulmonary diseases. Numerous investi-                    nasal septum is simple and straight-
gators have described the maintenance                    forward. This method increases the
of airway epithelial cells in a differen-                yield of respiratory epithelia 8-fold
tiated state in primary culture (6,9–11).                over tracheal cultures. The composition
However, the total number of cells                       of murine nasal epithelium compares
that can be isolated from the mouse                      favorably to the nearly 90% ciliated
trachea is very small. The tracheal                      respiratory epithelium we see in the
                                                                                                                Figure 5. Olfactory marker protein (OMP) and
epithelium of the mouse has ciliated                     developed air-liquid interface culture.                type IV staining. (A and B) Immunofluorescence
cells that occur only in scattered                          Septal air-liquid interface cultures                [septal olfactory epithelium (OE; panel A)] and
patches (7). In an attempt to increase                   exhibit full differentiation with cilia                immunofluorescence (septal air-liquid interface
the yield of tracheal epithelial cells,                  and confluence with tight junctions.                   culture; panel B) for both OMP (green) and
Kumar et al. (12) has recommended                        OE, which comprises approximately                      type IV β-tubulin (red). As expected, the septal
                                                                                                                OE stains heavily for OMP, while the air-liquid
expansion of the population through                      half of the murine nasal septum, is                    interface culture reveals staining only for type
serum-free growth media. However,                        absent on the differentiated air-liquid                IV β-tubulin. Blue staining for nuclei is present
the expansion and passage of murine                      interface. The reasons for this are                    [4′,6-diamidino-2-phenylindole (DAPI)].

202 ı BioTechniques ı www.biotechniques.com                                                                                               Vol. 43 ı No. 2 ı 2007
Short Technical Reports


                                                                                                               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:
                                                                                                                  S29-S37.
                                                                                                              11. Lankford, S.M., M. Macchione, A.L. Crews,
                                                                                                                  S.A. McKane, N.J. Akley, and L.D. Martin.
Figure 6. Representative short circiu current (ISC) tracing of polarized murine nasal epithelial                  2005. Modeling the airway epithelium in al-
cells. Murine nasal epithelial cells grown on Transwell permeable supports were mounted in Ussing                 lergic asthma: interleukin-13-induced effects
chambers under short-circuit conditions and sequentially exposed to amiloride, forskolin, and glyben-             in differentiated murine tracheal epithelial
clamide. Note that baseline ISC is predominantly inhibited by amiloride and that forskolin-stimulated ISC         cells. In Vitro Cell. Dev. Biol. Anim. 41:217-
is partially blocked by glybenclamide. These data are consistent with the presence of both electrogenic           224.
sodium absorption and anion secretion.                                                                        12. Kumar, R.K., S.E. Maronese, and R.
                                                                                                                  O’Grady. 1997. Serum-free culture of mouse
conductance regulator (CFTR)-                          ACKNOWLEDGMENTS                                            tracheal epithelial cells. Exp. Lung Res.
dependent mechanism.                                                                                              23:427-440.
    The absolute value of our measured                    M.B.A. and B.A.W. contributed                       13. Grubb, B.R., R.N. Vick, and R.C. Boucher.
                                                                                                                  1994. Hyperabsorption of Na+ and raised
ISC is less than that calculated by others             equally to this work. This work was sup-                   Ca(2+)-mediated Cl- secretion in nasal epi-
(13). This difference may be due to                    ported by the Young Investigator Award                     thelia of CF mice. Am. J. Physiol. 266:C1478-
differences in mouse strain or age, or                 from the Sinus and Allergy Health                          C1483.
to differences in culture conditions.                  Partnership (N.A.C.), and NIH grant                    14. Adler, K.B., P.W. Cheng, and K.C. Kim.
                                                       nos. AI065450 (A.J.R.), K08HL081080                        1990. Characterization of guinea pig tracheal
Also, our experiments were performed                                                                              epithelial cells maintained in biphasic organo-
essentially at room temperature, which                 (J.L.K.), and R01 DK58046 (R.C.R.).                        typic culture: cellular composition and bio-
may have further contributed to this                                                                              chemical analysis of released glycoconjugates.
variation. Nevertheless, the overall                                                                              Am. J. Respir. Cell Mol. Biol. 2:145-154.
phenotype of ion transport in these                    COMPETING INTERESTS                                    15. Gray, T.E., K. Guzman, C.W. Davis,
                                                       STATEMENT                                                  L.H. Abdullah, and P. Nettesheim. 1996.
cells is consistent with an electrically                                                                          Mucociliary differentiation of serially pas-
active, mucociliary airway epithelium                                                                             saged normal human tracheobronchial epi-
containing both a sodium-absorptive                       The authors declare no competing                        thelial cells. Am. J. Respir. Cell Mol. Biol.
pathway and a chloride-secretory                       interests.                                                 14:104-112.
pathway (14,15).
    The increased yield and percentage
of ciliated respiratory epithelium                     REFERENCES
makes the murine nasal septa air-liquid                 1. You, Y., E.J. Richer, T. Huang, and S.L.           Received 9 December 2006; accepted
interface model ideal for the study of                     Brody. 2002. Growth and differentiation of         23 May 2007.
sinonasal and pulmonary diseases and                       mouse tracheal epithelial cells: selection of a
ciliary physiology. Air-liquid interface                   proliferative population. Am. J. Physiol. Lung
cultures grown from nasal septa exhibit                    Cell. Mol. Physiol. 283:L1315-L1321.               Address correspondence to Bradford A.
                                                        2. Doty, R.L. 1986. Odor-guided behavior in           Woodworth, Department of Otorhino-
a transepithelial potential. Thus,                         mammals. Experientia 42:257-271.
studying the growth and differentiation                 3. Halpern, M. and A. Martinez-Marcos. 2003.          laryngology, 3400 Spruce Street, 5 Ravdin,
of septal respiratory epithelium will                      Structure and function of the vomeronasal sys-     Philadelphia, PA 19104, USA. e-mail:
enable more physiologically relevant                       tem: an update. Prog. Neurobiol. 70:245-318.       Bradford.Woodworth@uphs.upenn.edu
                                                        4. Storan, M.J. and B. Key. 2006. Septal organ
analysis of sinonasal and pulmonary                        of Gruneberg is part of the olfactory system. J.
diseases in vitro, and allow for the                       Comp. Neurol. 494:834-844.                         To purchase reprints of this article, contact:
evaluation of novel therapies.                                                                                Reprints@BioTechniques.com
204 ı BioTechniques ı www.biotechniques.com                                                                                               Vol. 43 ı No. 2 ı 2007

								
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