Cellular and Molecular Bioengineering, Vol. 3, No. 3, September 2010 (Ó 2010) pp.

307–318
DOI: 10.1007/s12195-010-0118-y

Air–Liquid Interface Culture of Nasal Epithelial Cells

on Denuded Amniotic Membranes
NURIT EVEN-TZUR,1 ARIEL JAFFA,2,3 ZOYA GORDON,2 RUTH GOTTLIEB,1 YOEL KLOOG,4 SHMUEL EINAV,1
MICHAEL WOLF,3,5 and DAVID ELAD1
1
Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, 69978 Tel Aviv, Israel; 2Ultrasound Unit
in Obstetrics and Gynecology, Lis Maternal Hospital, Tel Aviv Medical Center, 64239 Tel Aviv, Israel; 3Sackler Faculty of
Medicine, Tel Aviv University, 69978 Tel-Aviv, Israel; 4Department of Neurobiology, Faculty of Life Sciences,
Tel Aviv University, 69978 Tel Aviv, Israel; and 5Department of Otorhinolaryngology, The Sheba Medical Center,
52621 Tel Hashomer, Israel
(Received 22 August 2009; accepted 11 March 2010; published online 6 April 2010)

Associate Editor David Odde oversaw the review of this article.

Abstract—Laboratory models of the respiratory lining with
mucin secreting epithelial goblet cells have been obtained by
culturing epithelial cells under air–liquid interface (ALI)
conditions, which require specific wells with semi-permeable
substrates. In this study nasal epithelial cells (NEC) were
successfully cultured on denuded amniotic membrane (AM)
under ALI conditions. The cells adhered well on both sides of
the denuded AM (i.e., the basement membrane or spongy
layer) and proliferated to confluency at the same time as cells
grown on a synthetic membrane. The cytoskeleton structure
of cells grown on denuded AMs appeared to be denser and
firmer than that of cells cultured on synthetic membranes.
Cultures on the denuded AM differentiated to contain goblet
cells which produce and secrete mucins and ciliated cells.
Cells cultured on the denuded AM were more stable under
airflow conditions than cells on the synthetic membranes.
The results of this study suggest that NEC culture on
denuded AM and under ALI conditions creates a stable and
well-differentiated in vitro model of the nasal lining for
laboratory studies.
Keywords—Cell adhesion, Cell biomechanics, Cytoskeleton,
Cell differentiation, Mucus secretion, Respiratory epithe-
lium, Wall shear stress (WSS).
INTRODUCTION
The pseudostratified ciliated columnar epithelium in
the nose lies on a basement membrane. Whereas only
ciliated cells and goblet cells reach the epithelial sur-
face, all nasal epithelial cells (NEC) are attached to the
supporting membrane.36 The thickness of the basement
Address correspondence to Nurit Even-Tzur, Department of
Biomedical Engineering, Faculty of Engineering, Tel Aviv University,
69978 Tel Aviv, Israel. Electronic mail: nuritet@eng.tau.ac.il
307
membrane ranges between 1.6 to 10 lm and seems to
be homogenous under a light microscope.23 Electron
microscopy reveals fine collagen fibers lying parallel to
the surface but running in differing directions. Immu-
nohistochemical investigations of the basement mem-
brane of nasal mucosa from the inferior nasal conchae
revealed components such as types I, III, IV, V and VI
collagen, laminin, nidogen and heparan sulfate pro-
teoglycan. This structure of the basement membrane
was presumed to contribute to the mechanical stabil-
ization of the epithelium.3
Human NEC grow naturally (i.e., in vivo) under
polar conditions; they are fed on their basal stromal
side while exhibiting specialized differentiated proper-
ties at the opposite apical side. While the basal side of
the cells is attached to a basement membrane, the
apical side of the nasal ciliated and goblet cells is
exposed to the oscillating respired airflow. The peri-
ciliary liquid and the mucus layers constitute the only
protective interface between these cells and the air
medium. The nasal epithelium polarity highly con-
tributes to its primary role as a barrier between the
respiratory system and the environment by enabling
the efficient function of mucociliary clearance.
In the 1980s, the need for an in vitro model of the
respiratory epithelium in which the cells differentiate
into goblet and ciliated cells under polar conditions led
to the development of air–liquid interface (ALI) cul-
tures.50 In such cultures, epithelial cells are seeded on a
semi-permeable membrane in a special well (e.g.,
Millicells, Millipore, Billerica, MA) and are fed with
growth medium underneath the membrane, while only
humidified air is present above the cells, similar to the
in vivo situation of respiratory epithelial cells.2,16 The
semi-permeable synthetic PolyTetraFluoroEthylene
1865-5025/10/0900-0307/0 Ó 2010 Biomedical Engineering Society
308 EVEN-TZUR et al.
(PTFE) membrane with hole size of 0.4 lm is widely
used nowadays.5,16,20,40,51 Very often, these mem-
branes are coated with extracellular matrix proteins
such as collagen for enhancing cell adherence, and the
culture medium is supplemented with retinoids.30,53
The chorioamnion surrounds the amniotic cavity
and separates the fetus from the maternal endome-
trium. The innermost layer is the amniotic membrane
(AM) which is composed of a monolayer of cuboidal
epithelial cells uniformly arranged on a thick basement
membrane and an avascular stroma which contains
types I, III, V and VI collagen and fibronectin.32,47 The
AM dominates the mechanical responses of the chor-
ioamnion and is permeable to specific substances.6,35
The inferior layer of the AM is the intermediate or
spongy layer which is rich with proteoglycans and
glycoproteins and contains a nonfibrillar meshwork of
mainly collagen type III. The spongy layer is loosely
connected to the chorionic membrane and thus the two
membranes can be easily separated.32
The potential of using the AM as a substrate for cell
cultures was recognized during the 1980s in studies on
normal cell invasion and migration,17,19,29,38 cell–
matrix interactions and adhesion,10,46,48 and in studies
on tumor cell invasion.26,41 In these studies, the cells
were seeded on the denuded AMs that were stripped
off from the monolayer of amniotic epithelial cells.
Tissue engineering applications in ophthalmology were
focused on testing the growth and differentiation of
conjunctival, corneal or retinal epithelial cells cultured
on denuded AMs.9,21,28 The AM can be used as a
scaffold for cell culture with or without the amniotic
epithelium and it can be used fresh or after cryopres-
ervation in 50% glycerol.
In this work, an in vitro model was developed for
ALI culture of NEC on the denuded AM. Since ALI
culture of NEC requires collagenic or collagen-coated
substrates, the denuded AM membrane is an adequate
option due to its biological content and good
mechanical properties which provide excellent condi-
tions for cell adhesion and growth. Biological charac-
teristics of the cultured cells are demonstrated as well
as their response to the mechanical loading of nasal
airflows.
MATERIALS AND METHODS
Preparation of the Amniotic Membrane
Harvesting of human AMs from full term placentas
was approved by the ethical committee of Tel Aviv
Sourasky Medical Center (# 06/376). Immediately
after removal from the placenta, the AM was incu-
bated at 4 °C in minimum essential medium (MEM)
containing 2 mM L-glutamine, 0.04 mg/mL gentamicin,
100 U/mL penicillin and 100 lg/mL streptomycin
(Figs. 1a–1c). The amniotic epithelial cells were
removed from the AM within 4–5 h after delivery
according to the following protocol38: the AM was
rinsed 3 times for 5 min in 200 mL of ice-cold PBS-
Gentamicin (50 mg/L). Then, the AM was incubated in
1.7% NH4OH solution, at room temperature (RT), for
2 h on a rocker. The amniotic epithelial cells were then
mechanically removed from the AM using a scraper
(Fig. 1d) and the denuded AM was well washed in
double distilled water (DDW). The denuded AM was
either used immediately for cell culture or cryopreserved
in 50:50 DMEM and Glycerol at 70 °C until use.
Thawing the AMs was done at 37 °C bath and followed
by vigorous washing with DDW to remove glycerol
residues. The denuded AM was mounted in custom-
designed wells for cell seeding.
Custom-Designed Wells for ALI Cultures
In this study we used custom-designed wells for ALI
culturing of airway epithelial cells that can hold any
type of membrane including the denuded AM and
synthetic membranes (Figs. 1e–1i).14 The denuded AM
was stretched between two stainless steel rings and the
residues around the rings were cut with scissors
(Figs. 1f and 1g). The custom wells can be disassem-
bled into sub-units, i.e. medium-holder and well-bot-
tom (Fig. 1h). The well-bottom with the cultured cells
can be then installed in a flow chamber for application
of flow on the cell surface.14 Upon completion of a set
of experiments, the well can be re-assembled for either
biological treatments of the cells or for further culture
incubation for additional experiments.
Isolation and Culture of Nasal Epithelial Cells
Human NEC were isolated from specimens of nasal
turbinates of patients undergoing endonasal surgical
intervention as approved by the ethical committee of
The Sheba Medical Center (#2788/2002). The cells
were then cultured on the denuded AM under ALI
conditions as previously described.14–16,37 The epithe-
lial cells were dissociated from the excised turbinates
after incubation in pronase solution and scraping of
the cells from the tissues. The cell suspension was then
centrifuged, washed and resuspended in a serum-free
hormone culture bronchial epithelial growth medium
(BEGM) supplemented with additives.37 Primary cells
were plated in BEGM on collagen coated tissue culture
plastic dishes at density of 0.5–6 9 106 cells per dish.
After 24 h the dishes were washed with PBS and the
cells were fed with BEGM. Thereafter, the medium
was changed every 2–3 days. When primary cultures
reached 70–90% confluence (usually after 4–6 days),
 Culture of Nasal Cells on Amniotic Membranes 309

FIGURE 1. Detailed description of the preparation of the denuded amniotic membrane (AM) for air–liquid interface cell culture.
(a) A whole placenta with placental membranes, several minutes after term delivery; (b) Removal of the AM from the placenta;
(c) Washing the AM and incubation for removal of the amniotic epithelial cells; (d) Mechanical scraping of amniotic epithelial cells;
(e) The denuded AM and stainless steel rings of the custom-designed well; (e, f, g) Series of actions for mounting the denuded AM
in the well-bottom of the custom-designed well; (h) The disassembled custom-designed well. The well bottom allows performance
of experiments with physical stresses applied on the cells; (i) The assembled well in a tissue culture plastic dish.

the cells were trypsinized, washed, counted and plated
at density of 100–150 9 103 cells per 0.8 cm2 mem-
brane on the denuded AM or on collagen type IV
coated PTFE porous membranes which were cut from
30 mm Millicell inserts (Millipore, PICM03050). The
membranes were then installed in the custom-designed
wells for ALI culturing. The cells seeded in the wells
were fed with ALI medium.37 After 2–3 days, the
medium from the apical side of the membrane was
removed in order to allow ALI conditions, in which the
cells were fed only from the basal part of the mem-
brane, while the apical side was exposed to 5% carbon
dioxide enriched air.
Histology
Histological sectioning and Hematoxylin and Eosin
(H&E) staining were performed following fixation of
the cells on either the denuded AM or the synthetic
membrane with 4% paraformaldehyde and incubation
in formalin for 24 h. After several washes and solution
treatments, the well bottom was disassembled and the
membranes were embedded in paraffin. Sections of
3–5 lm thickness were cut from the paraffin block
using a microtome and mounted on a glass slide for
melting the paraffin and staining with H&E.
Immunofluorescent Stain of Microtubules, Actin
Filaments and Intracellular Mucins
Fixation of the cells for all the different immuno-
fluorescent stains was performed by incubation in 4%
paraformaldehyde for 10 min at RT followed by three
washes in PBS. Cell membrane perforation was
achieved by incubation in a 5% dilution of donkey
serum in 0.05% Tween 20 in PBS (PBST) for 4–5 h
while on a rocker (60 RPM) at RT. b-Tubulin fibers
were stained using 1:500 dilution of mouse monoclonal
anti-b-tubulin primary antibody, clone 2.1 (Sigma–
Aldrich, Rehovot, Israel). The secondary antibody was
donkey Rhodamine conjugated anti-mouse secondary
antibody (Jackson Immunoresearch Laboratories,
310 EVEN-TZUR et al.
West Grove, PA, USA), at a 1:400 dilution. Actin
filaments were stained with 1:1000 dilution of FITC
labeled Phalloidin in PBS.49 Intracellular mucins were
stained using 1:100 dilution of mouse monoclonal anti-
MUC5AC primary antibody, clone 45M1 (Thermo
Scientific, Runcorn, UK). The secondary antibody was
Cy-5 conjugated donkey anti-mouse secondary anti-
body (Jackson Immunoresearch Laboratories, West
Grove, PA, USA), at a 1:400 dilution. Cell nuclei were
stained using DAPI.
During the staining procedures, the custom-made
wells were mounted in 12-well plate filled with the
required solutions both inside and outside of the wells
in order to expose both basal and apical parts of the
cells to the liquids. Then, the custom-made wells were
disassembled and the membranes were mounted on
microscope glass slides. Cells were examined under a
Zeiss 410 confocal microscope, using a 940 magnify-
ing water objective.
Quantification of Mucin Secretion
Quantification of mucin secretion was performed
using an enzyme-linked lectinosorbent assay (ELLA)
that measured the reactivity of glycoproteins equiva-
lent to MUC5B.1 The assay was slightly modified as it
was based on peroxidase labeled wheat germ agglutinin
(WGA) lectin instead of soya-bean agglutinin lectin.
Mucin was collected by gently adding 150 lL of PBS
to the luminal surface of each culture in the custom-
designed wells. After incubation for 15 min at 37 °C all
the apical liquid, including mucin secretions, was col-
lected using a micropipettor with a thin tip. Calibra-
tion for absolute values of mucin concentration was
performed with MUC5B mucin standards that were
generously provided by Prof. C. W. Davis from The
University of North Carolina Cystic Fibrosis Pul-
monary Research and Treatment Center, NC, USA.
Serial dilutions of 0, 15.6, 31.3, 62.5, 125, 250, 500, and
1000 ng MUC5B per 1 mL PBS were used for the
standard curve. Mucin samples were diluted 1:400 in
PBS and 100 lL samples were transferred to a 96-well
plate with high binding property and incubated at
37 °C for 2 h. The wells were then washed four times
with 200 lL/well PBST. Then, 100 lL dilution of
0.5 lg peroxidase labeled WGA-lectin in 1 mL PBST
was added to each well and the plate was incubated for
1 h at 37 °C. Four washes of 200 lL/well PBST fol-
lowed. The color reaction was obtained by adding
150 lL of substrate (40 mg OPD in 100 mL Citrate
Phosphate Buffer, spiked with 40 lL of 30% Hydro-
gen Peroxide) to each well and keeping at RT for
15 min. The reaction was stopped by adding 50 lL
4 M sulfuric acid to each well. The absorbance
was read at 490 nm using a microtiter plate reader
(SpectraMax 340PC384, Molecular Devices Corp.).
Equivalent MUC5B reactivity was determined from a
polynomial curve fitted to the mucin standards mea-
surements. Mucin secretion results are expressed as
mean ± standard deviation of absolute concentration
in lg/mL. Comparison between groups was performed
by Student’s t test. p-Value of less than 0.05 was con-
sidered statistically significant.
Application of Flow Forces on ALI Cultured Cells
A special flow chamber was developed in our lab for
airflow studies with NEC cultured under ALI condi-
tions in the custom-designed wells.14,15 The chamber is
an 18 cm long conduit with a rectangular cross-sec-
tional area of 20 mm 9 10 mm that can be connected
to inlet and outlet tubes. The chamber base can host up
to 3 well-bottoms in carefully designed holes which are
filled with culture medium that is in contact with the
inferior plane of the membrane in the well-bottom
throughout the experiment. The flow chamber was
connected to an air source via a 2 m long silicon tube
to ensure fully developed airflow over the cultured
cells. The chamber’s exit was also connected to a 20 cm
long silicon tube in order to prevent exit disturbances.
A series of airflow experiments was conducted with
well-differentiated 7–14 days ALI cultured cells on
either the denuded AM or the synthetic PTFE mem-
branes. Two steady uniform airflows that generated
wall shear stresses (WSS) of 1 and 20 dyne/cm2 were
applied for 15 min on the surface of the cultured cells
at room environment. Immediately after the flow
experiments the cultures were fixated using 4% para-
formaldehyde for histological examination.
All the characterization studies and flow experi-
ments described in this paper were repeated three
times, each with cells from a different subject and
triplicate cultures were always considered. One excep-
tion is the cell density study which was performed with
cells from one subject only (as described and discussed
in the ‘‘Results’’ and ‘‘Discussion’’ sections), however,
triplicates were considered. All the cells used for a
specific study/experiment were seeded on a denuded
AM from one subject.
RESULTS
A histological section of an intact human AM is
presented in Fig. 2a where the monolayer of amniotic
epithelial cells lies on top of the basement membrane.
A section of a denuded AM is presented in Fig. 2b,
where the basement membrane can be identified as a
dark line with respect to the compact and spongy
layers. Examples of human NEC cultured on both
 Culture of Nasal Cells on Amniotic Membranes 311

FIGURE 2. Images of histological sections: (a) Intact human amniotic membrane (AM); (b) Denuded AM; (c) 7 days air–liquid
interface (ALI) culture of nasal epithelial cells (NEC) seeded on the basement membrane side of a denuded AM; (d) 7 days ALI
culture of NEC seeded on the spongy layer side of a denuded AM; (e) 7 days ALI culture of NEC seeded on a PolyTetraFluoro-
Ethylene (PTFE) synthetic membrane (SM). Bar = 10 lm.

sides of the denuded AM (i.e., the basement membrane
and the spongy layer sides) are shown in the histo-
logical sections in Figs. 2c and 2d in comparison to
cultures on a PTFE synthetic membrane (Fig. 2e).
These 7 days ALI cultures clearly demonstrate that the
cells adhered and grew well on both the basement
membrane (Fig. 2c) or the spongy layer sides (Fig. 2d)
of the denuded AM. In addition, the cells on the de-
nuded AM were more hypertrophic (Figs. 2c and 2d)
and in many cases, although not always, were more
columnar than the cells cultured on the synthetic
membranes, which in all cases appeared to be flat
(Fig. 2e). Light microscope images of cells cultured on
both the denuded AM and the synthetic PTFE mem-
brane over a time period of 14 days revealed that the
cultures became confluent after 4 days of ALI culture
(Fig. 3). These images also strengthen the observation
that the cells on the denuded AM are bigger than the
cells on the synthetic membranes (see enlargements in
Fig. 3).
Images of stained actin filaments and b-tubulin
fibers in NEC cultured under ALI conditions on either
denuded AM or on synthetic PTFE membranes are
depicted in Fig. 4. Both the b-tubulin and the actin
filaments are organized in a denser network and are
more intact in cells grown on the denuded AM than in
cells on the synthetic membranes. The b-tubulin and
the actin in cells on the AM were more filamentous
(Figs. 4a and 4c) whereas those in cells cultured on
the synthetic membrane seem to be grainier (Figs. 4b
and 4d). Moreover, actin staining in cells on the syn-
thetic membranes appeared mainly in the cortex of the
cells with only slight background staining in the cell
cytoplasm (Fig. 4d), while cortical actin and cyto-
plasmic stress fibers were strongly stained in cells cul-
tured on the denuded AM (Fig. 4c). The cells on the
denuded AM were organized as a confluent monolayer
of cells with clear cell–cell contacts, but their bound-
aries were less distinguishable in the actin images than
those on the synthetic membrane.
The number of NEC cultured under ALI conditions
on the denuded AMs was estimated by counting DAPI
stained nuclei in five fields of 210 lm 9 210 lm in
each culture under investigation. The averaged cell
number was 81 ± 20, 89 ± 10, 77 ± 13, and 85 ± 8
cells per field in 0, 7, 14, and 21 days ALI cultures,
respectively. The number of epithelial goblet cells over
time was estimated by counting cells that were posi-
tively stained for intracellular mucins in ten fields of
210 lm 9 210 lm and dividing by the total number of
cells in the field. The averaged percentage of goblet
cells per field in 0, 7, 14, and 21 days ALI cultures on
the denuded AMs is shown in Fig. 5a. It can be seen
that the percentage of goblet cells in the cultures
increased over time, reaching a plateau on the 14th day
of ALI culture. Representative images of intracellular
mucin stains in 0, 7, 14, and 21 days ALI cultures are
presented in Figs. 5b–5e.
Mucin secretion onto the epithelial surface during
ALI culture of NEC on the denuded AM increased
312
Nasal epithelial cells on Nasal epithelial cells on collagen
denuded amniotic membrane coated synthetic membrane
Day 0
Day 1
Day 2
D 4
Day
Day 7
Day 14
FIGURE 3. Light microscope images of cells cultured on the
denuded amniotic membrane and on synthetic PolyTetra-
FluoroEthylene (PTFE) membranes under air-liquid interface
conditions over a time period of 14 days. Enlargements are of
150%.
over time, reaching a plateau on the 14th day of ALI
culture (Fig. 5f). Mucin secretion over time was also
investigated in a single study as a function of cell
seeding density. Cells were seeded on the denuded AM
at densities of 50,000, 150,000, and 300,000 cells per
membrane and mucin secretion was measured over
time. In all cases mucin secretion was increased with
time until day 14 and then remained constant or
slightly decreased, except for one case where the cul-
ture with seeding density of 300,000 cells per mem-
brane reached a maximal secretion peak on day 7 and
then decreased slightly on day 14. This may suggest
that at a relatively high cell seeding density, the goblet
cells in the culture matured faster and reach their
maximal secretion ability earlier. There was no signif-
icant difference between mucin secretions on a specific
day from cultures with different cell seeding densities.
Cilia presence was detected using high magnification
scanning electron microscope images. Hundreds of
EVEN-TZUR et al.
ciliated cells were observed in fields of 1 mm 9 1 mm
in 14 days ALI cultures of NEC on the denuded AM.
Cilia on a single cell in a 14 days ALI culture on the
denuded AM are demonstrated in Fig. 6.
Flow experiments were performed with NEC cul-
tured under ALI conditions on denuded AMs and
synthetic membranes. Steady uniform WSS of 1 and
20 dyne/cm2 were applied on the cultured cells for
15 min. Histological sections of static and stimulated
cultures are shown in Fig. 7. It can be seen that the
cells remain attached both to the denuded AM and the
synthetic membrane after application of WSS of
1 dyn/cm2 for 15 min. However, when WSS was
increased to 20 dyn/cm2 and also applied for 15 min, it
resulted in detachment of the cells from the synthetic
membrane while cells on the denuded AM remained
attached.
DISCUSSION
Human NEC were successfully cultured on denuded
AMs under ALI conditions and proliferated to become
confluent at the same time as cells grown on PTFE
synthetic membranes. The cells adhered well on either
the basement membrane or the spongy layer sides of
the AMs. The cytoskeleton structure of cells grown on
the denuded AM appeared denser and firmer than that
of cells cultured on the synthetic membrane. Cultures
on the denuded AM differentiated to contain goblet
cells which produce and secrete mucins and ciliated
cells. After loading of steady airflow conditions the
cells were more stable on the denuded AM than on the
synthetic membrane.
Human NEC cultured on denuded AMs for
7–14 days had a hypertrophic appearance (Figs. 2c
and 2d), which resembles the morphology of these cells
in vivo.44 The cells on the denuded AMs were columnar
in most cases (Figs. 2c and 2d), although occasionally
they appeared flat (Fig. 7a). This outcome may result
from structural inhomogeneity of the AM due to dif-
ferent local molecular composition or fiber density, or
it may be related to the denuding process. Recently,
alternative methods of optimizing the preparation of
the AM were implemented,18,52 which may result in
more uniform or homogeneous morphology of the
cultured cells.
The actin filaments and b-tubulin fibers appeared
denser and more filamentous in cells cultured on the
denuded AM than in cells cultured on the synthetic
membrane. The fact that the cell boundaries were more
blurred in the images of cells on the denuded AM
(Figs. 4a, 4c, and 4e) than in cells on the synthetic
membrane (Figs. 4b, 4d, and 4f) might indicate on
better cell–cell adhesion in cells on the denuded AM.
 Culture of Nasal Cells on Amniotic Membranes 313

Nasal epithelial cells on Nasal epithelial cells on collagen

denuded amniotic membrane coated synthetic membrane

(a) (b)

β-tubulin

(c) (d)
Actin

(e) (f)
Merge

FIGURE 4. Stains of actin microfilaments (green) and b-tubulin fibers (red) in nasal epithelial cells cultured under air–liquid
interface conditions on the denuded amniotic membrane and on PolyTetraFluoroEthylene (PTFE) synthetic membranes. The cell
nuclei were stained with DAPI (blue). Bar = 10 lm.

In cultures on the denuded AM many more stress
fibers were observed whereas in cultures on the syn-
thetic membrane most of the actin filaments were
located underneath the cell membrane constituting the
cortical actin. Stress fibers are known to be involved in
cellular processes such as adhesion, motility and mor-
phogenesis.31 In epithelial cells, cadherin cell–cell
adhesion receptor complexes are essential for the
establishment of the epithelial cell shape and mainte-
nance of the differentiated epithelial phenotype. These
complexes are associated with actin filaments to
provide points of attachment for the cytoskeleton at
the membrane, which are important for the develop-
ment of mechanical stress during epithelial compaction
and polarization.7 The presence of stress fibers in cell
cultured on the denuded AMs may suggest their better
attachment to the substrate in comparison to the cells
on the synthetic membrane, as suggested by the results
of airflow experiments (Fig. 7).
Previously, it has been reported that actin microfil-
aments of the cytoskeleton are involved in agonist
induced mucin secretion from airway surface goblet
314 EVEN-TZUR et al.

(a)
100

Goblet Cells per Field [%]

80 *
60

40

20
*
0
0 7 14 21
Days of ALI Culture on Denuded AM

(b) (c) (d) (e)

(f) 12000 *
etion [µg/ml]

10000

8000

6000
*
Mucin Secre

4000

2000

0
0 1 2 4 7 14 21
Day of ALI Culture on Denuded AM

FIGURE 5. (a) Percentage of goblet cells per 210 lm 3 210 lm field in cultures of nasal epithelial cells on the denuded amniotic
membrane (AM) on days 0, 7, 14, and 21 of air–liquid interface cultures. * p < 0.05; (b–e) Images of intracellular mucins of the same
cultures in (a) on days 0, 7, 14, and 21, respectively; (f) Mucin secretion on different days from the same cultures in (a). * p < 0.05.

cells.39 Interactions of dephosphorylated myristoylated suggest that the epithelial phenotype of these cells is
alanine-rich C kinase substrate (MARCKS) with actin more substantial.
and myosin create the linkage of mucin granules to The elastic modulus of the AM is about 25 MPa
intracellular contractile apparatus, which is able to and its tensile failure strength is approximately
move them through the cell cytoplasm toward the 5 MPa.8 Recent studies have shown that the sub-
apical membrane. Moreover, actin filaments were strate’s elasticity affects the adhesion characteristics,
shown to maintain a barrier in mucin secretion from the dynamics of cell spreading, cell differentiation and
intestinal goblet cells by hindering mucin granule the cytoskeleton assembly.11,13 For example, fibro-
access to the plasma membrane.34 Microtubules are blasts have more organized F-actin and stress fibers
well known for having an important role in vesicular when cultured on increasingly stiffer substrates.
transport in cells in general27 and specifically in Neurons on the other hand branch more on softer
polarized epithelial cells.22,42 Thus, the fact that the substrates.11 Based on these results it may well be that
actin microfilaments and microtubules are more pro- the differences in the actin structure of cultured NEC
nounced in cells cultured on the denuded AM may on denuded AMs and synthetic membranes (Figs. 4c
 Culture of Nasal Cells on Amniotic Membranes 315

and 4d) are also due to the greater elasticity of the
AM.
The change in the total number of cells in ALI
cultures on the denuded AM was minor over 21 days.
On the other hand, the number of cells that were
positively stained for intracellular mucins increased
gradually until day 14, which reflects the increase
in goblet cell percentage in the cultures over time
(Figs. 5a–5e). Mucin secretion in cultures on the
denuded AM also increased gradually over time,
reaching a plateau on the 14th day of ALI culture
(Fig. 5f). The resemblance in the patterns of Figs. 5a
and 5f suggests that there is a good correlation between
the percentage of goblet cells in the culture over time
FIGURE 6. Cilia on a single cell in 14 days air–liquid inter-
face culture of nasal epithelial cells on the denuded amniotic
membrane. Bar = 1 lm.
and the amount of mucin secretion over time. The
percentage of goblet cells in the culture on the denuded
AM after 14 and 21 days are similar in magnitudes to
measurements performed by others in cultures of
human NEC on polyester membranes 14 and 28 days
after confluence.54 The results of mucin secretion
(Fig. 5f) are also in a very good agreement with mea-
surements performed on the same study, in which
mucin secretion increased over time and reached a
plateau on the 14th day of culture.54 In another study,
guinea pig tracheal epithelial cells were cultured on
denuded AMs under ALI conditions and presented
height and ratio of ciliated epithelial cells similar to
those in normal guinea pigs epithelium, however, no
goblet cells were identified.33 While the denuding
procedure in that work was similar to the method used
here (i.e. incubation of the intact AM in NH4OH and
mechanical scraping), in the current work goblet cells
have been identified using the intracellular mucin stain
and measurements of mucin secretion from the cultures.
Physiological WSS in the nasal cavity due to airflow
during quiet breathing were computed to be up to
15 dyn/cm2.12 Several studies investigated the biologi-
cal response of cultured airway and NEC to airflow
induced WSS, and specifically the changes in mucin
secretion and the cytoskeleton due to such stim-
uli.14,15,45 In the current study we examined the strength
of cells attachment to the substrate by applying differ-
ent levels of WSS on top of the different cultures. The
application of a relatively high WSS of 20 dyn/cm2
induced detachment of the cells from the synthetic
membrane while they remained attached to the denuded

Nasal epithelial cells on denuded Nasal epithelial cells on collagen

amniotic membrane coated synthetic membrane
(a) (b)
NEC
NEC

Denuded AM 10 μm 10 μm
SM

Static conditions (control)

(c) (d)
NEC
NEC

10 μm 10 μm
Denuded AM SM

After WSS of 1 dyne/cm2

(e) (f)
NEC
NEC

10 μm 10 μm
Denuded AM SM
After WSS of 20 dyne/cm2

FIGURE 7. Images of histological sections of cultured nasal epithelial cells that were exposed to airflow that generated steady
uniform WSS of 1 and 20 dyne/cm2 for 15 min in comparison to static conditions. Cell culture was done under air–liquid interface
conditions on both denuded amniotic membrane (AM) and synthetic membranes (SM). Bar = 10 lm.
316 EVEN-TZUR et al.
AM. This suggests that the adhesion of NEC to the
denuded AM is better than to the synthetic membrane.
While flow studies can be used for studying cells resis-
tance to detachment from substrates, further studies on
expression of adhesion molecules, location and number
of adhesion sites and more mechanical tests should be
performed to study the forces generated between the
cells and the denuded AM matrix.
Using the human AM as a substrate for cell cul-
turing for various applications has many advantages
due to its great mechanical integrity and excellent
molecular composition for development of cell adhe-
sion complexes. Specifically to our long-term goal of
studying the biological response of NEC to airflow
induced WSS, this new culture system is essential. The
need to develop this system emerged when application
of high WSS on cells cultured on synthetic membranes
resulted in detachment of the cells from the membrane
(as shown in Fig. 7f). Using the AMs allowed us to
perform experiments with realistic high WSS that exist
in vivo in the nasal cavity during different modes of
breathing. The utilization of the denuded AM as a
substrate for cell culture may also be beneficial for
other applications, especially when loading of a flow-
ing fluid is applied on the cell culture.
The collagen composition of the denuded AM is
naturally better than that of the collagen coated syn-
thetic membranes, yet the permeability of the denuded
AM, which has a key importance in ALI cultures, is
clearly not as uniform and repeatable as that of the
synthetic membranes. The thickness of the denuded
AM ranges from tens to hundreds of microns,6 and
thus, its permeability is expected to vary across the
membrane surface. Nevertheless, the observations of
this study show that NEC grew on most of the denuded
AMs area which indicates that permeability of nutrients
across the membrane is probably sufficient for cell
growth. The permeability of chorioamniotic mem-
branes was investigated for sodium, calcium, magne-
sium and thyrotrophin-releasing hormone,4,24,25 as well
as for the transfer of water across the AM due to
osmotic gradients.43 However, these studies were con-
ducted with intact membranes as opposed to denuded
AMs. During in vitro ALI culture of cells on denuded
AMs the entire supply of nutrition is from the medium
underneath the membrane. Thus, it will be essential to
perform comprehensive studies on permeability of
denuded AMs to culture medium components.
In conclusion, it has been demonstrated in this study
that denuded AM can serve as a very good substrate
for NEC. Cultures of NEC on the denuded AM dif-
ferentiated to contain goblet cells which produce and
secrete mucins and ciliated cells. The cells adhered well
on both sides of the denuded AM (either on
the basement membrane or the spongy layer) and
proliferated to become confluent at the same time as
cells grown on synthetic membranes. Cells on the
denuded AM were found to be more stable under
airflow conditions than cells on the synthetic mem-
branes. The results of this study suggest that using the
denuded AM as a substrate for NEC culture under
ALI conditions creates a well differentiated respiratory
epithelial cell model for in vitro biological or biome-
chanical studies.
ACKNOWLEDGMENTS
The authors thank Prof. Scott Randell and Prof.
C. William Davis from The University of North Car-
olina at Chapel Hill, NC, USA, for the useful advice
regarding nasal epithelial cell culture and mucin
quantification assays. We would also like to thank
Dr. Uri Zaretsky, Mrs. Dalit Shav and Mrs. Riki
Levkovitz from the Department of Biomedical Engi-
neering in Tel Aviv University for their valuable input
to this project. This work was partially supported by a
grant from the Ela Kodesz Institute for Biomedical
Engineering and Medical Physics at Tel Aviv Univer-
sity, by a generous donation from the Australian
Friends of Tel Aviv University (Vic) and by the Ber-
man Trust.
REFERENCES
1
Abdullah, L., S. W. Davis, L. Burch, M. Yamauchi, S. H.
Randell, P. Nettesheim, and C. W. Davis. P2u purinoceptor
regulation of mucin secretion in SPOC1 cells, a goblet cell
line from the airways. Biochem. J. 316:943–951, 1996.
2
Adler, K. B., P. W. Cheng, and K. C. Kim. Character-
ization of guinea pig tracheal epithelial cells maintained in
biphasic organotypic culture: cellular composition and
biochemical analysis of released glycoconjugates. Am. J.
Respir. Cell Mol. Biol. 2:145–154, 1990.
3
Agha-Mir-Salim, P., O. Rauhut, and H. J. Merker. Elec-
tron and fluorescence microscopic investigations on com-
position and structure of the epithelial basement
membrane of the human inferior concha. Eur. Arch. Oto-
laryngol. 250:401–407, 1993.
4
Bajoria, R., T. A. Ryedr, and N. M. Fisk. Transport and
metabolism of thyrotrophin releasing hormone across the
fetal membrane. J. Clin. Endocrinol. Metab. 82:3399–3407,
1997.
5
Booth, B. W., T. Sandifer, E. L. Martin, and L. D. Martin.
IL-13-induced proliferation of airway epithelial cells:
mediation by intracellular growth factor mobilization and
ADAM17. Respir. Res. 8:51–63, 2007.
6
Bourne, G. L. The anatomy of the human amnion and
chorion. Proc. R. Soc. Med. 59:1127–1128, 1960.
7
Braga, V. Epithelial cell shape: cadherins and small GTP-
ases. Exp. Cell Res. 261:83–90, 2000.
8
Calvin, S. E., and M. L. Oyen. Microstructure and
mechanics of the chorioamnion membrane with an
 Culture of Nasal Cells on Amniotic Membranes
emphasis on fracture properties. Ann. N. Y. Acad. Sci.
1101:166–185, 2007.
9 24
Capeáns, C., A. Piñeiro, M. Pardo, C. Sueiro-López, M. J.
Blanco, F. Domı́nguez, and M. Sánchez-Salorio. Amniotic
membrane as support for human retinal pigment epithe-
lium (RPE) cell growth. Acta Ophthalmol. Scand. 81:271–
277, 2003.
10
Davis, G. E., E. Engvall, S. Varon, and M. Manthorpe.
Human amnion membrane as a substratum for cultured
peripheral and central nervous system neurons. Brain Res.
430:1–10, 1987.
11
Discher, D. E., P. Janmey, and Y. L. Wang. Tissue cells feel
and respond to the stiffness of their substrate. Science
310:1139–1143, 2005.
12
Elad, D., S. Naftali, M. Rosenfeld, and M. Wolf. Physical
stresses at the air-wall interface of the human nasal cavity
during breathing. J. Appl. Physiol. 100:1003–1010, 2006.
13
Engler, A. J., H. L. Sweeney, D. E. Discher, and J. E.
Schwarzbauer. Extracellular matrix elasticity directs stem
cell differentiation. J. Musculoskelet. Neuronal Interact.
7:335, 2007.
14
Even-Tzur, N., D. Elad, U. Zaretsky, S. H. Randell,
R. Haklai, and M. Wolf. Custom-designed wells and flow
chamber for exposing air–liquid interface cultures to wall
shear stress. Ann. Biomed. Eng. 34:1890–1895, 2006.
15
Even-Tzur, N., Y. Kloog, M. Wolf, and D. Elad. Mucus
secretion and cytoskeletal modifications in cultured nasal
epithelial cells exposed to wall shear stresses. Biophys. J.
95:2998–3008, 2008.
16
Fulcher, M. L., S. Gabriel, K. A. Burns, J. R. Yankaskas,
and S. H. Randell. Well-differentiated human airway epi-
thelial cell cultures. Methods Mol. Med. 107:183–206, 2005.
17
Furie, M. B., M. C. Tancinco, and C. W. Smith. Mono-
clonal antibodies to leukocyte integrins CD11a/CD18 and
CD11b/CD18 or intercellular adhesion molecule-1 inhibit
chemoattractant-stimulated neutrophil transendothelial
migration in vitro. Blood 78:2089–2097, 1991.
18
Hopkinson, A., V. A. Shanmuganathan, T. Gray, A. M.
Yeung, J. Lowe, D. K. James, and H. S. Dua. Optimization
of amniotic membrane (AM) denuding for tissue engi-
neering. Tissue Eng. Part C: Methods 14:371–381, 2008.
19
Kallenbach, K., H. A. Fernandez, G. Seghezzi, F. G.
Baumann, S. Patel, E. A. Grossi, A. C. Galloway, and
P. Mignatti. A quantitative in vitro model of smooth
muscle cell migration through the arterial wall using the
human amniotic membrane. Arterioscler. Thromb. Vasc.
Biol. 23:1008–1013, 2003.
20
Kesimer, M., S. Kirkham, R. J. Pickles, A. G. Henderson,
N. E. Alexis, G. DeMaria, D. Knight, D. J. Thornton, and
J. K. Sheehan. Tracheobronchial air–liquid interface cell
culture: a model for innate mucosal defense of the upper
airways? Am. J. Physiol. Lung Cell. Mol. Physiol. 296:L92–
L100, 2009.
21
Koizumi, N., N. J. Fullwood, G. Bairaktaris, T. Inatomi,
S. Kinoshita, and A. J. Quantock. Cultivation of corneal
epithelial cells on intact and denuded human amniotic
membrane. Invest. Ophthalmol. Vis. Sci. 41:2506–2513,
2000.
22
Kreitzer, G., J. Schmoranzer, S. H. Low, X. Li, Y. Gan,
T. Weimbs, S. M. Simon, and E. Rodriguez-Boulan. Three-
dimensional analysis of post-Golgi carrier exocytosis in
epithelial cells. Nat. Cell Biol. 5:126–136, 2003.
23
Lang, J. Clinical Anatomy of the Nose, Nasal Cavity and
Paranasal Sinuses. Stuttgart–NewYork: George Thieme
317
Verlag; and New York: Thieme Medical Publisher, Inc.,
1989.
Lemancewicz, A., H. Laudanska, T. Laudanski,
A. Karpiuk, and S. Barta. Permeability of fetal membrane
to calcium and magnesium: possible role in preterm labor.
Hum. Reprod. 15:2018–2022, 2000.
25
Leontic, E. A., and J. E. Tyson. Prolactin and fetal
osmoregulation: water transport across isolated human
amnion. Am. J. Physiol. 232:R124–R127, 1977.
26
Liotta, L. A., C. W. Lee, and D. J. Morakis. New method
for preparing large surfaces of intact human basement
membrane for tumor invasion studies. Cancer Lett. 11:141–
152, 1980.
27
Lodish, H., D. Baltimore, A. Berk, S. L. Zipursky,
P. Matsudaira, and J. Darnell. Molecular Cell Biology.
New York: Scientific American, 1997.
28
Meller, D., and S. C. Tseng. Conjunctival epithelial cell
differentiation on amniotic membrane. Invest. Ophthalmol.
Vis. Sci. 40:878–886, 1999.
29
Mignatti, P., R. Tsuboi, E. Robbins, and D. B. Rifkin.
In vitro angiogenesis on the human amniotic membrane:
requirement for basic fibroblast growth factor-induced
proteinases. J. Cell Biol. 108:671–682, 1989.
30
Moller, P. C., L. R. Partridge, R. Cox, V. Pellegrini, and
D. G. Ritchie. An in vitro system for the study of tracheal
epithelial cells. Tissue Cell 19:783–791, 1987.
31
Naumanen, P., P. Lappalainen, and P. Hotulainen.
Mechanisms of actin stress fibre assembly. J. Microsc.
231:446–454, 2008.
32
Niknejad, H., H. Peirovi, M. Jorjani, A. Ahmadiani,
J. Ghanavi, and A. M. Seifalian. Properties of the amniotic
membrane for potential use in tissue engineering. Eur. Cell
Mater. 15:88–99, 2008.
33
Noguchi, Y., Y. Uchida, T. Endo, H. Ninomiya,
A. Nomura, T. Sakamoto, Y. Goto, S. Haraoka,
T. Shimokama, T. Watanabe, and S. Hasegawa. The
induction of cell differentiation and polarity of tracheal
epithelium cultured on the amniotic membrane. Biochem.
Biophys. Res. Commun. 210:302–309, 1995.
34
Oliver, M. G., and R. D. Specian. Cytoskeleton of intes-
tinal goblet cells: role of actin filaments in baseline secre-
tion. Am. J. Physiol. 259:G991–G997, 1990.
35
Oxlund, H., R. Helmig, J. T. Halaburt, and N. Uldbjerg.
Biomechanical analysis of human chorioamniotic mem-
branes. Eur. J. Obstet. Gynecol. Reprod. Biol. 34:247–255,
1990.
36
Proctor, D. F., and I. Andersen. The Nose, Upper Airway
Physiology and the Atmospheric Environment. Amster-
dam: Elsevier Biomedical Press, 1982.
37
Randell, S. H., D. L. Walstad, U. E. Schwab, B. R. Grubb,
and J. R. Yankaskas. Isolation and culture of airway epi-
thelial cells from chronically infected human lungs. In Vitro
Cell. Dev. Biol. Anim. 37:480–489, 2001.
38
Randolph, G. J., and M. B. Furie. Mononuclear phago-
cytes egress from an in vitro model of the vascular wall by
migrating across endothelium in the basal to apical direc-
tion: role of intercellular adhesion molecule 1 and the
CD11/CD18 integrins. J. Exp. Med. 183:451–462, 1996.
39
Rogers, D. F. The airway goblet cell. Int. J. Biochem. Cell
Biol. 35:1–6, 2003.
40
Ross, A. J., L. A. Dailey, L. E. Brighton, and R. B. Devlin.
Transcriptional profiling of mucociliary differentiation in
human airway epithelial cells. Am. J. Respir. Cell Mol. Biol.
37:169–185, 2007.
318 EVEN-TZUR et al.
41
Russo, R. G., C. M. Foltz, and L. A. Liotta. New invasion
assay using endothelial cells grown on native human
basement membrane. Clin. Exp. Metastasis 1:115–127,
1983.
42
Saunders, C., and L. E. Limbird. Disruption of microtu-
bules reveals two independent apical targeting mechanisms
for G-protein-coupled receptors in polarized renal epithe-
lial cells. J. Biol. Chem. 272:19035–19045, 1997.
43
Seeds, A. E. Water transfer across the human amnion in
response to osmotic gradients. Am. J. Obstet. Gynecol.
98:568–571, 1967.
44
Slomianka, L. Blue Histology—Respiratory System, 2006.
Available from www.lab.anhb.uwa.edu.au/mb140/.
45
Tarran, R., B. Button, M. Picher, A. M. Paradiso, C. M.
Ribeiro, E. R. Lazarowski, L. Zhang, P. L. Collins, R. J.
Pickles, J. J. Fredberg, and R. C. Boucher. Normal and
cystic fibrosis airway surface liquid homeostasis. The
effects of phasic shear stress and viral infections. J. Biol.
Chem. 280:35751–35755, 2005.
46
van der Linden, P. J., A. F. de Goeij, G. A. Dunselman,
H. W. Erkens, and J. L. Evers. Amniotic membrane as an
in vitro model for endometrium-extracellular matrix inter-
actions. Gynecol. Obstet. Invest. 45:7–11, 1998.
47
Vemuganti, G. K., S. Kashyap, V. S. Sangwan, and
S. Singh. Ex-vivo potential of cadaveric and fresh limbal
tissues to regenerate cultured epithelium. Indian J. Oph-
thalmol. 52:113–120, 2004.
48
Venaille, T. J., A. H. Mendis, A. Warton, L. Walker, J. M.
Papadimitriou, and B. W. Robinson. Study of human
epithelial cell detachment and damage: development of a
model. Immunol. Cell Biol. 67:359–369, 1989.
49
Werner, U., and T. Kissel. In vitro cell culture models of
the nasal epithelium: a comparative histochemical investi-
gation of their suitability for drug transport studies. Pharm.
Res. 13:978–988, 1996.
50
Whitcutt, M. J., K. B. Adler, and R. Wu. A biphasic
chamber system for maintaining polarity of differentiation
of cultured respiratory tract epithelial cells. In Vitro Cell.
Dev. Biol. 24:420–428, 1988.
51
White, S. R., B. M. Fischer, B. A. Marroquin, and
R. Stern. Interleukin-1b mediates human airway epithelial
cell migration via NF-jB. Am. J. Physiol. Lung Cell. Mol.
Physiol. 295:L1018–L1027, 2008.
52
Wilshaw, S. P., J. N. Kearney, J. Fisher, and E. Ingham.
Production of an acellular amniotic membrane matrix
for use in tissue engineering. Tissue Eng. 12:2117–2129,
2006.
53
Wu, R., M. Zhao, and M. M. Chang. Growth and differ-
entiation of conducting airway epithelial cells in culture.
Eur. Respir. J. 10:2398–2403, 1997.
54
Yoon, J. H., H. J. Moon, J. K. Seong, C. H. Kim, J. J. Lee,
J. Y. Choi, M. S. Song, and S. H. Kim. Mucociliary
differentiation according to time in human nasal epithelial
cell culture. Differentiation 70:77–83, 2002.