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

1Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, 69978 Tel Aviv, Israel; 2Ultrasound Unitin 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)

Associate Editor David Odde oversaw the review of this article.

Abstract—Laboratory models of the respiratory lining withmucin secreting epithelial goblet cells have been obtained byculturing epithelial cells under air–liquid interface (ALI)conditions, which require specific wells with semi-permeablesubstrates. In this study nasal epithelial cells (NEC) weresuccessfully cultured on denuded amniotic membrane (AM)under ALI conditions. The cells adhered well on both sides ofthe denuded AM (i.e., the basement membrane or spongylayer) and proliferated to confluency at the same time as cellsgrown on a synthetic membrane. The cytoskeleton structureof cells grown on denuded AMs appeared to be denser andfirmer than that of cells cultured on synthetic membranes.Cultures on the denuded AM differentiated to contain gobletcells which produce and secrete mucins and ciliated cells.Cells cultured on the denuded AM were more stable underairflow conditions than cells on the synthetic membranes.The results of this study suggest that NEC culture ondenuded AM and under ALI conditions creates a stable andwell-differentiated in vitro model of the nasal lining forlaboratory studies.

Keywords—Cell adhesion, Cell biomechanics, Cytoskeleton,

Cell differentiation, Mucus secretion, Respiratory epithe-

lium, Wall shear stress (WSS).

INTRODUCTION

The pseudostratified ciliated columnar epithelium inthe nose lies on a basement membrane. Whereas onlyciliated cells and goblet cells reach the epithelial sur-face, all nasal epithelial cells (NEC) are attached to thesupporting membrane.36 The thickness of the basement

membrane ranges between 1.6 to 10 lm and seems tobe homogenous under a light microscope.23 Electronmicroscopy reveals fine collagen fibers lying parallel tothe surface but running in differing directions. Immu-nohistochemical investigations of the basement mem-brane of nasal mucosa from the inferior nasal conchaerevealed components such as types I, III, IV, V and VIcollagen, laminin, nidogen and heparan sulfate pro-teoglycan. This structure of the basement membranewas presumed to contribute to the mechanical stabil-ization of the epithelium.3

Human NEC grow naturally (i.e., in vivo) underpolar conditions; they are fed on their basal stromalside while exhibiting specialized differentiated proper-ties at the opposite apical side. While the basal side ofthe cells is attached to a basement membrane, theapical side of the nasal ciliated and goblet cells isexposed to the oscillating respired airflow. The peri-ciliary liquid and the mucus layers constitute the onlyprotective interface between these cells and the airmedium. The nasal epithelium polarity highly con-tributes to its primary role as a barrier between therespiratory system and the environment by enablingthe efficient function of mucociliary clearance.

In the 1980s, the need for an in vitro model of therespiratory epithelium in which the cells differentiateinto goblet and ciliated cells under polar conditions ledto the development of air–liquid interface (ALI) cul-tures.50 In such cultures, epithelial cells are seeded on asemi-permeable membrane in a special well (e.g.,Millicells, Millipore, Billerica, MA) and are fed withgrowth medium underneath the membrane, while onlyhumidified air is present above the cells, similar to thein vivo situation of respiratory epithelial cells.2,16 Thesemi-permeable synthetic PolyTetraFluoroEthylene

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

Cellular and Molecular Bioengineering (� 2010)

DOI: 10.1007/s12195-010-0118-y

� 2010 Biomedical Engineering Society

(PTFE) membrane with hole size of 0.4 lm is widelyused nowadays.5,16,20,40,51 Very often, these mem-branes are coated with extracellular matrix proteinssuch as collagen for enhancing cell adherence, and theculture medium is supplemented with retinoids.30,53

The chorioamnion surrounds the amniotic cavityand separates the fetus from the maternal endome-trium. The innermost layer is the amniotic membrane(AM) which is composed of a monolayer of cuboidalepithelial cells uniformly arranged on a thick basementmembrane and an avascular stroma which containstypes I, III, V and VI collagen and fibronectin.32,47 TheAM 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 orspongy layer which is rich with proteoglycans andglycoproteins and contains a nonfibrillar meshwork ofmainly collagen type III. The spongy layer is looselyconnected to the chorionic membrane and thus the twomembranes can be easily separated.32

The potential of using the AM as a substrate for cellcultures was recognized during the 1980s in studies onnormal cell invasion and migration,17,19,29,38 cell–matrix interactions and adhesion,10,46,48 and in studieson tumor cell invasion.26,41 In these studies, the cellswere seeded on the denuded AMs that were strippedoff from the monolayer of amniotic epithelial cells.Tissue engineering applications in ophthalmology werefocused on testing the growth and differentiation ofconjunctival, corneal or retinal epithelial cells culturedon denuded AMs.9,21,28 The AM can be used as ascaffold for cell culture with or without the amnioticepithelium and it can be used fresh or after cryopres-ervation in 50% glycerol.

In this work, an in vitro model was developed forALI culture of NEC on the denuded AM. Since ALIculture of NEC requires collagenic or collagen-coatedsubstrates, the denuded AM membrane is an adequateoption due to its biological content and goodmechanical properties which provide excellent condi-tions for cell adhesion and growth. Biological charac-teristics of the cultured cells are demonstrated as wellas their response to the mechanical loading of nasalairflows.

MATERIALS AND METHODS

Preparation of the Amniotic Membrane

Harvesting of human AMs from full term placentaswas approved by the ethical committee of Tel AvivSourasky Medical Center (# 06/376). Immediatelyafter 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 wereremoved from the AM within 4–5 h after deliveryaccording to the following protocol38: the AM wasrinsed 3 times for 5 min in 200 mL of ice-cold PBS-Gentamicin (50 mg/L). Then, the AM was incubated in1.7% NH4OH solution, at room temperature (RT), for2 h on a rocker. The amniotic epithelial cells were thenmechanically removed from the AM using a scraper(Fig. 1d) and the denuded AM was well washed indouble distilled water (DDW). The denuded AM waseither used immediately for cell culture or cryopreservedin 50:50 DMEM and Glycerol at �70 �C until use.Thawing the AMs was done at 37 �C bath and followedby vigorous washing with DDW to remove glycerolresidues. 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 ALIculturing of airway epithelial cells that can hold anytype of membrane including the denuded AM andsynthetic membranes (Figs. 1e–1i).14 The denuded AMwas stretched between two stainless steel rings and theresidues 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 cellscan be then installed in a flow chamber for applicationof flow on the cell surface.14 Upon completion of a setof experiments, the well can be re-assembled for eitherbiological treatments of the cells or for further cultureincubation for additional experiments.

Isolation and Culture of Nasal Epithelial Cells

Human NEC were isolated from specimens of nasalturbinates of patients undergoing endonasal surgicalintervention as approved by the ethical committee ofThe Sheba Medical Center (#2788/2002). The cellswere then cultured on the denuded AM under ALIconditions as previously described.14–16,37 The epithe-lial cells were dissociated from the excised turbinatesafter incubation in pronase solution and scraping ofthe cells from the tissues. The cell suspension was thencentrifuged, washed and resuspended in a serum-freehormone culture bronchial epithelial growth medium(BEGM) supplemented with additives.37 Primary cellswere plated in BEGM on collagen coated tissue cultureplastic dishes at density of 0.5–6 9 106 cells per dish.After 24 h the dishes were washed with PBS and thecells were fed with BEGM. Thereafter, the mediumwas changed every 2–3 days. When primary culturesreached 70–90% confluence (usually after 4–6 days),

EVEN-TZUR et al.

the cells were trypsinized, washed, counted and platedat density of 100–150 9 103 cells per 0.8 cm2 mem-brane on the denuded AM or on collagen type IVcoated PTFE porous membranes which were cut from30 mm Millicell inserts (Millipore, PICM03050). Themembranes were then installed in the custom-designedwells for ALI culturing. The cells seeded in the wellswere fed with ALI medium.37 After 2–3 days, themedium from the apical side of the membrane wasremoved in order to allow ALI conditions, in which thecells were fed only from the basal part of the mem-brane, while the apical side was exposed to 5% carbondioxide enriched air.

Histology

Histological sectioning and Hematoxylin and Eosin(H&E) staining were performed following fixation ofthe cells on either the denuded AM or the syntheticmembrane with 4% paraformaldehyde and incubationin formalin for 24 h. After several washes and solution

treatments, the well bottom was disassembled and themembranes were embedded in paraffin. Sections of3–5 lm thickness were cut from the paraffin blockusing a microtome and mounted on a glass slide formelting the paraffin and staining with H&E.

Immunofluorescent Stain of Microtubules, ActinFilaments 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 threewashes in PBS. Cell membrane perforation wasachieved by incubation in a 5% dilution of donkeyserum in 0.05% Tween 20 in PBS (PBST) for 4–5 hwhile on a rocker (60 RPM) at RT. b-Tubulin fiberswere stained using 1:500 dilution of mouse monoclonalanti-b-tubulin primary antibody, clone 2.1 (Sigma–Aldrich, Rehovot, Israel). The secondary antibody wasdonkey Rhodamine conjugated anti-mouse secondaryantibody (Jackson Immunoresearch Laboratories,

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 AMin the well-bottom of the custom-designed well; (h) The disassembled custom-designed well. The well bottom allows performanceof experiments with physical stresses applied on the cells; (i) The assembled well in a tissue culture plastic dish.

Culture of Nasal Cells on Amniotic Membranes

West Grove, PA, USA), at a 1:400 dilution. Actinfilaments were stained with 1:1000 dilution of FITClabeled Phalloidin in PBS.49 Intracellular mucins werestained using 1:100 dilution of mouse monoclonal anti-MUC5AC primary antibody, clone 45M1 (ThermoScientific, Runcorn, UK). The secondary antibody wasCy-5 conjugated donkey anti-mouse secondary anti-body (Jackson Immunoresearch Laboratories, WestGrove, PA, USA), at a 1:400 dilution. Cell nuclei werestained using DAPI.

During the staining procedures, the custom-madewells were mounted in 12-well plate filled with therequired solutions both inside and outside of the wellsin order to expose both basal and apical parts of thecells to the liquids. Then, the custom-made wells weredisassembled and the membranes were mounted onmicroscope glass slides. Cells were examined under aZeiss 410 confocal microscope, using a 940 magnify-ing water objective.

Quantification of Mucin Secretion

Quantification of mucin secretion was performedusing an enzyme-linked lectinosorbent assay (ELLA)that measured the reactivity of glycoproteins equiva-lent to MUC5B.1 The assay was slightly modified as itwas based on peroxidase labeled wheat germ agglutinin(WGA) lectin instead of soya-bean agglutinin lectin.Mucin was collected by gently adding 150 lL of PBSto the luminal surface of each culture in the custom-designed wells. After incubation for 15 min at 37 �C allthe apical liquid, including mucin secretions, was col-lected using a micropipettor with a thin tip. Calibra-tion for absolute values of mucin concentration wasperformed with MUC5B mucin standards that weregenerously provided by Prof. C. W. Davis from TheUniversity 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, and1000 ng MUC5B per 1 mL PBS were used for thestandard curve. Mucin samples were diluted 1:400 inPBS and 100 lL samples were transferred to a 96-wellplate with high binding property and incubated at37 �C for 2 h. The wells were then washed four timeswith 200 lL/well PBST. Then, 100 lL dilution of0.5 lg peroxidase labeled WGA-lectin in 1 mL PBSTwas added to each well and the plate was incubated for1 h at 37 �C. Four washes of 200 lL/well PBST fol-lowed. The color reaction was obtained by adding150 lL of substrate (40 mg OPD in 100 mL CitratePhosphate Buffer, spiked with 40 lL of 30% Hydro-gen Peroxide) to each well and keeping at RT for15 min. The reaction was stopped by adding 50 lL4 M sulfuric acid to each well. The absorbancewas read at 490 nm using a microtiter plate reader

(SpectraMax 340PC384, Molecular Devices Corp.).Equivalent MUC5B reactivity was determined from apolynomial curve fitted to the mucin standards mea-surements. Mucin secretion results are expressed asmean ± standard deviation of absolute concentrationin lg/mL. Comparison between groups was performedby 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 forairflow studies with NEC cultured under ALI condi-tions in the custom-designed wells.14,15 The chamber isan 18 cm long conduit with a rectangular cross-sec-tional area of 20 mm 9 10 mm that can be connectedto inlet and outlet tubes. The chamber base can host upto 3 well-bottoms in carefully designed holes which arefilled with culture medium that is in contact with theinferior plane of the membrane in the well-bottomthroughout the experiment. The flow chamber wasconnected to an air source via a 2 m long silicon tubeto ensure fully developed airflow over the culturedcells. The chamber’s exit was also connected to a 20 cmlong silicon tube in order to prevent exit disturbances.

A series of airflow experiments was conducted withwell-differentiated 7–14 days ALI cultured cells oneither the denuded AM or the synthetic PTFE mem-branes. Two steady uniform airflows that generatedwall shear stresses (WSS) of 1 and 20 dyne/cm2 wereapplied for 15 min on the surface of the cultured cellsat room environment. Immediately after the flowexperiments 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 threetimes, each with cells from a different subject andtriplicate cultures were always considered. One excep-tion is the cell density study which was performed withcells from one subject only (as described and discussedin the ‘‘Results’’ and ‘‘Discussion’’ sections), however,triplicates were considered. All the cells used for aspecific study/experiment were seeded on a denudedAM from one subject.

RESULTS

A histological section of an intact human AM ispresented in Fig. 2a where the monolayer of amnioticepithelial 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 adark line with respect to the compact and spongylayers. Examples of human NEC cultured on both

EVEN-TZUR et al.

sides of the denuded AM (i.e., the basement membraneand the spongy layer sides) are shown in the histo-logical sections in Figs. 2c and 2d in comparison tocultures on a PTFE synthetic membrane (Fig. 2e).These 7 days ALI cultures clearly demonstrate that thecells adhered and grew well on both the basementmembrane (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 morecolumnar than the cells cultured on the syntheticmembranes, which in all cases appeared to be flat(Fig. 2e). Light microscope images of cells cultured onboth the denuded AM and the synthetic PTFE mem-brane over a time period of 14 days revealed that thecultures became confluent after 4 days of ALI culture(Fig. 3). These images also strengthen the observationthat the cells on the denuded AM are bigger than thecells on the synthetic membranes (see enlargements inFig. 3).

Images of stained actin filaments and b-tubulinfibers in NEC cultured under ALI conditions on eitherdenuded AM or on synthetic PTFE membranes aredepicted in Fig. 4. Both the b-tubulin and the actinfilaments are organized in a denser network and aremore intact in cells grown on the denuded AM than incells on the synthetic membranes. The b-tubulin andthe actin in cells on the AM were more filamentous(Figs. 4a and 4c) whereas those in cells cultured onthe 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 thecells with only slight background staining in the cellcytoplasm (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 thedenuded AM were organized as a confluent monolayerof cells with clear cell–cell contacts, but their bound-aries were less distinguishable in the actin images thanthose on the synthetic membrane.

The number of NEC cultured under ALI conditionson the denuded AMs was estimated by counting DAPIstained nuclei in five fields of 210 lm 9 210 lm ineach culture under investigation. The averaged cellnumber was 81 ± 20, 89 ± 10, 77 ± 13, and 85 ± 8cells per field in 0, 7, 14, and 21 days ALI cultures,respectively. The number of epithelial goblet cells overtime was estimated by counting cells that were posi-tively stained for intracellular mucins in ten fields of210 lm 9 210 lm and dividing by the total number ofcells in the field. The averaged percentage of gobletcells per field in 0, 7, 14, and 21 days ALI cultures onthe denuded AMs is shown in Fig. 5a. It can be seenthat the percentage of goblet cells in the culturesincreased over time, reaching a plateau on the 14th dayof ALI culture. Representative images of intracellularmucin stains in 0, 7, 14, and 21 days ALI cultures arepresented in Figs. 5b–5e.

Mucin secretion onto the epithelial surface duringALI culture of NEC on the denuded AM increased

FIGURE 2. Images of histological sections: (a) Intact human amniotic membrane (AM); (b) Denuded AM; (c) 7 days air–liquidinterface (ALI) culture of nasal epithelial cells (NEC) seeded on the basement membrane side of a denuded AM; (d) 7 days ALIculture 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.

Culture of Nasal Cells on Amniotic Membranes

over time, reaching a plateau on the 14th day of ALIculture (Fig. 5f). Mucin secretion over time was alsoinvestigated in a single study as a function of cellseeding density. Cells were seeded on the denuded AMat densities of 50,000, 150,000, and 300,000 cells permembrane and mucin secretion was measured overtime. In all cases mucin secretion was increased withtime until day 14 and then remained constant orslightly 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 andthen decreased slightly on day 14. This may suggestthat at a relatively high cell seeding density, the gobletcells in the culture matured faster and reach theirmaximal secretion ability earlier. There was no signif-icant difference between mucin secretions on a specificday from cultures with different cell seeding densities.

Cilia presence was detected using high magnificationscanning electron microscope images. Hundreds of

ciliated cells were observed in fields of 1 mm 9 1 mmin 14 days ALI cultures of NEC on the denuded AM.Cilia on a single cell in a 14 days ALI culture on thedenuded AM are demonstrated in Fig. 6.

Flow experiments were performed with NEC cul-tured under ALI conditions on denuded AMs andsynthetic membranes. Steady uniform WSS of 1 and20 dyne/cm2 were applied on the cultured cells for15 min. Histological sections of static and stimulatedcultures are shown in Fig. 7. It can be seen that thecells remain attached both to the denuded AM and thesynthetic membrane after application of WSS of1 dyn/cm2 for 15 min. However, when WSS wasincreased to 20 dyn/cm2 and also applied for 15 min, itresulted in detachment of the cells from the syntheticmembrane while cells on the denuded AM remainedattached.

DISCUSSION

Human NEC were successfully cultured on denudedAMs under ALI conditions and proliferated to becomeconfluent at the same time as cells grown on PTFEsynthetic membranes. The cells adhered well on eitherthe basement membrane or the spongy layer sides ofthe AMs. The cytoskeleton structure of cells grown onthe denuded AM appeared denser and firmer than thatof cells cultured on the synthetic membrane. Cultureson the denuded AM differentiated to contain gobletcells which produce and secrete mucins and ciliatedcells. After loading of steady airflow conditions thecells were more stable on the denuded AM than on thesynthetic membrane.

Human NEC cultured on denuded AMs for7–14 days had a hypertrophic appearance (Figs. 2cand 2d), which resembles the morphology of these cellsin vivo.44 The cells on the denuded AMs were columnarin most cases (Figs. 2c and 2d), although occasionallythey appeared flat (Fig. 7a). This outcome may resultfrom structural inhomogeneity of the AM due to dif-ferent local molecular composition or fiber density, orit may be related to the denuding process. Recently,alternative methods of optimizing the preparation ofthe AM were implemented,18,52 which may result inmore uniform or homogeneous morphology of thecultured cells.

The actin filaments and b-tubulin fibers appeareddenser and more filamentous in cells cultured on thedenuded AM than in cells cultured on the syntheticmembrane. The fact that the cell boundaries were moreblurred in the images of cells on the denuded AM(Figs. 4a, 4c, and 4e) than in cells on the syntheticmembrane (Figs. 4b, 4d, and 4f) might indicate onbetter cell–cell adhesion in cells on the denuded AM.

Nasal epithelial cells on denuded amniotic membrane

Nasal epithelial cells on collagen coated synthetic membrane

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FIGURE 3. Light microscope images of cells cultured on thedenuded amniotic membrane and on synthetic PolyTetra-FluoroEthylene (PTFE) membranes under air-liquid interfaceconditions over a time period of 14 days. Enlargements are of150%.

EVEN-TZUR et al.

In cultures on the denuded AM many more stressfibers were observed whereas in cultures on the syn-thetic membrane most of the actin filaments werelocated underneath the cell membrane constituting thecortical actin. Stress fibers are known to be involved incellular processes such as adhesion, motility and mor-phogenesis.31 In epithelial cells, cadherin cell–celladhesion receptor complexes are essential for theestablishment of the epithelial cell shape and mainte-nance of the differentiated epithelial phenotype. Thesecomplexes are associated with actin filaments to

provide points of attachment for the cytoskeleton atthe membrane, which are important for the develop-ment of mechanical stress during epithelial compactionand polarization.7 The presence of stress fibers in cellcultured on the denuded AMs may suggest their betterattachment to the substrate in comparison to the cellson the synthetic membrane, as suggested by the resultsof airflow experiments (Fig. 7).

Previously, it has been reported that actin microfil-aments of the cytoskeleton are involved in agonistinduced mucin secretion from airway surface goblet

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Nasal epithelial cells on denuded amniotic membrane

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

Culture of Nasal Cells on Amniotic Membranes

cells.39 Interactions of dephosphorylated myristoylatedalanine-rich C kinase substrate (MARCKS) with actinand myosin create the linkage of mucin granules tointracellular contractile apparatus, which is able tomove them through the cell cytoplasm toward theapical membrane. Moreover, actin filaments wereshown to maintain a barrier in mucin secretion fromintestinal goblet cells by hindering mucin granuleaccess to the plasma membrane.34 Microtubules arewell known for having an important role in vesiculartransport in cells in general27 and specifically inpolarized epithelial cells.22,42 Thus, the fact that theactin microfilaments and microtubules are more pro-nounced in cells cultured on the denuded AM may

suggest that the epithelial phenotype of these cells ismore substantial.

The elastic modulus of the AM is about 25 MPaand its tensile failure strength is approximately5 MPa.8 Recent studies have shown that the sub-strate’s elasticity affects the adhesion characteristics,the dynamics of cell spreading, cell differentiation andthe cytoskeleton assembly.11,13 For example, fibro-blasts have more organized F-actin and stress fiberswhen cultured on increasingly stiffer substrates.Neurons on the other hand branch more on softersubstrates.11 Based on these results it may well be thatthe differences in the actin structure of cultured NECon denuded AMs and synthetic membranes (Figs. 4c

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

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and 4d) are also due to the greater elasticity of theAM.

The change in the total number of cells in ALIcultures on the denuded AM was minor over 21 days.On the other hand, the number of cells that werepositively stained for intracellular mucins increasedgradually until day 14, which reflects the increasein goblet cell percentage in the cultures over time(Figs. 5a–5e). Mucin secretion in cultures on thedenuded 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. 5aand 5f suggests that there is a good correlation betweenthe percentage of goblet cells in the culture over time

and the amount of mucin secretion over time. Thepercentage of goblet cells in the culture on the denudedAM after 14 and 21 days are similar in magnitudes tomeasurements performed by others in cultures ofhuman NEC on polyester membranes 14 and 28 daysafter 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 whichmucin secretion increased over time and reached aplateau on the 14th day of culture.54 In another study,guinea pig tracheal epithelial cells were cultured ondenuded AMs under ALI conditions and presentedheight and ratio of ciliated epithelial cells similar tothose in normal guinea pigs epithelium, however, nogoblet cells were identified.33 While the denudingprocedure in that work was similar to the method usedhere (i.e. incubation of the intact AM in NH4OH andmechanical scraping), in the current work goblet cellshave been identified using the intracellular mucin stainandmeasurements of mucin secretion from the cultures.

Physiological WSS in the nasal cavity due to airflowduring quiet breathing were computed to be up to15 dyn/cm2.12 Several studies investigated the biologi-cal response of cultured airway and NEC to airflowinduced WSS, and specifically the changes in mucinsecretion and the cytoskeleton due to such stim-uli.14,15,45 In the current study we examined the strengthof cells attachment to the substrate by applying differ-ent levels of WSS on top of the different cultures. Theapplication of a relatively high WSS of 20 dyn/cm2

induced detachment of the cells from the syntheticmembrane while they remained attached to the denuded

FIGURE 6. Cilia on a single cell in 14 days air–liquid inter-face culture of nasal epithelial cells on the denuded amnioticmembrane. Bar = 1 lm.

Nasal epithelial cells on denuded amniotic membrane

Nasal epithelial cells on collagen coated synthetic membrane

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FIGURE 7. Images of histological sections of cultured nasal epithelial cells that were exposed to airflow that generated steadyuniform WSS of 1 and 20 dyne/cm2 for 15 min in comparison to static conditions. Cell culture was done under air–liquid interfaceconditions on both denuded amniotic membrane (AM) and synthetic membranes (SM). Bar = 10 lm.

Culture of Nasal Cells on Amniotic Membranes

AM. This suggests that the adhesion of NEC to thedenuded 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 onexpression of adhesion molecules, location and numberof adhesion sites and more mechanical tests should beperformed to study the forces generated between thecells and the denuded AM matrix.

Using the human AM as a substrate for cell cul-turing for various applications has many advantagesdue to its great mechanical integrity and excellentmolecular composition for development of cell adhe-sion complexes. Specifically to our long-term goal ofstudying the biological response of NEC to airflowinduced WSS, this new culture system is essential. Theneed to develop this system emerged when applicationof high WSS on cells cultured on synthetic membranesresulted in detachment of the cells from the membrane(as shown in Fig. 7f). Using the AMs allowed us toperform experiments with realistic high WSS that existin vivo in the nasal cavity during different modes ofbreathing. The utilization of the denuded AM as asubstrate for cell culture may also be beneficial forother applications, especially when loading of a flow-ing fluid is applied on the cell culture.

The collagen composition of the denuded AM isnaturally better than that of the collagen coated syn-thetic membranes, yet the permeability of the denudedAM, which has a key importance in ALI cultures, isclearly not as uniform and repeatable as that of thesynthetic membranes. The thickness of the denudedAM ranges from tens to hundreds of microns,6 andthus, its permeability is expected to vary across themembrane surface. Nevertheless, the observations ofthis study show that NEC grew on most of the denudedAMs area which indicates that permeability of nutrientsacross the membrane is probably sufficient for cellgrowth. The permeability of chorioamniotic mem-branes was investigated for sodium, calcium, magne-sium and thyrotrophin-releasing hormone,4,24,25 as wellas for the transfer of water across the AM due toosmotic gradients.43 However, these studies were con-ducted with intact membranes as opposed to denudedAMs. During in vitro ALI culture of cells on denudedAMs the entire supply of nutrition is from the mediumunderneath the membrane. Thus, it will be essential toperform comprehensive studies on permeability ofdenuded AMs to culture medium components.

In conclusion, it has been demonstrated in this studythat denuded AM can serve as a very good substratefor NEC. Cultures of NEC on the denuded AM dif-ferentiated to contain goblet cells which produce andsecrete mucins and ciliated cells. The cells adhered wellon both sides of the denuded AM (either onthe basement membrane or the spongy layer) and

proliferated to become confluent at the same time ascells grown on synthetic membranes. Cells on thedenuded AM were found to be more stable underairflow conditions than cells on the synthetic mem-branes. The results of this study suggest that using thedenuded AM as a substrate for NEC culture underALI conditions creates a well differentiated respiratoryepithelial 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 adviceregarding nasal epithelial cell culture and mucinquantification assays. We would also like to thankDr. Uri Zaretsky, Mrs. Dalit Shav and Mrs. RikiLevkovitz from the Department of Biomedical Engi-neering in Tel Aviv University for their valuable inputto this project. This work was partially supported by agrant from the Ela Kodesz Institute for BiomedicalEngineering and Medical Physics at Tel Aviv Univer-sity, by a generous donation from the AustralianFriends of Tel Aviv University (Vic) and by the Ber-man Trust.

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