respiratory diseases 
 such as asthma are becoming increasingly prevalent , 
 with reduced longevity and quality of life for those affected as well 
 as causing an economic burden upon healthcare systems worldwide .
consequently , there is a need to develop more 
 effective therapies to prevent and treat respiratory diseases .
developing 
 new therapies requires extensive testing to ensure efficacy and safety , 
 which is both time - consuming and costly .
therapies that show promise 
 during the first stage preclinical in vitro tests may be taken forward 
 for further studies .
for all new medications , regulatory authorities 
 insist upon acquiring information from animal studies because the 
 effect upon the whole body can be observed .
however , the limited biological 
 relevance of animal models to human diseases means that data obtained 
 from such studies could not always be relied on . in vitro models 
 of human tissues that are biomimetic and
closely 
 represent the functional properties of their respective tissues could 
 enable better understanding of disease processes , hence providing 
 more physiologically relevant platforms for identification of targets 
 for therapy as well as testing the efficacy and safety of new drug 
 leads . using such in vitro models in drug discovery cycle could in 
 turn substantially reduce the number of drug leads that need to be 
 taken forward to preclinical studies and , therefore , reducing the 
 number of animals required for such experiments .
in addition to providing scientific advantages ( e.g. , identification 
 of more efficacious targets for therapy ) , using biomimetic in vitro 
 tissue models also conforms with the  3rs 
principles 
 of refinement , replacement , and reduction of animal experimentations 
 in research wherever possible .
the 
 respiratory system is constantly exposed to potentially harmful 
 particles , allergens , and pathogens . to maintain sterility of the 
 lung
the respiratory system has a series of defense mechanisms and 
 the capability to respond to environmental challenges .
epithelial 
 cells are the predominant cell type in contact with the air and as 
 such the airway epithelium forms the first line of defense against 
 airborne insults .
epithelial cells are structurally arranged to form 
 a continuous layer and are joined via protein junctions to create 
 a paracellular barrier to shield interstitial tissue from the airway . 
 as well as a physical barrier , the epithelium forms a chemical barrier 
 via cellular secretions , for example , mucus that entraps infiltrating 
 particles .
furthermore , contact with invading pathogens prompts epithelial 
 cells to release lysozymes and phospholipase that destabilize bacterial 
 membranes , defensins that have antimicrobial activity , and surfactants 
 that promote phagocytosis of invading particles .
if the epithelial barrier is compromised , the epithelial 
 cells 
 not only change morphologically and functionally but also communicate 
 reciprocally via paracrine or contact - dependent 
 signaling with other cell types , such as underlying stromal and immune 
 cells including macrophages , dcs , lymphocytes , neutrophils , and mast 
 cells . summoning
support from underlying cells 
 can assist in restoring the epithelial barrier or initiate an immune 
 response through expression of adhesion molecules and release of mediators 
 including cytokines and chemokines .
the 
 synergistic interactions of cells within human lung tissue remains 
 largely understudied ; in particular , few in vitro lung models report 
 the inclusion of immune cells that are essential for sensing cellular 
 and environmental changes as well as exerting a crucial role in the 
 pathogenesis of lung diseases .
the tissue engineering of lung models 
 has largely focused toward engineering tracheal replacements due to 
 the simpler nature of this tissue .
the 
 robust architecture of the trachea can withstand the decellularization 
 process and subsequent repopulation , whereas it proves difficult to 
 repopulate decellularized tissue from deeper within the lung that 
 has a more complex construction .
the specific structural and cellular 
 architecture of complex lung tissue can be retained for experimentation 
 by using ex vivo tissue explants .
these biopsy samples are practical 
 for short - term experimentation , though interindividual variability 
 can have an impact upon the results and the availability of such tissue 
 is limited . to allow high - throughput screening of samples and ,
particularly , 
 longer - term experiments , it is preferable to have a sustainable source 
 of reproducible tissue models . the use of commercially available 
 two - dimensional ( 2d ) platforms 
 upon which epithelial cells can be cultured at the air  liquid 
 interface ( ali ) is widely practiced .
although information regarding 
 cellular interaction can be identified using these methods , the 2d 
 platforms fail to represent the cellular arrangement seen in vivo 
 and , therefore , are not amenable to direct cell
the use 
 of a 3d tissue equivalent is favorable over 2d cell culture providing 
 more in - vivo - like morphology , function , and intercellular interactions 
 enabling greater resemblance to physiological conditions . encapsulating cells within synthetic or natural hydrogels has been 
 widely used for culturing cells in a 3d environment and provide greater 
 cell  cell contact compared to culturing upon a solid 2d substrate .
in addition , hydrogels could provide a cellular 
 microenvironment resembling the native extracellular matrix ( ecm ) , 
 hence supporting key functional properties of different cell types .
although many cell types seem to thrive within the 3d environment 
 of hydrogels , encapsulating epithelial cells whose primary function 
 is barrier formation could be counterintuitive .
therefore , other types 
 of 3d matrix such as porous fiber sheets could be a favorable alternative 
 for culturing epithelial cells , providing closer morphological resemblance 
 to the basement membrane in barrier tissues such as skin and respiratory 
 epithelium . obviously , this does not 
 preclude use of hydrogel based scaffolds with the optimal topography 
 for 3d culture of epithelial cells . methods to create fibrous 
 3d platforms include phase separation , electrospraying , or electrospinning .
we show that the 
 ecm of lung tissue has a randomly arranged network of nanometer - sized 
 fibers , and as such , the electrospinning method proves a suitable 
 choice to create a matrix that mimics this arrangement for culturing 
 lung associated cells .
the porous network of polymer fibers that is 
 produced can be tailored in morphology and dimensions to mimic the 
 native ecm of the cells being cultured .
although there has been 
 some success in the construction of pure protein electrospun scaffolds 
 ( e.g. , collagen and fibrinogen ) , poor structural strength limits their use . 

synthetic polymers offer the choice of well - defined batches with a 
 greater range of mechanical and chemical properties than those of 
 natural materials .
furthermore , synthetic polymers may adsorb ecm 
 proteins in solution or can be surface modified for enhanced cell 
 attachment if necessary . synthetic polymers 
 pla and plga are the more extensively studied , having been explored 
 for both in vitro and in vivo research .
however , pla and plga are biodegradable and , 
 in our preliminary experiments , were found to become quite fragile 
 and difficult to handle after few days of cell culture , hence proving 
 unsuitable to support long - term cell cultures for the 3d lung model . 
 thus , use of other nonbiodegradable and biologically nonfouling polymers 
 such as poly(ethylene terephthalate ) ( pet ) , which was reported to 
 support cell culture , was considered .
subsequently , in this study , we have used electrospun fibers 
 of 
 pet to create a 3d model of airway epithelium , comprising epithelial 
 cells , dendritic cells , and fibroblasts , cultured at ali .
the model 
 has been characterized with regards to its barrier function , responses 
 to environmental stimuli , and migratory properties of the immune cells 
 after allergen challenge .
this model possesses reasonable cellular 
 and structural representation 
 of the airway epithelium and is amenable to in situ monitoring , and 
 as such , it presents an invaluable tool for academic and pharmaceutical 
 research within the fields of lung biology , disease modeling , and 
 drug discovery and delivery .
all 
 materials were purchased from sigma - aldrich , 
 u.k . , unless stated otherwise .
electrospun scaffolds were produced 
 by dissolving polyethylene terephthalate 
 ( pet ) in 1:1 trifluoroacetic acid ( tfa):dichloromethane ( dcm ) ( fisher 
 chemicals , u.k . ) to create a 10% ( w / v ) solution .
the polymer solution 
 was loaded into a syringe ( 20 ml ) , and an 18 gauge needle ( bd falcon , 
 u.k . )
the syringe was securely fitted to a syringe pump - driver 
 ( harvard apparatus ltd .
the pet solution was delivered 
 at a constant flow rate of 0.5 ml / hour at 14 kv for 4 h. the scaffolds 
 were air - dried in a fumehood for 24 h to allow residual solvent to 
 evaporate .
the 
 epithelial ( calu-3 ) and fibroblast ( mrc-5 ) cell lines ( lgc standards 
 cell , u.k . ) were routinely cultured at 37 c and 5% co2 in dmem - f12 ham or mem media , respectively .
both culture media were 
 supplemented with fetal calf serum ( fcs ) ( 10% ( v / v ) ) , l - glutamine 
 solution ( 2 mm ) ( 1% ( v / v ) ) , and an antibiotic / antimycotic solution 
 ( 1% v / v ) comprised of penicillin ( 10  000 units / ml ) , streptomycin 
 sulfate ( 100 mg / ml ) , and amphotericin b ( 25 g / ml ) .
dendritic cells ( dc ) 
 were generated from peripheral blood monocytes as we have previously 
 described .
briefly , peripheral blood mononuclear cells were isolated 
 from human blood buffy coat ( national blood transfusion service , u.k . ) 
 using histopaque density gradient centrifugation .
monocytes were isolated 
 using cd14 + magnetic beads ( milteny biotech , u.k . ) to the purity of 
 > 98% .
purified monocytes were cultured with gm - csf 
 ( 50 ng / ml ) and il-4 ( 250 iu / ml ) ( r&d systems ) for 6 days to generate 
 immature dcs .
dc phenotype was determined by flow cytometry after 
 staining for cell surface markers including cd11c , cd83 , cd83 , and 
 hla - dr .
electrospun scaffolds were cut to a size of 2 
 cm and 
 sterilized by irradiating with ultraviolet ( uv ) light at a distance 
 of 8 cm for 15 min each side .
the scaffolds were sterilely transferred 
 to a 12 well culture plate and a steel ring was placed on top to secure 
 the scaffold before further sterilization in an antibiotic / antimycotic 
 solution overnight ( 37 c , 5% co2 ) .
the sterilizing 
 solution was removed and the scaffold washed with pbs before submerging 
 the scaffold in the appropriate cell culture media to precondition 
 the scaffold .
calu-3 and mrc-5 cells were inoculated inside of the 
 steel ring onto separate pet scaffolds at a density of 3  10 cells / scaffold ( 1  10 cells / ml in 300 l ) 
 and incubated for 72 h ( 37 c , 5% co2 ) ( figure 1a ) .
steel rings are used to submerge the electrospun scaffolds 
 and 
 define the cell seeding area ( a ) .
the use of scaffholders ( b  e ) 
 allow the 3d tissue engineered constructs of lung tissue to be cultured 
 under appropriate conditions where epithelial cells are at the ali 
 ( e ) and fibroblast cells remain submerged , mimicking in vitro lung 
 conditions . following 72 h culture , 

single culture scaffolds were transferred 
 from steel rings into a polytetrafluoroethylene ( ptfe ) platform support 
 ( scaffholder ) , which was designed and fabricated in - house ( figure 1b  d ) . the epithelial scaffold monolayer was 
 placed on top of the fibroblast scaffold layer inside the scaffholder 
 to form the coculture model .
single culture controls were assembled 
 by combination of either a calu-3 or mrc-5 scaffold with an acellular 
 scaffold ( i.e. , without cells ) .
to ensure there is no possibility 
 of separation or movement during culture , scaffolds are secured in 
 place within the scaffholder .
alignment of cell layers is ensured 
 because the internal diameter of the steel ring matches that of the 
 scaffholder ( figure 1 ) .
the cells were submerged 
 in cell media for a further 12 h ; the cell media for cocultures comprised 
 a 50:50 mixture of the mrc-5 and calu-3 cell media .
media from the 
 apical surface of the scaffholder was removed to culture epithelial 
 cells at the ali ( figure 1e ) .
schematic figure showing 
 different steps of fabrication and configuration 
 of the 3d tissue engineered airway epithelium .
calu-3 epithelial cells 
 are seeded onto one pet scaffold and mrc-5 fibroblasts are seeded 
 onto a second , separate scaffold ( a ) . following 72 h culture ,
scaffolds 
 are combined by layering the epithelial scaffold monolayer on top 
 of the fibroblast scaffold layer to form the coculture model .
cells 
 are subsequently cultured for 2 weeks at the ali to allow for differentiation 
 of the epithelial cells , including establishment of tight junctions 
 ( b ) .
monocyte - derived dcs are seeded onto separate pet scaffolds and 
 then inserted into the coculture model .
the upper epithelial scaffold 
 is temporally lifted away from the lower mrc-5 fibroblast layer so 
 that the separate third scaffold containing dendritic cells may be 
 placed on - top of the mrc-5 scaffold layer ( c ) .
the calu-3 layer is 
 placed on - top of the dc scaffold layer , resulting in the dc layer 
 sandwiched between the epithelial and fibroblast scaffold layers to 
 form the triculture model ( d ) .
fibroblast cocultures were assembled and cultured 
 for 14 days at the ali prior to insertion of the dc layer .
the cell 
 culture media composition remained as a 50:50 mixture of calu-3 and 
 mrc-5 cell media .
immature dcs ( day 6 ) were prestained with 
 hoescht nuclear stain ( 5 g / ml ) ( invitrogen , u.k . ) and inoculated 
 onto pet electrospun scaffolds at a density of 2  10 cells / scaffold and incubated for 24 h ( 37 c , 5% co2 ) . the culture medium was aspirated to remove dcs that had not attached 
 prior to insertion in between calu-3 and mrc-5 layers in an established 
 coculture model to form an immunocompetent triculture model .
a schematic 
 describing the layer - on - layer approach to assembling epithelial and 
 fibroblast cocultures and subsequent insertion of the dc layer is 
 described in figure 2 .
triculture models were 
 stimulated with house dust mite extract ( hdm ) ( 10 g / ml ) ( greer , 
 u.s.a . ) and lipopolysaccharide ( lps ) ( 100 ng / ml ) ( sigma - aldrich , u.k . ) 
 or pbs control and incubated for 36 h prior to analysis .
the triculture 
 models were fixed with 4% ( v / v ) paraformaldehyde ( electron microscopy 
 sciences , u.s.a . ) in pbs , and the three scaffold layers were separated 
 and immunostained with pancytokeratin ( epithelial cell marker ) .
scaffolds 
 were then examined by confocal microscopy ( leica sp2 confocal laser 
 scanning microscope ) ( leica microsystems ltd .
, u.k . ) with postvisualization 
 performed using volocity software ( perkin - elmer , u.k . ) .
trans - epithelial electrical 
 resistance ( teer ) measurements were performed 
 across the epithelial cell monolayer of cells cultured at the ali . 

measurements were performed using an evom volt - ohm - meter and stx2 
 chopstick electrodes ( world precision instruments , u.k . ) .
prior to 
 recording teer , chopstick electrodes were sterilized ( 70% v / v ethanol 
 in distilled water ) and cell culture media was added to the upper 
 chamber ( 500 l ) and lower chamber ( 1.5 ml total volume ) and 
 allowed to equilibrate for 30 min ( 37 c , 5% co2 ) . 

cellular samples of pet 
 electrospun scaffold were placed onto carbon - coated electron microscope 
 stubs and sputter - coated with gold ( 5 min , blazers scd 030 blazers 
 union ltd . , liechtenstein ) under an argon atmosphere ( boc , u.k . ) prior 
 to analysis .
samples were imaged using sem ( scanning electron microscopy ) 
 analysis ( jeol jms-6060 lv microscope , jeol ltd . , u.k . )
cellular samples were fixed in 
 3% ( v / v ) glutaraldehyde overnight at 4
c before dehydration 
 through an ascending series of ethanol concentrations prior to sem 
 imaging .
briefly , 
 cellular scaffolds were fixed with 10% buffered formalin , excised , 
 and embedded in paraffin .
the paraffin embedded blocks were then sectioned 
 and stained with hematoxylin and eosin ( h&e ) before imaging .
scaffold samples were washed with 
 pbs prior to fixation with paraformaldehyde ( 4% ( w / v ) ) or methanol 
 ( 100% ( v / v ) ) for 15 min at room temperature ( rt ) .
samples were washed 
 in pbs ( 3  5 min each ) before being permeabilized using triton 
 x-100 ( 0.5% ( v / v ) ) for 5 min at room temperature . following a further 
 wash in pbs
( 3  5 min each ) , nonspecific antibody binding was 
 blocked with goat serum ( 10% ( v / v ) in pbs for 5 min at room temperature . 

primary antibodies used were anti - mucin 5ac [ 45m1 ] ( ab3649 , 
 abcam , u.k . ) , mouse anti - zo1 ( invitrogen , u.k . ) , anti - fibronectin 
 ( ab 23750 , abcam , u.k . ) , anti - ki67 ( ab15580 , abcam , u.k . ) , anti - collagen 
 ( ab34710 , abcam , u.k . ) , and pan - cytokeratin pk110 ( santacruz biotech , 
 u.k . ) .
samples were then washed with pbs ( 3  5 min each ) and 
 incubated with species - appropriate fluorescently labeled secondary 
 antibodies ( 1:100 ) for 30 min at room temperature .
secondary antibodies 
 included goat antimouse igg rhodamine red x ( invitrogen , u.k . ) , goat 
 antirabbit igg fitc ( invitrogen , u.k . ) , and alexa fluor 488 goat antimouse 
 igm (  chain ) ( invitrogen , u.k . ) .
samples were washed in pbs 
 ( 3  5 min each ) , incubated with hoechst ( 5 g / ml ) for 
 5 min at room temperature , and mounted using fluoromount mounting 
 medium .
immunostaining was observed using a confocal microscope ( leica 
 sp2 confocal laser scanning microscope , images processed with leica 
 confocal software ) with postvisualization performed using volocity 
 software .
a papain ( 60u / ml ) 
 solution was 
 prepared with l - cysteine ( 5 mm ) to reconstitute the cysteine 
 active site , and an aliquot ( 300 l ) was applied to the apical 
 surface of calu-3 cells .
the present study presents a multilayered 3d electrospun pet lung 
 model capable of incorporating multiple cell types each supported 
 upon their own individual electrospun layer .
the porous network of 
 electrospun pet fibers can permit cell interaction through both direct 
 cell  cell contact and paracrine factors within the 3d model . 

we report the incorporation of lung associated epithelial cells and 
 fibroblasts and monocyte - derived dcs within the 3d model and determine 
 how culturing these cells together influences their behavior .
sem analysis of 
 decellularised human lung tissue ( from healthy sections of lung tissue 
 obtained from patients undergoing surgical procedures ( nottingham 
 university hospitals nhs trust ) after informed consent and ethics 
 approval ) revealed a porous network of nanometer - sized fibers ( figure 3a ) .
accordingly , we used a nonbiodegradable polymer 
 ( namely , pet ) to fabricate a nanoscale porous scaffold ( figure 3b ) .
the mean fiber diameter of the pet scaffold 
 ( 240 nm 70 ) was similar to the mean diameter of lung ecm ( 245 
 nm 83 ) ( figure 3c ) .
the average thickness 
 of the pet scaffold , calculated using histological sections , was 60 
  10 m .
owing to its robust physical properties , the pet 
 electrospun scaffold is capable of withstanding repeat handling allowing 
 separation of individual cell layers for assessment following experimentation .
comparisons 
 of scanning electron micrographs of decellularised 
 lung tissue ( a ) with pet electrospun scaffold ( b ) showing morphological 
 similarities .
the fiber diameter of decellularised lung tissue and 
 pet electrospun scaffolds were measured and show comparable dimensions 
 ( c ) .
the main structural cell types 
 of the lung , epithelial , and fibroblast 
 cells were each cultured upon separate pet scaffolds . to incorporate 
 an immune component into the model ,
a triculture system was formed 
 by culturing dcs upon a third pet scaffold and inserting this between 
 the epithelial and fibroblast layers of an established epithelial 
epithelial 
 and fibroblast cells were each inoculated onto individual pet scaffold 
 layers and cultured submerged in media to allow cells to establish 
 growth upon the scaffold , following which the cell inoculated scaffolds 
 were assembled together within our developed scaffholders allowing 
 physiologically relevant positioning of the construct .
epithelial 
 cells were cultured upon the uppermost layer allowing culture at ali 
 where the upper cell surface is in contact with the air .
culturing 
 epithelial cells at the ali mimics in vivo conditions by providing 
 apical  basal polarity , which leads to full differentiation 
 of epithelial cells and development of a functional barrier .
the underlying fibroblasts were positioned directly 
 beneath the epithelial inoculated scaffold and remained submerged 
 in media much as they would in vivo .
the production and maintenance 
 of barrier integrity in epithelial cells cultured upon pet electrospun 
 scaffolds and positioned at the ali using scaffholders was assessed 
 using teer measurements and permeability studies .
barrier formation 
 was compared when epithelial cells were cultured alone ( single culture ) 
 or with fibroblasts ( coculture ) .
teer is widely used to monitor barrier 
 integrity , where an increase in resistance to flow of current is due 
 to greater integrity of the barrier , attributed to the formation of 
 cellular tight junctions .
an acellular 
 control scaffold was monitored in order to report 
 a control value , as it is known that teer values reportedly vary according 
 to the material upon which the epithelial cells are cultured .
in addition , a single culture of mrc-5 fibroblasts 
 was monitored as a control , as an increase in teer value was not expected 
 owing to mrc-5 fibroblasts characteristically not forming tight junctions .
statistical analysis was performed using two - way anova with sidaks 
 multiple comparison showed that teer measurements of cocultures produced 
 readings that were significantly greater than those of epithelial 
 cells cultured alone ( figure 4a ) .
the teer measurements 
 of cocultures are significantly higher than 
 those of epithelial cells cultured alone .
statistical analysis was performed using two - way 
 anova with sidaks multiple comparison with the difference between 
 coculture and single calu-3 culture having a p value 
 < 0.0001 ( a ) .
sem images of epithelial cells from single and coculture 
 where the upper surface that has been imaged is thought to be predominantly 
 formed from ecm protein presence .
the micrographs show that epithelial 
 cells from coculture have a smoother surface than those from single 
 culture ( b ) .
the teer measurements 
 show that after 14 days in culture epithelial 
 cells cultured alone , without fibroblasts , attained measurements of 
 130  cm .
the greatest increase was seen 
 for cocultures of epithelial and fibroblast cells attaining measurements 
 of 200  cm .
thus , epithelial cells cultured 
 together with fibroblasts appeared to achieve a confluent differentiated 
 state earlier than single cultures of epithelial cells as shown by 
 earlier and greater increases in teer measurements ( figure 4 a ) .
the teer values of the controls , an acellular 
 scaffold and mrc-5 single culture , did not increase , maintaining baseline 
 teer measurements of 100  cm .
although 
 human lung ex vivo teer results are not available in the literature , 
 ex vivo rabbit airway epithelium has been reported to be 260320 
  cm for the trachea and 266  cm for the bronchus .
thus , the maximal teer recorded in our 3d model are comparable 
 to the data available for animal ex vivo teers . the topography 
 of the confluent epithelial cell layer was observed 
 using sem .
the sem micrographs of epithelial cells from single culture , 
 coculture , and fibroblasts from cocultures showed what is thought 
 to be the presence of ecm protein deposition ( figure 4b ) .
h&e staining of the epithelial layer from single 
 and cocultures 
 indicated that single cultures formed a single thin layer of cells , 
 whereas the cocultured epithelial cells had a more dense and layered 
 arrangement after 14 days at ali ( figure 5 ) . 

furthermore , epithelial cells within cocultures appeared to produce 
 mucus earlier than single cultures as shown by immunocytochemical 
 staining ( figure 5 ) .
histological staining shows the presence 
 of epithelial cells on the uppermost surface of the pet scaffold with 
 a greater number of cells found in cocultures compared to single cultures . 

mucin production ( red ) was greater from coculture than single culture , 
 where cell nuclei are stained with dapi ( blue ) .
immunocytochemical staining of the deposition of ecm proteins 
 indicated 
 that the epithelial cells deposit collagen and fibronectin when cultured 
 upon pet electrospun scaffolds ( figure 6 ) . 

there appeared to be enhanced production of these ecm proteins from 
 cocultured epithelial cells .
the expression of the cell proliferation 
 marker ki67 showed that epithelial cells in coculture had lower levels 
 of ki67 , possibly indicating their tendency toward an earlier full 
 differentiated state as also evidenced by an accelerated increase 
 in teer readings in cocultures compared with single cultures of epithelial 
 cells ( figure 7a ) .
fibroblast cell growth has 
 not been adversely affected when positioned beneath the epithelial 
 cell layer during coculture experimentation ; the fibroblast cells 
 are still present and in a state of active growth as indicated by 
 the production of ki67 and have retained the ability to produce ecm 
 proteins collagen and fibronectin ( figure 6 ) .
ecm staining following 14 days culture at the ali shows the presence 
 of ecm proteins : collagen and fibronectin , predominantly found in 
 epithelial and fibroblast cells and also ki67 , showing that the cells 
 are in active growth .
epithelial cells in cocultures appear to express 
 lower levels of ki67 ( compared to single cultures ) , indicating their 
 tendency toward full differentiation .
following the 14 days at the ali , the enzyme papain was applied 
 to the epithelial surface of single and cocultures , which was shown 
 to disrupt tight junctions demonstrated by the measurement of teers 
 ( a ) and by confocal imaging of zo1 protein ( b ) .
the disruption to 
 tight junctions from coculture was observed to be less than that of 
 single cultures .
teer measurements were then performed for a further 
 14 days to monitor the healing process .
the cocultures were observed 
 to be recovering from the disruption with increase in teers and increase 
 in the presence of zo1 .
single cultures did not appear to recover 
 so well with teer measurements remaining around the same value as 
 when the cells were subjected to papain .
statistical analysis was 
 performed using two - way anova with sidaks multiple comparison , 
 showing that the difference between coculture and single calu-3 culture 
 that had been subjected to papain having a p value 
 < 0.0001 .
the 3d lung model was cultured at 
 the ali for 14 days before papain was applied to the epithelial surface 
 of single and cocultures .
papain is an allergen with cysteine protease 
 activity and is known to disrupt tight junctions in respiratory epithelium 
 using its enzymatic activity , mimicking the action of other airborne 
 allergens such as house dust mite , which also have a cysteine protease 
 activity .
the application of papain on 
 day 14 ali was shown to disrupt epithelial tight junctions in both 
 single and coculture as demonstrated by a sharp reduction in teer 
 values ( figure 7a ) and disintegration of zo1 
 protein visualized by immunostaining followed by confocal imaging 
 ( figure 7b ) .
the disruption to tight junctions 
 from coculture was thought to be less than that of single cultures , 
 where almost no zo1 could be observed post papain .
teer measurements 
 were performed for a further 14 days to monitor the epithelial repair 
 process , and further examination of zo1 expression was carried out . 

the cocultures were observed to be recovering from the disruption 
 with increased teers and increased presence of zo1 ( figure 7a and b ) .
however , teer measurements did not appear 
 to recover in single cultures during the same time frame and remained 
 at approximately the same value as when the cells were subjected to 
 papain ( figure 7a ) .
the confocal microscopy 
 images on day 15 ( post papain ) of single culture shows that the cell 
 growth has been disrupted by papain and that tight junctions are not 
 present over a wide area of the sample .
the cells from single cultures 
 recovered slightly as evidenced by the presence of nondisrupted nuclei 
 on day 28 ; however , the presence of tight junctions remain absent 
 ( figure 7b ) . in a proof - of - concept 
 experiment ,
an immunocompetent triculture model comprising of epithelial , 
 dendritic , and fibroblast layers was constructed and dc migration 
 within the model monitored .
the triculture model was stimulated with 
 hdm extract ( a common airborne allergen ) and toll - like 4 receptor 
 agonist lps ( abundant in most airborne bacterial pathogens ) , and subsequently , 
 the migration of dcs was assessed by confocal microscopy .
epithelial 
 cells were identified using pancytokeratin and dcs had been prestained 
 using the nuclear stain hoescht .
assessment 
 of the uppermost scaffold ( bearing epithelial cells ) revealed that 
 after 36 h in the triculture , most dcs seemed to have migrated to 
 this layer and were present in close proximity to the basal ( in unstimulated 
 samples ) or apical side ( in stimulated samples ) of the epithelial 
 cell layer ( figure 8a ) .
the dcs migration was 
 further confirmed by the fact that dcs were found to be largely absent 
 from the scaffold upon which they were originally inoculated ( middle 
 scaffold ) ( figure 8b ) .
typically , not many 
 dcs could be observed in the majority of lower scaffolds ( fibroblast 
 layer ) in either stimulated or unstimulated samples ( figure 8c ) .
confocal microscopy images ( 10 mag ) of dc migration 
 36 h 
 after allergen stimulation in 3d tricoculture .
the upper scaffold 
 contained the calu-3 epithelial layer ( a ) , a middle layer to which 
 dcs were seeded ( b ) , and a lower scaffold containing mrc-5 ( c ) .
dcs 
 were prestained with hoescht nuclear stain ( blue ) and calu-3 cells 
 were poststained with pancytokeratin ( green ) . in single culture ,
dcs 
 remained on the middle scaffold where they had been inoculated . in 
 the triculture , most dcs migrated from middle scaffold ( b ) to upper 
 scaffold ( a ) . upon treatment of the triculture with house dust mite 
 extract ( hdm ) ( 10 g / ml ) and lipopolysaccharide ( lps ) ( 100 ng / ml )
, 
 dcs appeared to primarily migrate to the apical surface of the epithelial 
 layer , whereas in unstimulated samples they appear to be mainly localized 
 in the basal region of the epithelial layer ( a ) .
using electrospun porous fibers , we have 
 engineered a 3d triculture 
 of airway epithelial cells , dendritic cells , and fibroblasts at ali 
 creating a modular construct that mimics the cellular orientation 
 and some functional properties of human airway epithelium .
the epithelium 
 in vivo provides the first line of defense against environmental insults , 
 and as such , it is capable of rapidly repairing any cellular damage 
 caused .
epithelial cells in vivo are thought to respond to insult 
 and injury through a cascade of events of which there are three distinct 
 stages : dedifferentiation , proliferation , and differentiation .
the first stage is the dedifferentiation of 
 underlying stromal cells , such as fibroblasts , that are initially 
 exposed to the environment following injury to epithelial cells .
the 
 stromal cells are thought to migrate to the wound site to assist repair 
 by dedifferentiation helping to cover the site of injury and assist 
 in restoring barrier integrity through proliferation .
the final stage in restoring barrier integrity is the differentiation 
 of cells involved in the repair process , including the restoration 
 of full function to epithelial cells , particularly their ability to 
 form junctional protein to  seal  the barrier .
the exact mechanisms controlling cell 
 fate during epithelium repair 
 are not clear ; however , it is thought that multiple paracrine factors 
 and direct cell  cell contact are key parameters required to 
 restore barrier integrity and function .
it is , therefore , important to consider these factors when configuring 
 tissue engineering strategies to create in vitro models of epithelial 
 tissue .
this study developed a 
 3d multicellular lung model that is capable 
 of forming a more physiologically relevant representation of lung 
 tissue than culturing cells alone or in 2d systems .
our studies corroborate 
 the relevance of culturing multiple cell types found in lung tissue 
 together , as results demonstrate that when epithelial cells were subjected 
 to chemical insult ( i.e. , an enzymatically active allergen ) , they 
 recovered earlier in cocultures than single cultures .
this supports 
 the relevance of cocultures as it is thought that the cells signal 
 and respond in alliance .
this study also assembled a triculture 
 model in a proof - of - concept 
 experiment to examine whether dc migration could occur within the 
 lung model following stimulation .
a large number of dcs were 
 found to migrate from the scaffold in 
 which they were initially inoculated ( middle layer ) through to the 
 uppermost scaffold bearing the epithelial cells .
this occurred both 
 in the presence and absence of stimulation and may suggest that dcs 
 were responding to epithelial secretions .
however , in samples stimulated with lps and hdm , most dcs were located 
 closer to the apical side of the epithelial barrier , whereas in unstimulated 
 samples they were mainly located close to the basal side .
these results 
 prove that the 3d lung model is amenable to cell migration and that 
 cell  cell contact is feasible .
individual scaffolds are typically 
 60  10 m thick and the results of the dc migration study 
 demonstrate that scaffold thickness does not seem to impede dc migration . 

the pattern of migration observed in this experiment substantiates 
 dc migration in vivo , where upon activation , dcs migrate to the epithelium 
 and survey the immune environment before migrating through the subepithelial 
 compartment before traveling to lymph nodes .
this enables future studies focused on inhaled 
 drug delivery systems as well as allergen and inhaled drugs uptake 
 by dcs .
we would estimate the triculture would be limited to one week 
 due to migration of dcs and their phenotypical / functional changes 
 upon stimulation , which should provide enough time for performing 
 drug uptake / delivery experiments .
limitations to in vitro models , 
 particularly those generated from commercially available 2d inserts , 
 are that the surface upon which cells grow is planar , so cell  cell 
 interaction is restricted .
cell interactions can occur through the 
 pores of the insert ; however , these are thought to be constrained 
 to paracrine interactions because the pore size of the substrate upon 
 which the epithelial cells are commonly cultured to allow barrier 
 formation are too small , thus preventing cell migration .
a 3d human 
 skin triculture model comprising of keratinocytes , dcs , and fibroblasts 
 has been reported .
this model shares 
 similarities with the model developed in this work , including a physiologically 
 relevant arrangement of the cell layers each supported on separate 
 3d scaffolds and the establishment of a differentiated epithelial 
the 
 keratinocytes and fibroblasts were supported on 3d microfiber - scale 
 scaffolds ; however , the dc layer was inserted into the model in an 
 agarose gel rather than on a fibrous scaffold .
migration of dcs in 
 the skin model was reported from the agarose gel layer to only the 
 fibroblast layer .
furthermore , the degree of migration appeared to 
 be far less than that observed in the 3d model developed in this study . 

hence , direct seeding of dcs on the porous scaffolds as opposed to 
 encapsulation within a hydrogel seems to facilitate cell migration .
epithelial cell lines such as calu-3 , the cell line we have used 
 in this study , are widely used as surrogates for primary cells . despite many similarities between calu-3 and 
 primary cells , particularly in features like barrier formation and 
 mucus production , there are also considerable functional differences 
 ( e.g. , their cytokine profile )
. therefore , use of cell lines somewhat 
 limits the physiological relevance of the model and future efforts 
 should focus on replacing cell lines with human primary cells .
the 3d immunocompetent lung model presented in this study successfully 
 supports the culture of multiple cell types on electrospun pet scaffolds 
 that structurally resemble the native lung ecm .
the porous network 
 of electrospun pet fibers can permit cell interaction through both 
 direct cell  cell contact and through paracrine factors within 
 the 3d model as shown by the enhanced formation of a differentiated 
 epithelial layer , enhanced epithelial repair , and migration of immune 
 cells incorporated within the model .
furthermore , the modular nature 
 of our approach means that other relevant structural ( e.g. , smooth 
 muscle cells ) and immune cells ( e.g. , mast cells or eosinophils ) could 
 be included into the model with relative ease .
collectively , 
 we believe this model possesses adequate cellular 
 and structural representation of the airway epithelium and is amenable 
 to in situ monitoring ; as such , it presents an invaluable tool for 
 academic and pharmaceutical research within the fields of lung biology , 
 disease modeling , and drug discovery and delivery with the potential 
 of reducing the need for some animal experimentation in this area .
 the development of more complex in 
 vitro models for the assessment 
 of novel drugs and chemicals is needed because of the limited biological 
 relevance of animal models to humans as well as ethical considerations . 
 
 although some human - cell - based assays exist , they are usually 2d , 
 consist of single cell type , and have limited cellular and functional 
 representation of the native tissue . in this study , we have used biomimetic 
 porous electrospun scaffolds to develop an immunocompetent 3d model 
 of the human respiratory tract comprised of three key cell types present 
 in upper airway epithelium . 
 the three cell types , namely , epithelial 
 cells ( providing a physical barrier ) , fibroblasts ( extracellular matrix 
 production ) , and dendritic cells ( immune sensing ) , were initially 
 grown on individual scaffolds and then assembled into the 3d multicell 
 tissue model . 
 the epithelial layer was cultured at the air  liquid 
 interface for up to four weeks , leading to formation of a functional 
 barrier as evidenced by an increase in transepithelial electrical 
 resistance ( teer ) and tight junction formation . 
 the response of epithelial 
 cells to allergen exposure was monitored by quantifying changes in 
 teer readings and by assessment of cellular tight junctions using 
 immunostaining . 
 it was found that epithelial cells cocultured with 
 fibroblasts formed a functional epithelial barrier at a quicker rate 
 than single cultures of epithelial cells and that the recovery from 
 allergen exposure was also more rapid . 
 also , our data show that dendritic 
 cells within this model remain viable and responsive to external stimulation 
 as evidenced by their migration within the 3d construct in response 
 to allergen challenge . 
 this model provides an easy to assemble and 
 physiologically relevant 3d model of human airway epithelium that 
 can be used for studies aiming at better understanding lung biology , 
 the cross - talk between immune cells , and airborne allergens and pathogens 
 as well as drug delivery . 
Introduction
Experimental Section
Results
Discussion