The research leading to these AirPROM results has received funding from the European Union under grant agreement&#160;n&#176; 270194.This work was also funded by the National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs), and the Engineering and Physical Research Centre (EPSRC) Doctoral Training Centre (DTC) in Regenerative Medicine, U.K.

Primary ASM cells from non-asthmatic individuals were isolated from bronchial biopsies at the Glenfield Hospital (Leicester, UK) as described previously. The research was approved by the Leicestershire Ethics Committee and patients gave their written, informed consent.
1. Electrospinning PET Scaffolds (Figure 2)
<ol>
	<li>Prepare a solution (10 ml) of drink bottle grade PET in a 1:1 dichloromethane (DCMro) &#8211; trifluoroacetic acid (TFA) solution. PET concentrations of 8%, 30% and 10% weight/volume (wt/vol) are required for nanofiber, microfiber and aligned scaffolds respectively and then stir the solution O/N at RT for the PET to dissolve.</li>
	<li>Transfer the PET solution into a syringe and attach a 23-G (for nanofiber) or an 18-G (for microfiber and aligned) needle to the syringe and place the syringe into a motorised syringe pump. Make sure the point of the needle is rotated to the bottom.</li>
	<li>Position the mandrel 15 cm away from the tip of the needle, ensuring the needle is pointed at the centre of the drum.</li>
	<li>Attach the electricity supply to the needle tip with crocodile clips and ground the mandrel (via banana socket) to earth. Switch on the power supply to the mandrel and set speed to 60 rpm (equivalent to approximately 13.2 m&#183;min-1) for nanofiber and microfiber scaffolds. Set speed to 2,000 rpm (equivalent to approximately 440 m&#183;min-1) for aligned scaffolds.</li>
	<li>Set the syringe pump to a flow rate of 0.5 ml&#183;hr-1 for nanofiber random and aligned scaffolds or 2.0 ml&#183;hr-1 for microfiber scaffolds. Allow the pump to run until solution is extruded from the needle tip in order to remove any air in the needle. Then stop the pump.</li>
	<li>On the syringe pump, set the total volume of solution to be expelled to 2 ml&#160;and start the pump.</li>
	<li>Set voltage supply to 14 kV, and switch on the power supply.</li>
	<li>Electrospin until 2 ml&#160;of solution has been electrospun (4 hr&#160;for nanofiber and aligned scaffolds, 1 hr&#160;for microfiber scaffolds).</li>
	<li>Cut the scaffold with a blade along the width of the mandrel. This produces a 2D sheet of scaffold the size of the mandrel surface area (in this case, a rectangular sheet 22 cm x 11 cm) which can be carefully peeled off the mandrel). Store the scaffold in aluminium foil to reduce electrostatic charge). 
	Note: Biphasic scaffolds are produced by sequential electrospinning a nanofiber scaffold directly onto a microfiber scaffold.</li>
</ol>
2. Sterilization of Scaffolds before Use in Cell Culture
<ol>
	<li>From the scaffold sheet, punch out discs of biphasic or aligned scaffolds using a 0.8 mm diameter biopsy pen and stick to gasket using non-toxic aquarium glue.</li>
	<li>Sterilize scaffolds using ultra violet irradiation for 30 min&#160;on each side of the scaffolds before soaking in a 20% vol/vol antibiotic/ antimycotic solution (2x105 units ml-1 penicillin G, 2,000 mg ml-1 streptomycin sulphate and 500 &#181;g ml-1 amphotericin B) O/N at 4 &#176;C.</li>
	<li>Wash scaffolds with PBS 3 times, and then store scaffolds in TCP well plates in PBS at 4 &#176;C until use.</li>
</ol>
3. Cell Culture Media Constituents
<ol>
	<li>Culture CALU3 epithelial cells in DMEM-F12 media supplemented with 10% vol/vol foetal calf serum (FCS), 2 nM L-glutamine solution, 1% vol/vol antibiotic/ antimycotic solution (10,000 units ml-1 penicillin G, 100 mg ml-1 streptomycin sulphate and 25 &#181;g ml-1 amphotericin B).</li>
	<li>Culture MRC5 fibroblast and ASM cells in DMEM media supplemented with 10% vol/vol foetal calf serum (FCS), 2 nM L-glutamine solution, 1% vol/vol antibiotic/ antimycotic solution (10,000 units ml-1 penicillin G, 100 mg ml-1 streptomycin sulphate and 25 &#181;g ml-1 amphotericin B).</li>
	<li>Culture epithelial-fibroblast-ASM cell co-cultures in a 70:30 DMEM-F12:DMEM media mixture.</li>
</ol>
4. Seeding of Fibroblast and Epithelial Cells onto Biphasic Scaffold
<ol>
	<li>In a tissue culture plate, soak the scaffolds in DMEM-supplemented media and incubate at 37 &#176;C for 1 hr&#160;before cell seeding.</li>
	<li>Remove the DMEM-supplemented media, place the microfiber phase of scaffold apically and seed 1.5 x104 MRC5 fibroblast cells in 30 &#181;l&#160;of DMEM-supplemented media. Agitate plate on an orbital shaker for 2 hr, before leaving in an incubator at 37 &#176;C 5% CO2 in air O/N.</li>
	<li>Turn the scaffold over so the nanofiber phase of scaffold faces apically, seed 3.0 x104 CALU3 epithelial cells in 30 &#181;l&#160;of DMEM&#8211;F12-supplemented media onto the nanofiber phase of the scaffold and incubate for 2 hr&#160;at 37 &#176;C, 5% CO2 in air before submerging the scaffold in 70:30 DMEM-F12:DMEM-supplemented media O/N prior to transferring to the bioreactor.</li>
</ol>
5. Seeding of ASM Cells onto the Aligned Scaffold
<ol>
	<li>In a tissue culture plate, soak the scaffolds in DMEM-supplemented media and incubate for 1 hr at 37 &#176;C before seeding 2.5 x104 cells in 30 &#181;l&#160;of DMEM-supplemented media and incubating for 2 hr&#160;(37 &#176;C, 5% CO2 in air). Submerge scaffold in DMEM-supplemented media return to incubator and leave O/N before setting up a tri-culture in the bioreactor.</li>
</ol>
6. Setting-up the Tri-culture using the Bioreactor System (Figure 7)
<ol>
	<li>Place the biphasic scaffold gasket into the groove within one chamber with the epithelial phase facing up into the chamber.</li>
	<li>Place the aligned scaffold underneath the biphasic scaffold (so it is adjacent to the microfiber phase of the biphasic scaffold) and lock the two chambers of the bioreactor together.</li>
	<li>Assemble two perfusion flow circuits connected to two medium reservoirs (DMEM-F12-supplemented media in apical reservoir, DMEM-F12/DMEM-supplemented media in basal reservoir) and pump media around the two circuits at approximately 0.1 ml/min&#160;using a peristaltic pump (40 ml&#160;media is recycled through each circuit). House all parts of the system within an incubator at 37 &#176;C, 5% CO2 in air.</li>
	<li>After one week, remove the media from the apical chamber, and culture the epithelial cells at the ALI for a further week before analysis.</li>
</ol>

<ol>
	<li>Macchiarini, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+transplantation+of+a+tissue-engineered+airway.">Clinical transplantation of a tissue-engineered airway.</a> Lancet. 372 (9655), 2023-2030 (2008).</li><li>Song, J., Guyette, J., Gilpin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+experimental+orthotopic+transplantation+of+a+bioengineered+kidney.">Regeneration and experimental orthotopic transplantation of a bioengineered kidney.</a> Nature medicine. 19 (5), 646-651 (2013).</li><li>Wenzel, S., Holgate, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+trap:+it+still+yields+few+answers+in+asthma.">The mouse trap: it still yields few answers in asthma.</a> American journal of respiratory and critical. 174 (11), 1171-1173 (2006).</li><li>Zosky, G. R., Sly, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+asthma.">Animal models of asthma.</a> Clinical and experimental allergy journal of the British Society for Allergy and Clinical Immunology. 37 (7), 973-988 (2007).</li><li>Bates, J. H. T., Rincon, M., Irvin, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+asthma.+American+journal+of+physiology.">Animal models of asthma. American journal of physiology.</a> Lung cellular and molecular physiology. 297 (3), L401-L410 (2009).</li><li>Johansson, F., Carlberg, P., Danielsen, N., Montelius, L., Kanje, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+outgrowth+on+nano-imprinted+patterns.">Axonal outgrowth on nano-imprinted patterns.</a> Biomaterials. 27 (8), 1251-1258 (2006).</li><li>Paik, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+micropatterning+of+cell+lines+and+human+pluripotent+stem+cells+on+elastomeric+membranes.">Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.</a> Biotechnology and bioengineering. 109 (10), 2630-2641 (2012).</li><li>Williams, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+Cell+Sheets+Grown+on+Thermo-Responsive+Substrates+with+Microcontact+Printed+Protein+Patterns.">Aligned Cell Sheets Grown on Thermo-Responsive Substrates with Microcontact Printed Protein Patterns.</a> Advanced Materials. 21 (21), 2161-2164 (2009).</li><li>Schindler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+nanofibrillar+matrix+promotes+in+vivo-like+organization+and+morphogenesis+for+cells+in+culture.">A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture.</a> Biomaterials. 26 (28), 5624-5631 (2005).</li><li>Schindler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+in+Three+Dimensions.">Living in Three Dimensions.</a> Cell Biochemistry and Biophysics. 45 (14), 215-227 (2006).</li><li>Ahmed, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology,+cytoskeletal+organization,+and+myosin+dynamics+of+mouse+embryonic+fibroblasts+cultured+on+nanofibrillar+surfaces.">Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces.</a> Molecular and Cellular Biochemistry. 301 (1-2), 241-249 (2007).</li><li>Flemming, R., Murphy, C., Abrams, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synthetic+micro-and+nano-structured+surfaces+on+cell+behavior.">Effects of synthetic micro-and nano-structured surfaces on cell behavior.</a> Biomaterials. 20, 573-588 (1999).</li><li>Sun, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+3D+Cell+Culture+System+for+Investigating+Cell+Interactions+With+Electrospun+Fibers.">Development of a 3D Cell Culture System for Investigating Cell Interactions With Electrospun Fibers.</a> Biotechnol. Bioeng. 97 (5), 1318-1328 (2007).</li><li>Kim, D. -. H., Provenzano, P. P., Smith, C. L., Levchenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+nanotopography+as+a+regulator+of+cell+function.">Matrix nanotopography as a regulator of cell function.</a> The Journal of cell biology. 197 (3), 351-360 (2012).</li><li>Jeffery, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+in+Asthma+and+Chronic+Obstructive+Lung+Disease.">Remodeling in Asthma and Chronic Obstructive Lung Disease.</a> American Journal of Respiratory and Critical Care Medicine. 164 (10 pt 2), S28-S38 (2001).</li><li>Jungebluth, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracheobronchial+transplantation+with+a+stem-cell-seeded+bioartificial+nanocomposite:+a+proof-of-concept+study.">Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study.</a> Lancet. 378 (9808), 1997-2004 (2011).</li><li>Miller, C., George, S., Niklason, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+a+tissue-engineered+model+of+the+human+bronchiole.">Developing a tissue-engineered model of the human bronchiole.</a> Journal of Tissue Engineering and Regenerative. (July), 619-627 (2010).</li><li>West, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+3D+multicell+microtissue+culture+model+of+airway+smooth+muscle.+American+journal+of+physiology.">Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle. American journal of physiology.</a> Lung cellular and molecular physiology. 304 (1), L4-L16 (2013).</li><li>Nesmith, A. P., Agarwal, A., McCain, M. L., Parker, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+airway+musculature+on+a+chip:+an+in+vitro+model+of+allergic+asthmatic+bronchoconstriction+and+bronchodilation.">Human airway musculature on a chip: an in vitro model of allergic asthmatic bronchoconstriction and bronchodilation.</a> Lab on a chip. , (2014).</li><li>Hadjizadeh, A., Ajji, A., Bureau, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano/micro+electro-spun+polyethylene+terephthalate+fibrous+mat+preparation+and+characterization.">Nano/micro electro-spun polyethylene terephthalate fibrous mat preparation and characterization.</a> Journal of the mechanical behavior of biomedical materials. 4 (3), 340-351 (2011).</li><li>Morris, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+airway+smooth+muscle+maintain+in+situ+cell+orientation+and+phenotype+when+cultured+on+aligned+electrospun+scaffolds.">Human airway smooth muscle maintain in situ cell orientation and phenotype when cultured on aligned electrospun scaffolds.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 307 (1), L38-L47 (1999).</li><li>Morris, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+electrospun+biphasic+scaffold+provides+optimal+three-dimensional+topography+for+in+vitro+co-culture+of+airway+epithelial+and+fibroblast+cells.">A novel electrospun biphasic scaffold provides optimal three-dimensional topography for in vitro co-culture of airway epithelial and fibroblast cells.</a> Biofabrication. 6 (3), 035014 (2014).</li><li>Sell, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+Natural+Polymers+in+Tissue+Engineering:+A+Focus+on+Electrospun+Extracellular+Matrix+Analogues.">The Use of Natural Polymers in Tissue Engineering: A Focus on Electrospun Extracellular Matrix Analogues.</a> Polymers. 2 (4), 522-553 (2010).</li><li>Subbiah, T., Bhat, G. S., Tock, R. W., Parameswaran, S., Ramkumar, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+nanofibers.">Electrospinning of nanofibers.</a> Journal of Applied Polymer Science. 96 (2), 557-569 (2005).</li><li>Dalton, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+and+additive+manufacturing:+converging+technologies.">Electrospinning and additive manufacturing: converging technologies.</a> Biomaterials Science. 1 (2), 171 (2013).</li><li>Boland, E. D., Wnek, G. E., Simpson, D. G., Pawlowski, K. J., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+tissue+engineering+scaffolds+using+electrostatic+processing+techniques:+A+study+of+poly(glycolic+acid)+electrospinning.">Tailoring tissue engineering scaffolds using electrostatic processing techniques: A study of poly(glycolic acid) electrospinning.</a> Journal of Macromolecular Science-Pure and Applied Chemistry. 38 (12), 1231-1243 (2001).</li><li>Stitzel, J. D., Bowlin, G. L., Mansfield, K., Wnek, G. E., Simpson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospraying+and+electrospinning+of+polymers+for+biomedical+applications.+Poly(lactic-co-glycolic+acid)+and+poly(ethylene-co-vinylacetate).">Electrospraying and electrospinning of polymers for biomedical applications. Poly(lactic-co-glycolic acid) and poly(ethylene-co-vinylacetate).</a> Revolutionary Materials: Technology And Economics. 32, 205-211 (2000).</li><li>Liu, W., Thomopoulos, S., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibers+for+regenerative+medicine.">Electrospun nanofibers for regenerative medicine.</a> Advanced healthcare materials. 1 (1), 10-25 (2012).</li><li>Veleirinho, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+and+concentration+effects+on+the+properties+of+electrospun+poly+(ethylene+terephthalate)+nanofiber+mats.">Solvent and concentration effects on the properties of electrospun poly (ethylene terephthalate) nanofiber mats.</a> Journal of Polymer. , 460 (2008).</li><li>Balguid, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+Fiber+Diameter+in+Electrospun+Poly(epsilon-Caprolactone)+Scaffolds+for+Optimal+Cellular+Infiltration+in+Cardiovascular+Tissue+Engineering.">Tailoring Fiber Diameter in Electrospun Poly(epsilon-Caprolactone) Scaffolds for Optimal Cellular Infiltration in Cardiovascular Tissue Engineering.</a> Tissue Engineering Part A. 15 (2), 437-444 (2009).</li><li>Shalumon, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+three-dimensional+nano,+micro+and+micro/nano+scaffolds+of+porous+poly(lactic+acid)+by+electrospinning+and+comparison+of+cell+infiltration+by+Z-stacking/three-dimensional+projection+technique.">Fabrication of three-dimensional nano, micro and micro/nano scaffolds of porous poly(lactic acid) by electrospinning and comparison of cell infiltration by Z-stacking/three-dimensional projection technique.</a> IET nanobiotechnology / IET. 6 (1), 16-25 (2012).</li><li>Thevenot, P., Nair, A., Dey, J., Yang, J., Tang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+to+Analyze+Three-Dimensional+Cell+Distribution+and+Infiltration+in+Degradable+Scaffolds.">Method to Analyze Three-Dimensional Cell Distribution and Infiltration in Degradable Scaffolds.</a> Tissue Engineering Part C-Methods. 14 (4), 319-331 (2008).</li><li>Rose, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelatin-Based+Materials+in+Ocular+Tissue+Engineering.">Gelatin-Based Materials in Ocular Tissue Engineering.</a> Materials. 7 (4), 3106-3135 (2014).</li><li>Liliensiek, S. J., Nealey, P., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Endothelial+Basement+Membrane+Nanotopography+in+Rhesus+Macaque+as+a+Guide+for+Vessel+Tissue+Engineering.">Characterization of Endothelial Basement Membrane Nanotopography in Rhesus Macaque as a Guide for Vessel Tissue Engineering.</a> Tissue Engineering Part A. 15 (9), (2009).</li><li>Abrams, G. A., Murphy, C. J., Wang, Z. -. Y., Nealey, P. F., Bjorling, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+basement+membrane+topography+of+the+bladder+epithelium.">Ultrastructural basement membrane topography of the bladder epithelium.</a> Urological research. 31 (5), 341-346 (2003).</li><li>Abrams, G. A., Bentley, E., Nealey, P. F., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Microscopy+of+the+Canine+Corneal+Basement+Membranes.">Electron Microscopy of the Canine Corneal Basement Membranes.</a> Cells Tissues Organs. 170 (4), 251-257 (2002).</li><li>Reichl, S., Bednarz, J., Muller-Goymann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+equivalent+as+cell+culture+model+for+in+vitro+drug+permeation+studies.">Human corneal equivalent as cell culture model for in vitro drug permeation studies.</a> British Journal of Ophthalmology. 88 (4), 560-565 (2004).</li></ol>

The Authors have nothing to disclose.

The ability to electrospin polymeric fibers with structural properties comparable to natural ECM has led to numerous natural or synthetic polymers, or polymer blends being electrospun to recreate these environments,. Manipulation of the process parameters (including polymer choice, polymer concentration, solvent, needle tip distance and temperature) can all influence scaffold characteristics; however some parameters can have greater impact on scaffold properties than others25,. When modifying PET fiber diameter, we found the key parameters to be the concentration of the polymer used, the rate at which the polymer solution was electrospun, and the needle&#8217;s diameter. When electrospinning aligned fibers the key parameters are the collection method used (a rotating mandrel), and the speed at which the mandrel rotates. Through reducing the mandrel speed to 60 rpm, we found the randomly aligned scaffolds produced showed greater scaffold uniformity compared to electrospinning onto a flat collector plate.
The characteristics of decellularized airway tissue were analysed to provide guidance for the various scaffold topographies developed for the culture of each airway cell-type. Electrospun nanofibers are an attractive scaffold to replicate basement membrane structures due to the small pore size but high overall porosity that allows for increased metabolite diffusion whilst restricting cellular movement.9 Whilst optimising the electrospinning parameters, a range of PET concentrations and flow rates were tested. The thinnest fibers were achieved using a 6% wt/vol PET solution, previously used by other groups. However, high levels of beading, and a lack of uniformity were constant problems. Initial attempts to remove the beading included adding a cationic surfactant, cetyltrimethylammonium bromide (CTAB), to the solution to lower the surface tension, and testing various sources of PET. The addition of surfactant reduced the amount of beading, but not fully. After trying a number of commercial sources of PET pellets, food grade drink bottle PET was used, by cutting up drinks bottles and dissolving these in the DCM:TFA solvent solution. Using this as the PET source resulted in an increase in fiber uniformity and a reduction in fiber beading. By increasing the solution concentration slightly to 8% wt/vol and reducing the needle diameter (18G to 23G) we consistently produced defect free nanofibers with a fiber diameter of approximately 250 nm. Electrospun nanofiber scaffolds can be highly electrostatic upon collection from the mandrel making manual handling of the scaffolds difficult. This was improved by storing the scaffolds in aluminium foil after spinning and soaking in 70% IMS before use. This appeared to help dissipate the residual electrostatic charge left on the scaffold from the electrospinning process.
To provide topographies similar to the individual microenvironments encountered by the three main airway cell types within the airway wall, a basic electrospinning protocol was adapted to produce three unique scaffolds for these cell types: by electrospinning a low concentration PET solution slowly, randomly aligned nanofibers were generated, onto which epithelial cells were seeded (mimicking the RBM). By electrospinning a higher concentration PET solution at a faster rate, randomly aligned microfibers were generated (producing a more porous scaffold), into which fibroblasts were seeded (mimicking the sub-mucosal area immediately beneath the RBM). By electrospinning a low concentration PET solution onto a high velocity rotating mandrel aligned nanofibers were produced, onto which smooth muscle cells orientated in the fiber direction, producing aligned sheets of cells.
Increased fiber diameters lead to increased pore size, allowing greater cell penetration into scaffolds and so help create a true 3D environment,. Microfiber scaffolds were employed for the culture of fibroblasts in the study to ensure that the cells resided on multiple planes within the scaffold. By increasing the PET concentration to 30% wt/vol, PET fibers with an average diameter of 2.5 &#181;m were produced. Greater diameter fibers (&#8776; 4 &#181;m) were produced using a 35% wt/vol PET solution, but scaffold thickness uniformity was lost, and higher variability between individual fibers was apparent. Pores in the microfiber scaffold were over 7 times greater than those measured in the nanofiber scaffolds (10.45 &#181;m vs 1.43 &#181;m respectively), but still static seeding of cells provided limited cellular penetration. This was improved by dynamically seeding the cells using an orbital mixer, a method shown to be effective previously.
Highly aligned nanofiber scaffolds were created by electrospinning a 10% wt/vol PET solution onto the mandrel rotating at 2,000 rpm (&#8776; 440 m&#183;min-1). The PET concentration was increased from 8% to prevent an irregular wave-like morphology that fibers exhibited at lower concentrations. Despite the increase in concentration, aligning the fibers reduced the average fiber diameter (216 nm vs. 255 nm, aligned vs. random). The high speed of the mandrel drawing the fibers out before they have dried is likely to cause this effect. The alignment of the fibers influenced the alignment of ASM cells, and also has other physiological effects on the ASM cells which have been characterized elsewhere21.
The major limitation of this protocol is the inability to image live cells on/in the scaffolds making initial cell-attachment or differentiation over an extended time period impossible to determine without sacrificing samples. This means most optimization occurs after the culture period; i.e., fixing samples and then measuring whether cell culture was successful after not during cell culture. Observing a color change in scaffolds incubated in alamarBlue (blue to pink) indicates cells are present and viable, but is not ideal. Electrospinning more transparent polymers such as gelatin could aid in visualizing cells on the scaffolds. The electrospinning of a nanofibrous scaffold within our model allowed the replication of the airway RBM, similarly comprised of dense nanofibers on which the epithelial cells reside. It has previously been shown that structural cells cannot migrate through the nanofiber scaffold22, although immune cells are able to (data not shown). Whilst advantageous for the culturing of specific cell-types on a 3D surface (such as epithelial and endothelial cells), this prevention of structural cell migration through the nanofibers may be detrimental for the culturing of other cell types. As smooth muscle is unable to migrate through the aligned nanofiber scaffold we assembled the tri-layered coculture with the smooth muscle and fibroblast layers facing each other (opposed to the aligned scaffold separating the two cell types). Whilst this allows the cells to be in close apposition, the current study cannot conclude whether one cell type (either the fibroblast or smooth muscle) would overtake the whole layer over a more prolonged culture period. Despite these limitations, a viable tri-layer model of the airway wall has been developed that provides an alternative platform to investigate the interactions between these multiple cell-types. A similar tri-layered study has recently been published where fibroblasts were embedded within a cylindrical collagen I hydrogel with smooth muscle seeded on the outer surface and epithelial cells within the luminal surface17. The mechanical stability of the electrospun scaffolds developed here would allow similar tubular constructs to be formed, and remains a current research aim within our group. Whilst the study has focused on the airway, several organs within the body share this basic mucosal structural unit, and through modifying the tenants highlighted in this study, similar platforms could be developed for tissues containing a basement membrane unit including blood vessels, the bladder and the cornea, where collagen based multi-layered models have been employed.

The field of regenerative medicine and tissue engineering is rapidly advancing, with breakthroughs in tracheal and kidney regeneration two notable recent achievements. The biomaterials developed in tissue engineering are becoming more accessible, with opportunities to transfer such protocols to less specialized laboratories. One field primed to benefit through increasing use of biomaterials is in vitro diagnostics.
In vitro studies are an important platform to investigate intra-cellular signaling pathways within single cell-types, and have helped delineate mechanisms underlying numerous disease pathophysiologies. These studies generally rely on a single cell-type being cultured as a monolayer on tissue culture plastic (TCP); a two-dimensional (2D) rigid surface far less elastic and porous than the three-dimensional (3D) environment cells are exposed to within a tissue or organ. Animal models have traditionally been employed to confirm effects found in vitro also translate to whole tissue, and can also be used as pre-clinical platforms to investigate human diseases. However, inter-species differences undermine this reliance on animal models in our understanding of human disease-. For example, much understanding of the lung disease asthma is based upon a mouse model despite inherent differences between the human condition and this model including little evidence of mast cell infiltration of the airway smooth muscle bundle, or the capacity for spontaneous disease development within the animal model3,4. There are also ethical considerations regarding the use of animal models, with the &#8220;3Rs&#8221; standard of &#8220;replacement, refinement, and reduction&#8221; in animal experimentation being encouraged in the UK and other countries.
An attractive alternative would be the recapitulation of human tissue in vitro creating structures to investigate the cooperative nature between adult human cell types in a single unit. Cells exist in 3D multi-cellular structures within tissue, each within a unique micro-environment. The culturing of cells on TCP only allows the culture of cell monolayers, unable to replicate this environment, or provide the capacity for multi-cell culture. Biomaterials advancement provides the opportunity to develop both natural and synthetic 3D platforms for cell culture. Decellularized ECM can be used for 3D cell culture when recellularized with other cell-types including stem cells1, however such protocols can be complex and time consuming, with the availability of tissue limited and largely of non-human origin. Other protocols allow greater control over the cellular environments created such as nanoimprinting, ECM deposition, cell sheet technologies, or electrospinning. Electrospinning creates non-woven 3D porous mats of fibers with diameters ranging from nanometer to micrometer, replicating natural ECM dimensions. Electrospun scaffolds are increasingly being employed as 3D cell culturing platforms-. Manipulation of the electrospinning parameters allows intricate control over scaffold characteristics such as pore size, fiber diameter, topography and alignment, and surface chemistry. Alterations of such parameters have been shown to directly affect cell adhesion and growth, when cells were cultured in isolation.
These advantages have been exploited in the present study to allow the culturing of multiple cell types as a single 3D tissue construct, using the airway bronchiole as a model 3D tissue structure. The bronchiole wall consists of three main regions (Figure 1). The mucosa is where airway epithelial cells sit at the air-liquid interface (ALI), providing an important barrier to the external environment. They reside on the reticular basement membrane (RBM), a tightly compact ECM comprised mainly of collagen IV, perlecans, and laminins. The sub-mucosal layer is found directly below the mucosa, a more porous region comprised of multiple cell types including fibroblasts, myofibroblasts and infiltrating leukocytes under disease conditions. Finally smooth muscle bundles wrap around the airway in a helical fashion, and are comprised of aligned sheets of airway smooth muscle (ASM). It is the smooth muscle&#8217;s relative contractile state that controls airway tone. The employment of electrospun scaffolds within the pulmonary system had until recently been limited to regenerative purposes; with an electrospun tracheal replacement being successfully transplanted. Whilst successful, such treatments are limited in numbers, and are focused upon the trachea due to the relative simple nature of the tissue&#8217;s function. Limited examples of airway models used for in vitro diagnostics exist, and are mainly focused on smooth muscle contraction measurements,. The non-toxic and non-degradable polymer polyethylene terephthalate (PET) has previously been electrospun, and was employed in the present study to ensure scaffolds produced could be stored for long periods without degrading (allowing them to be used &#8220;off the shelf&#8221;), and also be suitable for stable cell culture over extended time periods. Previous studies from our group have shown that employing three variations of a basic electrospinning protocol, three individual PET electrospun scaffolds can be produced to provide optimal topographies for culturing airway epithelial, fibroblast, and smooth muscle cells in isolation21,22 and the coculture of airway epithelial and fibroblast cells22. Fiber diameter was found to greatly affect epithelial functionality22, and fiber alignment allowed the generation of aligned sheets of smooth muscle21. These studies were performed independently of each other under static conditions. In the present study, these different scaffolds, containing fully differentiated adult human cells have been cocultured for one week at the ALI in a commercially available bioreactor as a 3D section of the airway wall, providing a physiologically relevant in vitro model to investigate airway inter-cellular responses. Whilst this protocol is using the airway bronchiole as a model system, it could be adapted as a platform for the 3D coculture of mucosal units from other organs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polyethylene terephthalate (PET)</td>
			<td>Lucozade (GSK) bottles</td>
			<td>N/A</td>
			<td>Source of PET for electrospinning. Cut into small pieces and weigh out as necessary</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td></td>
			<td></td>
			<td>Solvent for PET</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Sigma</td>
			<td></td>
			<td>Solvent for PET</td>
		</tr>
		<tr>
			<td>Rotating Mandrel</td>
			<td>Built in house</td>
			<td></td>
			<td>Used to collect electrospun fibres. By rotating at different speeds, fibres can be aligned or randomly oriented</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Harvard apparatus</td>
			<td></td>
			<td>used in the electrospinning process</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td></td>
			<td>Culture medium for CALU3 cells</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td></td>
			<td>Culture medium for HASM cells</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Gibco</td>
			<td></td>
			<td>Culture medium for MRC5 cells</td>
		</tr>
		<tr>
			<td>Antibiotic/ antimycotic solution</td>
			<td>Gibco</td>
			<td></td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Gibco</td>
			<td></td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>Orbital mixer (Orbital shake 503)</td>
			<td>Stuart Scientific</td>
			<td></td>
			<td>For dynamic seeding of cells onto microfibre scaffolds</td>
		</tr>
		<tr>
			<td>Peristaltic Pump&#160;</td>
			<td>Watson Marlow</td>
			<td></td>
			<td>For providing media flow through bioreactor</td>
		</tr>
		<tr>
			<td>3DKube</td>
			<td>Kiyatec</td>
			<td></td>
			<td>Bioreactor for 3D cell culture</td>
		</tr>
	</tbody>
</table>

adapting the electrospinning process to provide three unique environments for a tri-layered in vitro model of the airway wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Scanning electron microscope images of decellularized airway tissue or immunohistological images from airway biopsies identified desirable characteristics of the airway ECM we wished to introduce into electrospun scaffold topographies: Native RBM matrix consists of fibers approximately 153 nm &#177; 30.6 nm (mean &#177; SD n=50 measurements) in diameter (Figure 3A &#38; 5A), whilst the matrix surrounding the RBM was more porous in nature (Figure 3C). Immunohistological sections through smooth muscle bundles show ASM present as aligned sheets of cells21 (Figure 3E). This information was used to guide the characteristics introduced into the electrospun scaffolds. A rotating mandrel was able to stably rotate at high speeds (&#62;2,000 rpm/ 440 m&#183;min-1) to produce scaffolds with aligned fibers. Slow mandrel rotation (60 rpm 13.2 m&#183;min-1) did not affect the fiber orientation, but produced scaffolds with more uniform thickness than when electrospinning onto a static plate. By altering the electrospinning parameters (PET concentration and solution flow rate), it was possible to create scaffolds fibers with diameters of several micrometers or hundreds of nanometers (electrospinning parameters are stated in Table 1).
Electrospun fibers with equivalent fiber diameters to natural RBM fibers (150 nm) were produced using a 6% PET solution, however at such low PET concentrations, beading of the fibers occurred (Figure 4A). This was eliminated by increasing the concentration of the PET solution to 8% which produced uniform nanofibers possessing an average diameter of 255 nm &#177; 2.4 nm (mean &#177; SEM) and average pore size of 1.43 &#181;m &#177; 0.02 &#181;m (Figures 4B, 5A&#38;B). To electrospin nanofibers it was necessary to reduce the needle size from 18 G to 23 G, allowing slower flow rates whilst maintaining a higher flow velocity and reducing needle blockage, a recurring problem with the larger needle. When electrospinning microfibers, an increase in PET solution to &#62;30 % PET lead to a lack of uniformity in fiber diameter (Figure 4F) in addition to multiple blockages at the needle tip due to the high solution viscosity. Uniform microfiber mats possessing an average fiber diameter of 2.50 &#181;m &#177; 0.02 &#181;m (mean &#177; SEM) and average pore size of 10.45 &#181;m &#177; 0.13 &#181;m were produced when electrospinning a 30% wt/vol PET solution at a flow rate of 2 mL/h-1 (Figures 4F, 5A&#38;B). Sequential electrospinning of the nanofiber scaffold directly onto a microfiber scaffold (still attached to the mandrel) created a biphasic scaffold (Figure 3D). The average fiber diameters were 280 nm &#177; 20 nm and 2.30 &#181;m &#177; 0.06 &#181;m for the nanofiber and microfiber phases respectively, comparable to dimensions of individual nanofiber and microfiber scaffolds. Additionally, the thickness of the biphasic scaffold was similar to the sum of the individual nanofiber and microfiber scaffolds (Figure 5C). Through increasing the mandrel speed (2,000 rpm/440 m&#183;min-1), highly aligned nano- or microfiber scaffolds were produced. The 8% wt/vol PET solution was not optimal for producing aligned nanofibers and produced fibers possessing a wave-like morphology (Figure 3F). An increase to 10% PET nullified this effect, but still resulted in a reduced fiber diameter (216 nm &#177; 2.2 nm (mean &#177; SEM)) compared to the randomly aligned nanofiber scaffolds. Fiber alignment was calculated by determining the deviation of each fiber&#8217;s angle from the mean fiber angle. In aligned nanofiber scaffolds 79% of fibers were aligned (&#177; 10&#176; mean fiber angle) compared to 10.2% of fibers in randomly aligned nanofiber scaffolds (Figure 5D).
The 8% nanofiber scaffold was used to recapitulate the RBM on which epithelial cells reside, and was used to support the culture of CALU3 cells (an airway epithelial cell-line). The 30% wt/vol microfiber scaffold, being more porous in nature was used to mimic the sub-mucosal region, and was cultured with MRC5 cells (an airway fibroblast cell-line). The 10% wt/vol aligned nanofiber scaffold provided topological referencing to ensure ASM cell alignment when cultured on the scaffold. All three cell-types showed an increase in viability when cultured on their individual scaffolds over a 2 week period (Figure 6A), and expressed cell-type specific proteins after the 2 week culture period (Figure 6B, 6C, &#38; 6D). Further characterization of individual cell-scaffold interactions have been reported elsewhere21,. The sequential electrospinning of the nanofiber scaffold onto the microfiber scaffold produced a biphasic scaffold for the coculture of the CALU3 epithelial cells and MRC5 fibroblast cells onto the nanofiber and microfiber phases respectively. The culturing of the two cells together under static conditions has been reported elsewhere22. Further work attempting to add other cell layers or extend biphasic culturing times beyond 2 weeks under static conditions proved unsuccessful (data not shown). To culture a tri-layered model over an extended period, we used a perfusion flow bioreactor. The CALU3 and MRC5 cells were seeded onto the biphasic scaffold, and ASM cells seeded onto an aligned scaffold, and both were cultured separately for 2 days. The two scaffolds were then bought together to form a tri-layer model of the airway wall within the bioreactor (Figure 7). Both chambers were perfused with media for 7 days before the apical epithelial chamber had its media removed, and the epithelial cells were cultured at the ALI for a further 7 days. Scaffolds were fixed and either sectioned and stained, or whole scaffolds were immunostained for cell-specific markers. Sections through the tri-layer culture showed cell nuclei distributed through all three layers of the coculture (Figure 8B). When cells were immunostained for cell-specific markers, epithelial cells populated the apical nanofiber phase as a confluent cell-layer and stained positive for cytokeratin (Figure 8C), on the microfiber phase, fibroblasts stained positive for S100A4 (Figure 8D), and on the aligned 10% scaffold ASM cells stained positive for SM22&#945; (Figure 8E), indicating good survival of each cell-type within the 3D model of the airway wall.
<img alt="Figure 1" src="/files/ftp_upload/52986/52986fig1.jpg" /> 
Figure 1.&#160;The airway bronchiole. Bronchial biopsy from a severe asthmatic airway showing the epithelium on the reticular basement membrane (RBM), with the underlying submucosal region populated with mesenchymal cells, and the surrounding smooth muscle bundles populated with smooth muscle cells stained for smooth muscle alpha-actin (brown). Scale bar indicates 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52986/52986fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52986/52986fig2.jpg" /> 
Figure 2.&#160;A schematic of the electrospinning equipment. A syringe containing the polymer/solvent solution (with needle attached) is placed on a syringe pump facing the mandrel. An electric potential is established between the needle and mandrel causing fibers to be ejected from the needle tip and deposited on the rotating mandrel. As the fiber passes through the atmosphere, the solvent evaporates causing deposition of polymer fibers on the mandrel. <a href="https://www.jove.com/files/ftp_upload/52986/52986fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52986/52986fig3.jpg" /> 
Figure 3.&#160;Comparison of electrospun scaffolds to native airway ECM. Scanning electron microscope images of decellularized basement membrane (A), decellularized airway bronchiole cross section (C) and a histological section of an airway smooth muscle bundle (stained with haematoxylin and eosin, scale bar 40 &#181;m) (E) compared with nanofiber (B) biphasic (D) and aligned (F) PET scaffolds. <a href="https://www.jove.com/files/ftp_upload/52986/52986fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1.&#160;Parameters used for electrospinning the microfiber, nanofiber and aligned scaffolds individually and for the biphasic scaffold.
<img alt="Figure 4" src="/files/ftp_upload/52986/52986fig4.jpg" /> 
Figure 4.&#160;Alteration in PET concentrations affects fiber characteristics. Scanning electron microscope images of electrospun scaffolds spun from 6%, 8%, 10% (A-C), 25%, 30% and 35% (E-G) (wt/vol) PET solutions. Also shown are aligned scaffolds produced from 8% (D) and 30% (H) wt/vol PET solutions. A-D imaged at 5,000X magnification, E-H imaged at 1,000X magnification. <a href="https://www.jove.com/files/ftp_upload/52986/52986fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52986/52986fig5.jpg" /> 
Figure 5.&#160;Properties of electrospun PET scaffolds. (A) Distribution curves showing fiber diameter distribution from the reticular basement membrane extracellular matrix (RBM ECM), randomly aligned nano- and micro-fiber scaffolds, and the aligned nanofiber scaffold. (B) Histogram showing the relative pore size distribution in nano- and micro-fiber scaffolds. (C) Average thickness of the nanofiber, microfiber, biphasic and aligned scaffolds (mean&#177;standard deviation, n=6). (D) Histogram plot showing the deviation from mean fiber orientation in random or aligned nanofiber scaffolds Scaffold fiber analysis was carried out using ImageJ software. <a href="https://www.jove.com/files/ftp_upload/52986/52986fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52986/52986fig6.jpg" /> 
Figure 6.&#160;The culture of the individual cell types on the individual scaffolds. (A) alamarBlue cell viability assay results for ASM cells on aligned scaffolds, CALU3 cells on nanofiber scaffolds and MRC5 cells on microfiber scaffolds (mean&#177;SEM, n=3-20). Scanning electron microscope images of the nanofiber, microfiber and aligned fiber scaffolds (B,C and D respectively) and immunofluorescent images of CALU3 cells stained for E-cadherin (red), MRC5 cells stained for vinculin (red) and ASM cells stained for SM22&#945; (red) all on their specialised scaffold (E, F and G respectively). Hoechst was used to stain nuclei (blue), scale bar indicates 40 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52986/52986fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52986/52986fig7.jpg" /> 
Figure 7.&#160;Perfusion system for tri-layer airway wall model. (A) Photograph of bioreactor vessel connected to peristaltic pump and 2x media reservoirs. (B) Schematic of the tri-layered scaffolds. (C) Schematic of the two bioreactor circuits when the airway model is held at the air-liquid interface. <a href="https://www.jove.com/files/ftp_upload/52986/52986fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/52986/52986fig8.jpg" /> 
Figure 8.&#160;Complete&#160;3D airway wall model. (A) Bronchial biopsy of airway wall with the mucosal layer highlighted in red, the submucosal region highlighted in green, and the smooth muscle bundle highlighted in gold. (B) Section through tri-layered airway wall consisting of biphasic and aligned scaffolds populated with epithelial, fibroblast and smooth muscle cells fixed after 2 weeks coculture. Cell nuclei were stained with DAPI (blue) with scaffold reflectance (grey) concurrently imaged. Scaffolds were also fixed and immunostained for cell specific markers after 2 weeks, with CALU3 cells stained for cytokeratin (green) (C), MRC5 cells stained for S100A4 (green) (D), and smooth muscle cells stained for SM22&#945; (red) (E). Scale bar indicates 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52986/52986fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/52986/52986table1.jpg" /> 
Table 1.

Watch this Scientific Journal Video about Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall at JoVE.com

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

adapting-electrospinning-process-to-provide-three-unique-environments

Research

JoVE Journal

Bioengineering

12.4K Aufrufe.  University of Nottingham. Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall. 

Serie: Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.

The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.