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.

从非哮喘个体初级ASM细胞分离自支气管活检在格林菲尔德医院（莱斯特，英国），如前所述。这项研究是经莱斯特伦理委员会和患者给他们的书面知情同意书。 1.静电PET支架（图2） <ol><li>制备的饮料瓶级PET在1中的溶液（10毫升）:1二氯甲烷（DCMro） - 三氟乙酸（TFA）溶液。所需的8％，30％和10％重量/体积（重量/体积）的PET浓度为纳米纤维，微纤维及分别对准支架，然后搅拌该溶液O / N在RT将PET溶解。 </li><li>转移的PET溶液放入注射器并附加一个23-G（对于纳米纤维）或18-G（对于极细纤维和对齐）针注射器和将注射器插入电动注射器泵。确保针头的点旋转到下方。 </li><li>放置马ndrel15厘米远离针的尖端，以确保针指向该鼓的中心。 </li><li>附加的电力供应，针尖与鳄鱼夹和接地芯棒（ 通过插座香蕉）地球。上的电源向芯轴和设定速度以每分钟60转开关（相当于约13.2米·分钟-1），纳米纤维和微纤维支架。设定速度为2000转（相当于约440米·分钟-1）对齐支架。 </li><li>注射泵设定为0.5毫升的流速·小时-1为纳米纤维无规和对准支架或2.0毫升·小时-1为超细纤维支架。允许泵运行，直到溶液从针尖挤出以除去任何空气中的针。然后停泵。 </li><li>在注射器泵中，设置的溶液的总体积被驱逐至2ml，并启动泵。 </li><li>设置电源电压为14千伏，并接通电源联党LY。 </li><li>静电纺丝直到凌晨2毫升溶液已被静电纺丝（4小时的纳米纤维和对准支架，1小时为超细纤维支架）。 </li><li>切断该支架具有沿着所述心轴的宽度的叶片。这产生脚手架的2D片芯棒表面区域的大小（在此情况下，矩形片22厘米×11厘米），它可以仔细剥离心轴）。存储在铝箔的支架，以减少静电荷）。 注:双相支架是由连续直接静电纳米纤维支架上超细纤维支架生产。 </li></ol>支架的使用在细胞培养前2灭菌<ol><li>从脚手架板，切出双相或对准支架盘使用直径0.8mm活检笔，并坚持使用无毒胶水水族馆向垫片。 </li><li>浸泡在20消毒前用紫外线照射对支架的每侧30分钟支架％ 体积/体积的抗生素/抗 ​​真菌溶液（2×10 5单位毫升-1青霉素G，2000毫克毫升-1链霉素硫酸盐和500微克毫升-1两性霉素B）O / N在4℃。 </li><li>用PBS洗支架3次，然后存储支架TCP中孔板的PBS在4℃下直到使用。 </li></ol> 3.细胞培养基成分<ol><li>培养CALU3上皮细胞在DMEM-F12培养基补充有10％ 体积/体积的胎牛血清（FCS），为2nM L-谷氨酰胺溶液，1％ 体积/体积的抗生素/抗 ​​真菌溶液（10,000单位毫升-1青霉素G，100毫克毫升-1链霉素硫酸盐和25微克毫升-1两性霉素B）。 </li><li>培养MRC5成纤维细胞和ASM细胞在DMEM培养基补充有10％ 体积/体积的胎牛血清（FCS），为2nM L-谷氨酰胺溶液，1％ 体积/体积的抗生素/抗 ​​真菌溶液（10,000单位毫升-1青霉素G，100毫克毫升-1链霉素硫酸盐和25微克毫升-1两性霉素B）。 </li><li>培养上皮的成纤维细胞的ASM细胞共培养物在70:30的DMEM-F12:DMEM培养基混合物。 </li></ol> 4.播种成纤维细胞和上皮细胞的双相上脚手架<ol><li>在组织培养板，浸泡在DMEM补充的介质上的支架和孵育在37℃进行细胞接种前1小时。 </li><li>除去DMEM为辅媒体，放置支架的超细纤维相顶部和种子1.5×10 4 MRC5成纤维细胞的30微升DMEM补充的媒体。搅动板在定轨振荡器2小时，之前，在37℃，5％CO 2在培养箱中留在空气中O / N。 </li><li>转动支架上以便支架的纳米纤维相面对顶部，种子3.0×10 4 CALU3上皮细胞中加入30μl的DMEM-F12补充的介质上支架的纳米纤维相和孵化FOR 2小时，在37℃，5％CO 2的空气中浸没该支架在70:30的DMEM-F12之前:转移至生物反应器的DMEM-补充培养基O / N之前。 </li></ol> 5.播种ASM细胞到不结盟脚手架<ol><li>在组织培养板，浸泡在DMEM补充的介质上的支架和幼苗2.5×10 4个细胞的30μlDMEM-补充培养基和温育2小时（37℃，5％的CO之前孵育1小时，在37℃下2在空气中）。淹没在脚手架DMEM-补充媒体回归到孵化器，并留下O / N设立三培养在生物反应器前。 </li></ol> 6.设置，使用起来的生物反应器系统的三文化（图7） <ol><li>将双相支架垫圈进凹槽一个室内与上皮相朝上进入腔室。 </li><li>将双相支架下方对准的支架（因此它是相邻的英里双相支架crofiber相）和锁定生物反应器的两个腔室一起。 </li><li>装配连接到两个介质水库2灌注流动回路（DMEM-F12-补充培养基在心尖水库，DMEM-F12 /基底储层的DMEM补充的培养基）在大约为0.1毫升/分钟使用蠕动泵周围的两个电路和泵媒体（40毫升介质通过每个回路再循环）。房子的培养箱内的系统在37℃，5％CO 2的空气中的所有部件。 </li><li>一周后，从心尖室分析前移除媒体和文化的上皮细胞在ALI的进一步星期。 </li></ol>

<ol><li>Macchiarini, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+transplantation+of+a+tissue-engineered+airway%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">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/pubmed?term=((Regeneration+and+experimental+orthotopic+transplantation+of+a+bioengineered+kidney%5BTitle%5D)+AND+%22Nature+medicine%22%5BJournal%5D)">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/pubmed?term=((The+mouse+trap%3A+it+still+yields+few+answers+in+asthma%5BTitle%5D)+AND+%22American+journal+of+respiratory+and+critical%22%5BJournal%5D)">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/pubmed?term=((Animal+models+of+asthma%5BTitle%5D)+AND+%22Clinical+and+experimental+allergy+journal+of+the+British+Society+for+Allergy+and+Clinical+Immunology%22%5BJournal%5D)">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/pubmed?term=((Animal+models+of+asthma.+American+journal+of+physiology%5BTitle%5D)+AND+%22Lung+cellular+and+molecular+physiology%22%5BJournal%5D)">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/pubmed?term=((Axonal+outgrowth+on+nano-imprinted+patterns%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Rapid+micropatterning+of+cell+lines+and+human+pluripotent+stem+cells+on+elastomeric+membranes%5BTitle%5D)+AND+%22Biotechnology+and+bioengineering%22%5BJournal%5D)">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/pubmed?term=((Aligned+Cell+Sheets+Grown+on+Thermo-Responsive+Substrates+with+Microcontact+Printed+Protein+Patterns%5BTitle%5D)+AND+%22Advanced+Materials%22%5BJournal%5D)">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/pubmed?term=((A+synthetic+nanofibrillar+matrix+promotes+in+vivo-like+organization+and+morphogenesis+for+cells+in+culture%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Living+in+Three+Dimensions%5BTitle%5D)+AND+%22Cell+Biochemistry+and+Biophysics%22%5BJournal%5D)">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/pubmed?term=((Morphology%2C+cytoskeletal+organization%2C+and+myosin+dynamics+of+mouse+embryonic+fibroblasts+cultured+on+nanofibrillar+surfaces%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Effects+of+synthetic+micro-and+nano-structured+surfaces+on+cell+behavior%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Development+of+a+3D+Cell+Culture+System+for+Investigating+Cell+Interactions+With+Electrospun+Fibers%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">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/pubmed?term=((Matrix+nanotopography+as+a+regulator+of+cell+function%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">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/pubmed?term=((Remodeling+in+Asthma+and+Chronic+Obstructive+Lung+Disease%5BTitle%5D)+AND+%22American+Journal+of+Respiratory+and+Critical+Care+Medicine%22%5BJournal%5D)">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/pubmed?term=((Tracheobronchial+transplantation+with+a+stem-cell-seeded+bioartificial+nanocomposite%3A+a+proof-of-concept+study%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">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/pubmed?term=((Developing+a+tissue-engineered+model+of+the+human+bronchiole%5BTitle%5D)+AND+%22Journal+of+Tissue+Engineering+and+Regenerative%22%5BJournal%5D)">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/pubmed?term=((Development+and+characterization+of+a+3D+multicell+microtissue+culture+model+of+airway+smooth+muscle.+American+journal+of+physiology%5BTitle%5D)+AND+%22Lung+cellular+and+molecular+physiology%22%5BJournal%5D)">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/pubmed?term=((Human+airway+musculature+on+a+chip%3A+an+in+vitro+model+of+allergic+asthmatic+bronchoconstriction+and+bronchodilation%5BTitle%5D)+AND+%22Lab+on+a+chip%22%5BJournal%5D)">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/pubmed?term=((Nano%2Fmicro+electro-spun+polyethylene+terephthalate+fibrous+mat+preparation+and+characterization%5BTitle%5D)+AND+%22Journal+of+the+mechanical+behavior+of+biomedical+materials%22%5BJournal%5D)">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/pubmed?term=((Human+airway+smooth+muscle+maintain+in+situ+cell+orientation+and+phenotype+when+cultured+on+aligned+electrospun+scaffolds%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">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/pubmed?term=((A+novel+electrospun+biphasic+scaffold+provides+optimal+three-dimensional+topography+for+in+vitro+co-culture+of+airway+epithelial+and+fibroblast+cells%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">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/pubmed?term=((The+Use+of+Natural+Polymers+in+Tissue+Engineering%3A+A+Focus+on+Electrospun+Extracellular+Matrix+Analogues%5BTitle%5D)+AND+%22Polymers%22%5BJournal%5D)">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/pubmed?term=((Electrospinning+of+nanofibers%5BTitle%5D)+AND+%22Journal+of+Applied+Polymer+Science%22%5BJournal%5D)">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/pubmed?term=((Electrospinning+and+additive+manufacturing%3A+converging+technologies%5BTitle%5D)+AND+%22Biomaterials+Science%22%5BJournal%5D)">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/pubmed?term=((Tailoring+tissue+engineering+scaffolds+using+electrostatic+processing+techniques%3A+A+study+of+poly%28glycolic+acid%29+electrospinning%5BTitle%5D)+AND+%22Journal+of+Macromolecular+Science-Pure+and+Applied+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Electrospraying+and+electrospinning+of+polymers+for+biomedical+applications.+Poly%28lactic-co-glycolic+acid%29+and+poly%28ethylene-co-vinylacetate%29%5BTitle%5D)+AND+%22Revolutionary+Materials%3A+Technology+And+Economics%22%5BJournal%5D)">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/pubmed?term=((Electrospun+nanofibers+for+regenerative+medicine%5BTitle%5D)+AND+%22Advanced+healthcare+materials%22%5BJournal%5D)">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/pubmed?term=((Solvent+and+concentration+effects+on+the+properties+of+electrospun+poly+%28ethylene+terephthalate%29+nanofiber+mats%5BTitle%5D)+AND+%22Journal+of+Polymer%22%5BJournal%5D)">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/pubmed?term=((Tailoring+Fiber+Diameter+in+Electrospun+Poly%28epsilon-Caprolactone%29+Scaffolds+for+Optimal+Cellular+Infiltration+in+Cardiovascular+Tissue+Engineering%5BTitle%5D)+AND+%22Tissue+Engineering+Part+A%22%5BJournal%5D)">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/pubmed?term=((Fabrication+of+three-dimensional+nano%2C+micro+and+micro%2Fnano+scaffolds+of+porous+poly%28lactic+acid%29+by+electrospinning+and+comparison+of+cell+infiltration+by+Z-stacking%2Fthree-dimensional+projection+technique%5BTitle%5D)+AND+%22IET+nanobiotechnology+%2F+IET%22%5BJournal%5D)">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/pubmed?term=((Method+to+Analyze+Three-Dimensional+Cell+Distribution+and+Infiltration+in+Degradable+Scaffolds%5BTitle%5D)+AND+%22Tissue+Engineering+Part+C-Methods%22%5BJournal%5D)">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/pubmed?term=((Gelatin-Based+Materials+in+Ocular+Tissue+Engineering%5BTitle%5D)+AND+%22Materials%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+Endothelial+Basement+Membrane+Nanotopography+in+Rhesus+Macaque+as+a+Guide+for+Vessel+Tissue+Engineering%5BTitle%5D)+AND+%22Tissue+Engineering+Part+A%22%5BJournal%5D)">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/pubmed?term=((Ultrastructural+basement+membrane+topography+of+the+bladder+epithelium%5BTitle%5D)+AND+%22Urological+research%22%5BJournal%5D)">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/pubmed?term=((Electron+Microscopy+of+the+Canine+Corneal+Basement+Membranes%5BTitle%5D)+AND+%22Cells+Tissues+Organs%22%5BJournal%5D)">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/pubmed?term=((Human+corneal+equivalent+as+cell+culture+model+for+in+vitro+drug+permeation+studies%5BTitle%5D)+AND+%22British+Journal+of+Ophthalmology%22%5BJournal%5D)">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.

到静电纺丝聚合物纤维相媲美的天然细胞外基质的结构特性的能力已经导致了许多天然或合成的聚合物，或聚合物共混物是静电重建这些环境中，。工艺参数（包括聚合物的选择，聚合物浓度，溶剂，针尖的距离和温度）都可以影响脚手架特性的操纵;然而，一些参数可以有比其他25载体性能影响更大。当改性的PET纤维的直径，我们发现的关键参数是聚合物的所用浓度，的速率将聚合物溶液进行电纺丝，并且针的直径。当静电排列的纤维的关键参数是用于（一个转动心轴）的收集方法，其速度在该心轴旋转。通过降低心轴转速每分钟60转，我们发现所产生的随机排列支架表现出更大的脚手架单formity相比静电到平坦集电板。 脱细胞的气道组织的特性进行了分析，为每个气道细胞类型的培养发展了各种支架形貌提供指导。纳米纤维是一个有吸引力的脚手架复制由于小孔径但高的总孔隙率，其允许增加代谢物扩散，同时限制蜂窝移动基底膜结构。9虽然优化静电参数，一个范围的PET浓度和流速进行了测试。最薄纤维使用6％ 重量/体积的PET溶液，以前使用的其它基团来实现。然而，高含量的成珠，以及缺乏均匀性是恒定的问题。最初试图消除卷边收录加入阳离子表面活性剂，十六烷基三甲基溴化铵（CTAB），向溶液中以降低表面张力，和测试各种来源宠物。在加入表面活性剂的减少成珠的量，但不完全。在尝试了许多PET颗粒的商业来源，食品级饮料瓶使用PET，通过切割饮料瓶，并在DCM中溶解这些:TFA溶剂溶液。以此作为在PET源导致增加纤维的均匀性和在纤维成珠的降低。通过稍微增加溶液的浓度至8％ 重量/体积 ，降低了针直径（18G至23G），我们一致地生产无缺陷的纳米纤维具有约250纳米的纤维直径。静电纳米纤维支架可以在领取高度静电作出支架困难的手工处理的芯棒。这是通过存储在铝箔支架纺纱机和在70％的IMS浸泡在使用前后好转。这显然​​有助于消散留下的静电过程中支架的残留静电荷。 提供顶级ographies类似于由气道壁内的三个主要的气道细胞类型所遇到的个体的微环境，碱性电纺丝协议是适于产生用于这些细胞类型三个独特的支架:通过静电缓慢，产生随机排列的纳米纤维浓度低的PET溶液，其上的上皮细胞接种（模仿RBM）。通过电纺丝以更快的速度较高浓度的PET溶液，产生随机排列微纤维（产生更多孔支架）， 成纤维细胞接种（立即RBM的下方模仿子粘膜区域）。通过电纺丝的低浓度的PET溶液到一个高速旋转的心轴对准纳米纤维生产的，在其上取向的纤维方向，平滑肌细胞产生对准细胞片层。 增加纤维直径导致增加的孔径，从而允许更大的细胞penetratioN转换支架，从而帮助创造一个真实的3D环境。超细纤维的支架被用于成纤维细胞的培养在研究中，以确保细胞驻留在该支架内的多个平面。通过增加的PET浓度为30％ 重量/体积 ，PET纤维具有2.5微米的平均直径被生产。更大直径的纤维（≈4微米）用35％ 重量/体积的PET溶液生产，但支架厚度均匀性丧失，和单个纤维之间较高的变异是明显的。在超细纤维支架毛孔均较纳米纤维支架测量（10.45微米VS分别为1.43微米），更大的7倍，但电池还是静态播种提供了有限的细胞渗透。这是通过使用轨道混合器，显示先前是有效的方法，动态地接种细胞提高。 高度对准的纳米纤维支架被通过电10％创建重量/体积的PET溶液到芯轴转速为2000转（≈440米·分钟-1）。在PET浓度从8％增至以防止纤维显示出在较低浓度不规则的波浪状的形态。尽管提高浓度，对准纤维减少的平均纤维直径（216nm处相对于255纳米，对准与随机的）。心轴将纤维拉伸出来他们已经干燥前的高速是容易造成这种效果。的纤维的取向的影响的ASM细胞的对准，并且还具有关于在ASM细胞已在别处21特征在于其他生理效应。 这个协议的主要限制是不能图象活细胞上/中制备的初始细胞附着或分化在延长的时间期间不可能确定在不牺牲样品的支架。这意味着文化时期后最优化的发生; 即，修复荷兰国际集团的样本，然后测量细胞培养是否成功后没有细胞培养过程中 。观察支架在培养一个的AlamarBlue颜色变化（蓝色为粉红色）表示细胞存在和可行的，但效果不理想。静电更透明的聚合物如明胶可能有助于对支架可视化细胞。我们模型中的纳米纤维支架的电纺丝允许的气道RBM的复制，同样由致密纳米纤维上的上皮细胞存在的。先前已表明，结构的细胞可以通过纳米纤维支架22不迁移，虽免疫细胞能够（数据未显示）。虽然有利的特定细胞类型上的三维表面的培养（如上皮细胞和内皮细胞），这种预防结构性细胞迁移通过纳米纤维可以是有害的其它细胞类型的培养。由于平滑肌是无法通过迁移第Ë对齐纳米纤维支架我们组装的三层共培养的平滑肌和成纤维细胞层彼此面对（相对的对准脚手架分隔两个细胞类型）。虽然这可以使细胞在接近并置，目前的研究不能断定一种细胞类型（无论是成纤维细胞或平滑肌）是否会超过整层过更长时间的培养期。尽管有这些限制，已经开发了气管壁的一个可行的三 - 层模型，它提供了一个替代的平台来调查这些多种细胞类型之间的相互作用。类似的三层研究最近已经公布了，其中成纤维细胞内嵌入圆筒状胶原I水凝胶与平滑肌接种于腔表面17内的外表面和上皮细胞。这里开发将允许类似管状的静电支架的机械稳定性构造来形成，并且仍然是一个电流resea我们集团内RCH目标。虽然研究已集中在气道，内体份额几种器官这个基本的粘膜结构单元，并且通过修改所述居民在这项研究中强调的，类似的平台可以为含有基底膜部组织，包括血管，膀胱和开发角膜，其中，胶原为基础的多层次的模型已经使用。

再生医学和组织工程领域正在快速推进，在气管和肾脏再生两个显着的成就近期突破。在组织工程开发的生物材料变得更容易，有机会这样的协议转让给少专业实验室。一个字段底漆通过增加使用生物材料的受益是体外诊断。 体外研究是调查范围内的单细胞类型的细胞内信号通路的重要平台，并帮助划定潜在疾病的众多机制病理生理学。这些研究通常依靠单一细胞类型是作为组织培养塑料（TCP）的单层中培养;二维（2D）的刚性表面少得多的弹性和多孔比三维（3D）环境的细胞的组织或器官内暴露。动物模型历来Ëmployed确认体外发现也转化为整个组织的效果，而且还可以用来作为临床前平台来调查人类疾病。然而，物种间的差异破坏这种依赖动物模型在我们人类疾病的认识- 。例如，肺部疾病哮喘太多了解基于尽管人的条件和该模型之间的内在差异，包括几乎没有证据的气道平滑肌束的肥大细胞浸润，或用于动物内自发疾病发展的能力的小鼠模型模型3,4。也有关于使用动物模型被鼓励在英国和其他国家的伦理方面的考虑，与"更换，细化和减少"在动物实验的"3R原则"的标准。 一个有吸引力的替代办法是人体组织体外创作结构再演s至调查在单个单元成人细胞类型之间的合作性质。在3D多细胞结构存在于组织细胞中，每个内独特的微环境。细胞在TCP培养只允许细胞单层，无法复制这样的环境，或提供用于多细胞培养的能力的培养。生物材料发展提供了发展的机会，为细胞培养天然和合成的3D平台。脱细胞的ECM可用于三维细胞培养物时与其它细胞类型，包括干细胞1 recellularized，但是这样的协议可以是复杂的和耗时的，与组织有限很大程度上非人类来源和的可用性。其他协议允许更大的控制权产生如纳米压印，ECM沉积，细胞片技术，静电或细胞环境。创建静电纤维的直径范围从纳米到微米，R无纺布3D多孔垫eplicating天然细胞外基质的尺寸。静电支架越来越多地被用作3D细胞培养平台- 。静电纺丝参数的操纵允许复杂的控制脚手架特性，例如孔径大小，纤维直径，地形和对准，以及表面化学。这种参数的变化已经显示出直接影响细胞的粘附和生长，当细胞培养在隔离。 这些优点已被利用在本研究中，以允许多种细胞类型的培养作为单个三维组织构建物，使用气道支气管作为模型的三维组织结构。细支气管壁由三个主要区域（ 图1）。粘膜是哪里呼吸道上皮细胞坐在空气 - 液体界面（ALI），提供了一个重要的屏障到外部环境。它们位于网状基底膜（RBM）的，紧密紧凑的ECM主要由collag恩四，perlecans和层粘连蛋白。子粘膜层被发现正下方的黏膜，由多种细胞类型，包括成纤维细胞，肌成纤维细胞和疾病状况下白细胞浸润的更多孔的区域。最后平滑肌束周围气道包裹以螺旋方式，并且由气道平滑肌（ASM）的对齐纸张的。这是平滑肌的控制气道张力相对收缩的状态。静电支架的肺系统内的就业人数，直到最近被限制在再生的目的;与静电气管更换被成功移植。虽然成功，这种治疗方法是有限的标号，其集中在气管由于组织的功能的相对简单的性质。用于体外诊断的气道模型的有限实施例的存在，并且主要集中在平滑肌收缩测量 。无毒和无正降解聚合物聚对苯二甲酸乙酯（PET）的先前已静电纺丝，并被用于本研究，以确保产生的可长期保存的支架，而不会降低（允许它们被使用的"现成的"），并且还适用超过更长的时间内稳定的细胞培养。我们小组先前的研究已经表明，采用基本静电协议三种变型中，三个单独的PET静电支架可制造以提供最佳的形貌用于培养呼吸道上皮，成纤维细胞和平滑肌细胞中隔离21,22及气道的共存上皮细胞和成纤维细胞22。纤维直径被认为大大地影响上皮功能22，以及纤维取向允许平滑肌21对准片材的产生。这些研究是在静态条件下彼此独立地执行。在本研究中，这些各色nt的支架，含完全分化成人细胞已被共培养一周在ALI在市售的生物反应器作为气道壁的三维部分，提供了生理学相关的体外模型来研究的气道细胞间的反应。虽然该协议是使用气道支气管作为模型系统，它可适于作为用于从其它器官的粘膜单元的三维共培养的平台。

<table><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&amp;nbsp;</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.

脱细胞气道组织或免疫组化图像识别距离我们希望引入静电支架地形呼吸道ECM的理想特性气道活检的扫描电子显微镜图像:母语RBM矩阵包括纤维约153纳米±30.6纳米（平均值±SD N = 50测量）在直径（ 图3A和5A），而RBM的周围的基质是在自然界中（ 图3C）更多孔。通过平滑肌束免疫组织学切片显示ASM表现为细胞21（ 图3E）的对齐纸张。这个信息被用来引导引入静电支架的特性。一种旋转心轴是能够稳定地以高速旋转（&gt; 2000转/440米·分钟-1），以产生支架具有对准的纤维。慢心轴旋转（60rpm下13.2米·分钟-1）不影响纤维取向，但生产与支架厚度更加均匀，当静电到静止板比。通过改变静电参数（PET浓度和溶液流速），有可能创建支架的纤维的直径为几微米或几百纳米（静电参数在表1中表示）。 电纺纤维具有等效纤维直径到天然成果管理纤维（150纳米），使用6％的PET溶液被生产，但是在这样低的PET浓度，纤维的成珠出现（ 图4A）。这是通过增加的PET溶液至8％，这产生均一的纳米纤维具有255的平均直径纳米±2.4纳米的浓度消除（平均值±SEM）和1.43微米±0.02微米（ 图4B，图5A及B）的平均孔尺寸。到静电纺丝纳米纤维，有必要减少从18G的针头大小至23 G，允许较慢的流速whils牛逼保持较高流速，减少堵塞针头，一个经常发生的问题与较粗的针。当静电微纤维，增加的PET溶液至&gt; 30％的PET导致缺乏统一性纤维直径（ 图4F）除了多个堵塞在针尖由于高溶液粘度。均匀的超细纤维垫具有2.50微米±0.02微米的平均纤维直径（平均值±SEM）和10.45微米±0.13微米的平均孔径时静电以2mL / h的流速在30％ 重量/体积的PET溶液被生产-1（ 图4F，5A＆B）。直接纳米纤维支架上超细纤维支架（仍附着于心轴）的顺序静电产生的双相骨架（ 图3D）。的平均纤维直径为280纳米分别±20纳米和2.30微米±0.06微米的纳米纤维和微纤维的阶段，媲美个别纳米纤维和超细纤维支架的尺寸。此外，双相支架的厚度为类似于个人纳米纤维和微纤维的支架（ 图5C）的总和。通过增加芯棒速度（2000转/ 440·M·分-1），高度一致纳米超细纤维或支架制作。的8％ 重量/体积的PET溶液不是最佳的制造对齐的纳米纤维和制备纤维具有波浪状的形态（ 图3F）。增加至10％的PET废止这种效果，但仍产生了约化纤维直径（216纳米±2.2纳米（平均值±SEM））相比，随机排列的纳米纤维支架。纤维取向进行了计算确定每个纤维的角度的偏差从平均纤维角度。在排列的纳米纤维支架的纤维79％被对齐（±10°的平均纤维角度）相比，在随机排列的纳米纤维斯卡夫纤维10.2％年轻人（ 图5D）。 的8％的纳米纤维支架被用来概括在其上的上皮细胞所在的成果管理，并被用来支持CALU3细胞的培养（呼吸道上皮细胞系）。的30％ 重量/体积的超细纤维的支架，在自然状态下更多的多孔被用来模仿子粘膜区域，并且是与MRC5细胞（气道成纤维细胞系）中培养。提供拓扑引用10％ 重量/体积排列纳米纤维支架，以确保ASM单元格对齐方式时，在支架上培养。所有三种细胞类型显示出增加的存活力，当他们在2周的时间内（ 图6A）个别支架中培养，并表示2周培养期间（ 图6B，6C，和6D）后细胞类型特异性的蛋白质。单个细胞支架相互作用的进一步鉴定已在别处21日报道，。纳米纤维支架的连续静电到微纤维支架产生的双相骨架为CALU3上皮细胞和MRC5成纤维细胞的共培养到纳米纤维和微纤维的阶段分别。两个小区的共同静态条件下培养已在别处22日报道。进一步的工作试图在静态条件下添加其他细胞层或延伸双相培养时间超出2周证明是不成功的（数据未显示）。培养在较长时间内的三分层模型，我们使用了灌注流的生物反应器。的CALU3和MRC5细胞接种到双相支架和ASM细胞接种到一个对准的支架，和两个分别培养2天。两个支架然后购买在一起以形成生物反应器内的气道壁的三-层模型（ 图7）。两个腔室灌注经媒体7天前心尖上皮室有其媒体除去，将上皮细胞培养在阿里进一步的7天。支架固定并任切片，染色，或整个支架进行免疫染色对细胞特异性标记。通过三层培养切片显示通过共培养（ 图8B）的所有三层分布细胞核。当进行免疫染色的细胞为细胞特异性标记，上皮细胞填充心尖纳米纤维相作为会合的细胞层和染色阳性细胞角蛋白（ 图8C），在超细纤维相，成纤维细胞染色呈阳性，S100A4（ 图8D），并在染色呈阳性SM22α（ 图8E）的对准的10％的支架的ASM细胞，指示每个细胞类型的气道壁的三维模型中的良好的生存。 <img alt="图1" src="/files/ftp_upload/52986/52986fig1.jpg" /> 图1.气管支气管，从严重的支气管活检哮喘气道表现出对网状基底膜（RBM）的上皮细胞，以填充细胞间质底层粘膜下区，并填充染色平滑肌的α-肌动蛋白（棕色）平滑肌细胞周围的平滑肌束。比例尺为200微米。 <a href="https://www.jove.com/files/ftp_upload/52986/52986fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52986/52986fig2.jpg" /> 图2.电纺丝设备的示意图。将含有聚合物/溶剂溶液（用针连接的）注射器置于注射器泵对着心轴。将电势针和心轴使纤维从针尖​​喷出并附着在旋转的芯轴之间建立。作为纤维穿过大气层，溶剂挥发造成的芯棒聚合物纤维的沉积。 <a href="https://www.jove.com/files/ftp_upload/52986/52986fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52986/52986fig3.jpg" /> 静电支架的图3比较，以天然气道的ECM。的脱细胞的基底膜（A）中 ，脱细胞的气道细支气管截面（C）和一个气道平滑肌束的组织切片的扫描电子显微镜图像（用苏木精和曙红，规模巴40微米）（E）与纳米纤维（B相比）双相（D）和对齐（F）PET支架。 <a href="https://www.jove.com/files/ftp_upload/52986/52986fig3large.jpg" target="_blank">请点击此处查看</a>一个更大的版本这个数字。 用于电纺丝的极细纤维，纳米纤维表1参数和单独地和用于双相支架对准支架。 <img alt="图4" src="/files/ftp_upload/52986/52986fig4.jpg" /> 图4.维修在PET浓度会影响光纤特性。从6％，8％，10％（AC），25％，30％和35％（EG）（ 重量/体积 ）的PET溶液纺丝静电支架的扫描电子显微镜图像。也显示对准支架从8％（D）和30％的生产（H）， 重量/体积 PET解决方案。AD在成像放大5000倍，EH在成像放大1000倍。 <a href="https://www.jove.com/files/ftp_upload/52986/52986fig4large.jpg" target="_blank">请点击此处查看大图</a>版本这个数字。 <img alt="图5" src="/files/ftp_upload/52986/52986fig5.jpg" /> 的静电的PET支架图5.性质。（A）的分布曲线示出了从网状基底膜细胞外基质（RBM，ECM），随机排列纳米和微纤维的支架，并对准的纳米纤维支架的纤维直径分布。 （B）的直方图表示在纳米和微纤维支架的相对孔径分布。 （C）的纳米纤维，微纤维，双相和对准支架的平均厚度（平均值±标准偏差，N = 6）。 （D）中的柱状图示出了从平均纤维取向无规或对准的纳米纤维支架脚手架纤维进行分析使用ImageJ软件的偏差。 <a href="https://www.jove.com/files/ftp_upload/52986/52986fig5large.jpg" target="_blank">P</a>租赁点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/52986/52986fig6.jpg" /> 图6.对个人支架的单个细胞类型的文化。（A）的AlamarBlue细胞活力检测结果ASM细胞上的对齐支架，在支架的纳米纤维细胞CALU3和MRC5细胞的支架材料超细纤维（平均值±SEM，N = 3- 20）。纳米纤维，微纤维的扫描电子显微镜图像和对齐纤维支架（B，C和 D分别）和染色E-钙粘蛋白（红色）CALU3细胞，MRC5细胞染色纽蛋白（红色）和ASM细胞染色SM22α的免疫荧光图像（红色）都在其专业支架（E，F和G分别）。 Hoechst的被用于染色细胞核（蓝色），比例尺为40＃181;米<a href="https://www.jove.com/files/ftp_upload/52986/52986fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/52986/52986fig7.jpg" /> 图7.灌注系统，三层气道壁模型。连接到蠕动泵和2倍的媒体水库生物反应器容器（A）的照片。 （B）示意图三层状支架。当气道模型保持在气-液界面两种生物反应器电路（C）示意图。 <a href="https://www.jove.com/files/ftp_upload/52986/52986fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/52986/52986fig8.jpg" /> 图8.完成3ð气道壁模型。（A）的气道壁以红色突出显示粘膜层支气管活检，黏膜下区域突出显示为绿色，并且在金平滑肌束突出。 （B）中 ，通过三分层气道壁部分包括填充了上皮，成纤维细胞和平滑肌细胞固定2周后共存双相和对准支架。细胞核用DAPI染色（蓝色）与支架反射率（灰色）同时成像。支架也被固定和2周后免疫染色细胞特异性标记物，用染色角蛋白（绿色）CALU3细胞（C），MRC5细胞染色S100A4（绿色）（D）和染色SM22α（红色）平滑肌细胞（ E）。比例尺为200微米。 <a href="https://www.jove.com/files/ftp_upload/52986/52986fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 表1中。

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.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

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

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

12.4K 次观看.  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. 

视频: 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.

三层次的静电，以模仿母语动脉结构采用聚己内酯，弹性和胶原的初步研究

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

科研

生物工程

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

将二维电纺纳米纤维垫扩展到三维支架中

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

遗传学

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

静电生长因子释放到微纤维支架

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

静电纤维聚合物支架用于组织工程和细胞培养

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

生物学

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

评估流和拉伸下生物材料的炎症和再生能力的多奎生物反应器

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

工程学

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Controlling Electrospun Polymer Morphology for Tissue Engineering Demonstrated Using hepG2 Cell Line

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of electrospun polymers is regarded as a structural imitation of the extracellular matrix (ECM). Hence, electrospun fibers fabricated from biocompatible polymers have been widely investigated by tissue engineering researchers for their potential role as an artificial ECM for guiding tissue growth both in vitro and in vivo. All cells are acutely sensitive to their mechanical environment. This has been demonstrated by the discovery of multiple mechanotransduction pathways intrinsically linked to the cytoskeletal actin filaments. The cytoskeleton acts as a mechanical sensor that can direct the functionality and differentiation of the host cell depending on the stiffness and morphology of its substrate. Electrospun fibers can be tuned both in terms of fiber size and morphology to easily modulate the mechanical environment within a fibrous polymer scaffold. Here, methods for electrospinning polycaprolactone (PCL) for three distinct morphologies at two different fiber diameters are described. The morphological fiber categories consist of randomly oriented fibers, aligned fibers, and porous cryogenically spun fibers, with 1 &#181;m and 5 &#181;m diameters. The methods detailed within this study are proposed as a platform for investigating the effect of electrospun fiber architecture on tissue generation. Understanding these effects will allow researchers to optimize the mechanical properties of electrospun fibers and demonstrate the potential of this technology more thoroughly.

<meta charset="utf8"></head><body>使用 hepG2 细胞系演示控制组织工程的静电纺丝聚合物形态

This method provides the means to test different polycaprolactone fiber morphologies and topographies for the purpose of tissue engineering. Small and large fibers are fabricated with random orientations, aligned orientations, and also porous cryogenically electrospun structures and used as platforms for cell culture.

使用 hepG2 细胞系演示控制组织工程的静电纺丝聚合物形态

Electrospinning affords researchers the opportunity to fabricate reproducible micro to nanoscale polymer fibers. The 3D fibrous architecture of ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

成像引导的生物反应器，用于生成生物工程气道组织

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

适应静电过程中提供三个独特的环境为一个三层次

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使用 hepG2 细胞系演示控制组织工程的静电纺丝聚合物形态

成像引导的生物反应器，用于生成生物工程气道组织