我们感谢罗杰先生艾克罗伊德，黄宏黄伟文先生和克里斯·斯托达德，顾问外科医生在谢菲尔德教学医院NHS信托基金会，他们在获得食管组织样本，并支持我们的工作帮助。我们感谢Ashraful哈克对他的帮助下结合肿瘤细胞系到模型中。我们非常感谢从巴尔坦研究和教育信托基金（BRET）和约克郡癌症研究（YCR）资助这项研究的财政支持。

人食管癌细胞是从发生胃或食管手术的患者获得。获得知情同意被用于研究目的的组织，并在适当的伦理批准（SSREC 165/03，人类研究组织库牌照12179）的组织所使用匿名。 1.隔离人食管上皮细胞<ol><li>比1:工作在层流组织培养罩，并使用无菌技术，通过混合的Dulbecco氏改良的Eagle氏培养基（DMEM）和火腿的F12在3制备上皮培养基（体积:体积）。加10％（体积/体积）FCS，10纳克/毫升的表皮生长因子，0.4微克/ ml氢化可的松，1.8×10 -4 M腺嘌呤，5微克/毫升胰岛素，5微克/毫升转铁蛋白，2×10 -7 M的甲状腺素，1×10 -10 M霍乱毒素，2×10 -3 M谷氨酰胺，100IU / ml青霉素，100微克/ ml链霉素，和0.625微克/毫升两性霉素B. </li><li>使用标准无菌操作技术，把11毫升的DMEM，10％FCS，2×10 -3 M L-谷氨酰胺，100IU / ml青霉素，100微克/ ml链霉素，和0.625微克/毫升两性霉素B成T75组织培养瓶中。加1×10 6个致死剂量照射小鼠3T3成纤维细胞12在1ml相同的培养基中，在5％的CO 2，湿润气氛孵育过夜，在37℃。这将产生一个饲养层上皮细胞的后续培养。 </li><li>用手术刀和以下一个组织病理学家的建议，解剖组织的约2平方厘米的样品从食管鳞状黏膜从接受胃或食管手术的患者获得的无病背景区域。运送至实验室在50ml容器无菌运输培养基（PBS 100国际单位/毫升青霉素，100微克/毫升链霉素和0.625微克/毫升两性霉素B）在室温下。 </li><li>在这一点上，存储所述组织中的传输在4℃的培养基进行长达12小时（过夜），为方便起见，然而不使用较长的贮存期，因为它们导致降低细胞活力。 </li><li>工作在一个层流组织培养罩，并使用无菌技术，通过浸泡在70％乙醇5分钟消毒手术刀刀片。冲洗在无菌PBS中。 </li><li>放置在无菌培养皿的组织并使用手术刀切它成0.5厘米的条。加0.1％5 ml（重量/体积）胰蛋白酶的PBS至培养皿中，放置盖子上的菜，孵育1小时，在37℃。 </li><li>添加5毫升的FCS到组织中的培养皿，以抑制胰蛋白酶。保持组织用无菌镊子的带材，轻轻刮去上皮表面用手术刀刀片为每条1-2分钟以去除上皮细胞到周围介质中。卸下刮组织，待用。重复该过程为所有的组织带。 </li><li>收集含分离的上皮细胞的培养基入15ml离心管离心（5分钟，200×g离心）。倒出上清，悬浮于12ml上皮介质的粒料（在步骤1.1中所述）。 </li><li>倒出并丢弃从在步骤1.2中制备的T75烧瓶中的饲养细胞层的介质。使用无菌移液管向含有新鲜分离的上皮细胞中的细胞悬浮液添加到烧瓶中并在37℃下，在5％的CO 2，湿润气氛。 </li><li> 24小时后倒出并弃去培养基，这也将含有细胞碎片，并更换用12ml新鲜上皮介质。继续在37℃下，在5％的CO 2，湿润气氛温育。菌落将开始出现在未来24至48小时，并且可以通过将培养瓶中用标准光学显微镜下观看。倾掉废培养基并更换为新鲜培养基等体积每2至3天。 </li><li>一旦烧瓶已达到80％汇合，流路单元13。斯普利特的C以1:1的比率ELLS:5，以增加细胞数为模型中的用途。按照步骤1.2建立照射3T3细胞的饲养层在将要接收的上皮细胞24小时前传代的细胞的每个烧瓶中。 注意:使用上皮细胞在下述段落1和仅4之间的食管模型。 </li></ol> 2.隔离人食管癌成纤维细胞<ol><li>工作在层流柜使用无菌技术，以组织预留在步骤1.7。放置在无菌培养皿中，并通过用无菌解剖刀刀片切碎剁碎细。加入10毫升0.5％（重量/体积）胶原酶的溶液，然后将盖培养皿上，并在5％的CO 2，湿润气氛孵育在37℃下过夜。 </li><li>转移消化至15ml离心管中并离心（10分钟，200×g离心），小心倒出并弃去上清液和重悬沉淀在10ml成纤维细胞培养培养基（DMEM 10％FCS的，2×10 -3 M L-谷氨酰胺，100IU / ml青霉素，100微克/ ml链霉素，和0.625微克/毫升两性霉素B）。 </li><li>将悬浮液注入一个T75烧瓶中并在5％的CO 2，湿润气氛在37℃。 24小时后倒出培养基，将含有碎屑和替换用12ml新鲜的成纤维细胞培养基。更换培养基，每2至3天，并传代细胞，一旦他们达到80％汇合13，分裂细胞以1:5的比例，以增加细胞数。 注意:使用的成纤维细胞在下述通路4和仅10之间的模型。 </li></ol> 3.准备去细胞化食管脚手架<ol><li>获得完整的猪食管的新鲜屠宰长白猪的屠宰场被用于食品生产。一个单一的食道将提供足够的支架大约30个不同的结构。 注:由于大量的时间和合作nsuming需要灭菌协议，它通常是值得获得并在同一时间处理至少10食管。 </li><li>立即将新鲜切除食道到含有约150毫升10％聚维酮 - 碘溶液中5分钟，以减少任何污染性微生物负荷180毫升无菌锅。食道转移到含有含200国际单位/毫升青霉素，200微克/毫升链霉素和1.25微克/毫升两性霉素B约100毫升无菌PBS中的第二锅<ol><li>更换盖在容器上，并运送食管回实验室室温。两个或三个食管可以在每个容器进行运输，这取决于它们的大小。 </li></ol></li><li>一旦在实验室中，处理中使用无菌技术，以减少微生物污染层流罩的组织。浸泡5分钟，在70％乙醇，并冲洗在无菌PBS消毒镊子，剪刀和解剖刀刀片。 </li><li>办理ESophagus使用无菌镊子。使用剪刀，剪开食道纵向。通过将食道到含有约100毫升无菌PBS的180毫升无菌锅，轻轻晃动，以消除任何碎屑冲洗组织。 </li><li>通过喷洒宽松用70％乙醇清洁软木的夹层板并晾干。 </li><li>从PBS中取出食管，并用消毒针头，针从食道到电路板上，粘膜表面最上面。抓住粘膜表面，用无菌镊子食管的一端，并拉离板。此将分离的粘膜从底层粘膜下层。 <ol><li>然后，使用无菌手术刀刀片纵向解剖沿食管粘膜固有层，去除引脚作为解剖的进展，使粘膜被解除了。 </li></ol></li><li>保留解剖粘膜和丢弃底层粘膜下层。 </li><li>切断并丢弃最近端和远端2厘米吨的他粘膜是有更多的粘膜下腺体和较厚的固有层在这些领域，分别为7。 </li><li>用手术刀切割剩余组织为5个平方厘米。将所有的切割组织到含有约150毫升无菌PBS的180毫升无菌锅，轻轻搅动以冲洗组织。如果在处理超过五个食管在同一时间，可能有必要使用多个罐，这和三个随后的步骤。 </li><li>使用无菌镊子，转移切割组织成180毫升锅含有150mM毫升无菌的1M的NaCl，200国际单位/毫升青霉素，200微克/毫升链霉素和1.25微克/毫升两性霉素B温育72小时，在37℃。 </li><li>将组织到一个新的180毫升锅含有150mM毫升无菌PBS中。上皮将已开始明显从下面的组织中分离出来。轻轻地从猪的食道上皮细胞分离使用无菌镊子和丢弃果皮。 <ol><li>将剩余的牛逼问题变成一个新的180毫升锅内增加150个毫升无菌PBS中。轻轻搅动5分钟以洗涤。倒出PBS中，并用新鲜的PBS重复再两次。 </li></ol></li><li>将所有的组织在含有24小时，在室温温度为400毫升的无菌80％（体积/体积）甘油溶液的500毫升玻璃瓶中。转移到400毫升的无菌90％（体积/体积）甘油溶液另外24小时。 </li><li>储存在400ml的在室温下的无菌100％的甘油溶液为至少4个月到脱水和灭菌的组织，同时保持基底膜的完整性。 </li></ol> 4.生产文化传媒<ol><li>制备螯合新生小牛血清的培养液中使用。 <ol><li>倾100克CHELEX 100到玻璃1升瓶中，并添加去离子水。确切的量并不严格，但必须加入足量的水来覆盖CHELEX粉末。加磁力搅拌器和通过高压灭菌（121℃下消毒15分钟）。 </li><li>允许瓶冷却并CHELEX沉降。在层流柜倒出多余的水，加500毫升新生小牛血清（NCS）和离开搅拌过夜，在4℃。 </li><li>停止搅拌，让CHELEX解决，这可能需要长达30分钟。在层流柜用无菌移液管，注意不要在-20℃扰乱CHELEX，并储存在10毫升等份倒出螯合血清。 </li></ol></li><li>准备三种不同的媒体在层流柜，开始与亲增殖和促分化配方结束。 <ol><li> 1比（ν::体积），4×10 -3 M L-谷氨酰胺，0.5微克/ ml氢化可的松，1×10 -4 M O对-phosphorylethanolamine，2×制备复合培养基I在一个3由DMEM和火腿F12 10 -12 M三碘甲状腺氨酸，1.8×10 -4 M腺嘌呤，1.88×10 -3 M的CaCl 2，4×10 -12 M孕酮，在10μg/ ml的胰岛素，10μg/ ml的转铁蛋白，10纳克/毫升硒，1×10 -3 M乙醇胺和0.1％（体积/体积）螯合的NCS。 </li><li>制备复合介质二作为复合培养基I除了代替螯合的血清用0.1％（体积/体积）的非螯合的NCS。 </li><li>制备复合介质三作为复合介质二除了省略孕酮，增​​加血清至2％（体积/体积）的非螯合的NCS。 </li></ol></li></ol> 5.生产人类食管黏膜模型<ol><li>执行所有工作在层流罩，使用无菌技术来保持无菌环境。通过浸泡在70％乙醇5分钟，漂洗在无菌PBS之前消毒的所有工具。 </li><li>这一天则需要之前，补充水分的猪去细胞支架食管。需要一个片支架的每个构建体来制备。 <ol><li>为了补充水分组织足够的地方猪件支架装有100ml无菌PBS的锅，搅拌，浸泡10分钟。德卡新台币PBS和更换新鲜PBS。重复洗涤过程5次，以确保除去所有甘油痕迹。 </li></ol></li><li>过夜的100ml DMEM中温育再水化的组织在37℃下测试该脚手架无菌。如果没有变化到培养基中的颜色或混浊在所述支架被假定为无菌培养期结束。一旦无菌已被证实，最上放置5 cm 2的脱细胞的支架进6孔板，粘膜下侧。 </li><li>放置一个无菌医用级不锈钢环（内径10mm时，外径20毫米）到每个支架的中心，并轻轻地用无菌镊子，以保证足够的密封与组织按下。 </li><li>收获用胰蛋白酶-EDTA 13以产生细胞悬浮液的成纤维细胞。计数使用血球14细胞。离心细胞悬浮液（10分钟，200×g离心）。重悬细胞沉淀成appropria成纤维细胞培养基忒体积，以产生一个最终细胞每毫升2.5×10 6个细胞的计数。 </li><li>中加入0.2ml的成纤维细胞培养基中含有5×10 5人食管癌成纤维细胞，每个环内。用约2 ml成纤维细胞培养基中的泛洪围绕环的外侧的区域。在5％的CO 2，湿润气氛在37℃。 </li><li> 24小时后取出圈和成纤维细胞培养基中，并添加至少5毫升新鲜的成纤维细胞培养基到每个孔中，以确保该支架被完全沉浸在介质。在5％的CO 2培养1周，在37℃，湿润气氛更换培养基每2至3天。 </li><li> 1周后删除包含支架，以层状洪水罩的6孔培养板，除去培养基，用无菌镊子反转每个支架，将粘膜表面最上面。这个时间点被称为第0天。 </li><li>放置在粘膜表面上的钢环中的中心组织如在步骤5.4。 </li><li>制备上皮细胞的T75烧瓶进行胰蛋白酶消化13。后的PBS洗涤，并在加入的胰蛋白酶-EDTA溶液，加入2无菌0.02％ml（重量/体积）的EDTA溶液每个烧瓶中。孵育2分钟，在37℃。轻按瓶轻轻选择性分离i3​​T3饲养层，同时使附着的上皮细胞。 注:这通常是不实际或需要病人配合上皮细胞前一周模型已经纳入了成纤维细胞。 </li><li>倒出含有饲养层的EDTA溶液，并添加胰蛋白酶-EDTA溶液收获上皮细胞13。计数使用血球14细胞。离心细胞悬浮液（10分钟，200×g离心）。重悬细胞沉淀成复合培养基1的适当体积，以产生一个最终细胞每毫升5×10 6个细胞的计数。 </li><li>添加复合介质我为0.2ml，含1×10 6上皮细胞，在环内。洪水周围环用约2mL复合培养基I的外侧的区域，然后将构建体进入孵化器在37℃，在5％的CO 2，湿润气氛。 </li><li> 24小时后（第1天）去掉环和介质，并添加至少5毫升新鲜复合培养基I，确保了支架被完全淹没，并返回到培养箱中。另外24小时（2天）后除去培养基，并用至少5毫升复合培养基Ⅱ的取代，从而也确保了支架被完全淹没，并返回到培养箱中。 </li><li>第4天发生无菌医疗级不锈钢网格（2厘米宽×2厘米长×0.5厘米高），到新6孔板，每个构建之一。使用无菌镊子对构建体转移到钢网格，粘膜表面最上层的顶部。 <ol><li>添加足够复合介质三使液体到达复合材料的下侧，但表面暴露在空气中，这确保了样品保持在空气 - 液体界面。需要将取决于支架，阱的体积和不锈钢网格的精确高度的厚度而变化介质的精确量。 </li></ol></li><li>保持复合材料在空气 - 液体界面之间10和20天（第14天至第24天），根据实验的要求。用新鲜培养基更换复合介质三，每2〜3天（周一，周三，周五是一个用户友好的政权）。 注意:5天后在空气 - 液体界面（9日）的构建体具有足够成熟食管上皮中的环境因素对食管上皮的影响的实验中使用。 </li><li>在实验结束时，从上皮进一步analys修复构建体用于组织学分析和免疫组织化学染色7，或提取物蛋白质15或RNA 11,16是。如果需要的话，保持经调节的培养基对细胞外信号分子17的分析。 </li></ol>

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Pathol. 205 (4), 468-475 (2005).</li><li>Okawa, 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=The+functional+interplay+between+EGFR+overexpression,+hTERT+activation,+and+p53+mutation+in+esophageal+epithelial+cells+with+activation+of+stromal+fibroblasts+induces+tumor+development,+invasion,+and+differentiation.">The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation.</a> Genes &amp; Development. 21 (21), 2788-2803 (2007).</li><li>Green, N., 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+development+and+characterization+of+an+organotypic+tissue-engineered+human+esophageal+mucosal+model.">The development and characterization of an organotypic tissue-engineered human esophageal mucosal model.</a> Tissue Eng Part A. 16 (3), 1053-1064 (2010).</li><li>Harada, H., 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=Telomerase+induces+immortalization+of+human+esophageal+keratinocytes+without+p16INK4a+inactivation.">Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation.</a> Mol. Cancer Res. 1 (10), 729-738 (2003).</li><li>Bhargava, S., Chapple, C. R., Bullock, A. J., Layton, C., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+buccal+mucosa+for+substitution+urethroplasty.">Tissue-engineered buccal mucosa for substitution urethroplasty.</a> BJU. Int. 93 (6), 807-811 (2004).</li><li>Ralston, D. 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=Keratinocytes+contract+human+dermal+extracellular+matrix+and+reduce+soluble+fibronectin+production+by+fibroblasts+in+a+skin+composite+model.">Keratinocytes contract human dermal extracellular matrix and reduce soluble fibronectin production by fibroblasts in a skin composite model.</a> Br. J. Plast. Surg. 50 (6), 408-415 (1997).</li><li>Green, N. H., 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=Pulsatile+exposure+to+simulated+reflux+leads+to+changes+in+gene+expression+in+a+3D+model+of+oesophageal+mucosa.">Pulsatile exposure to simulated reflux leads to changes in gene expression in a 3D model of oesophageal mucosa.</a> Int. J. Exp. Pathol. 95 (3), 216-228 (2014).</li><li>Wang, C. 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=Selective+culture+of+epithelial+cells+from+primary+breast+carcinomas+using+irradiated+3T3+cells+as+feeder+layer.">Selective culture of epithelial cells from primary breast carcinomas using irradiated 3T3 cells as feeder layer.</a> Pathol. Res. Pract. 197 (3), 175-181 (2001).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsinizing+and+subculturing+mammalian+cells.">Trypsinizing and subculturing mammalian cells.</a> J. Vis. Exp. (16), e755 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+Hemacytometer+to+Count+Cells.">Using a Hemacytometer to Count Cells.</a> Basic Methods in Cellular and Molecular Biology. , (2014).</li><li>Mahmood, T., Yang, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blot:+technique,+theory,+and+trouble+shooting.">Western blot: technique, theory, and trouble shooting.</a> N. Am. J. Med. Sci. 4 (9), 429-434 (2012).</li><li>Streit, S., Michalski, C. W., Erkan, M., Kleeff, J., Friess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Northern+blot+analysis+for+detection+and+quantification+of+RNA+in+pancreatic+cancer+cells+and+tissues.">Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues.</a> Nat. Protoc. 4 (1), 37-43 (2009).</li><li>Morgan, 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=Cytometric+bead+array:+a+multiplexed+assay+platform+with+applications+in+various+areas+of+biology.">Cytometric bead array: a multiplexed assay platform with applications in various areas of biology.</a> Clin. Immunol. 110 (3), 252-266 (2004).</li><li>Xu, 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=The+abnormal+expression+of+retinoic+acid+receptor-beta,+p+53+and+Ki67+protein+in+normal,+premalignant+and+malignant+esophageal+tissues.">The abnormal expression of retinoic acid receptor-beta, p 53 and Ki67 protein in normal, premalignant and malignant esophageal tissues.</a> World J. Gastroenterol. 8 (2), 200-202 (2002).</li><li>Moll, R., Divo, M., Langbein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+keratins:+biology+and+pathology.">The human keratins: biology and pathology.</a> Histochem. Cell Biol. 129 (6), 705-733 (2008).</li><li>Rice, R. H., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+in+human+epidermal+cells+of+a+soluble+protein+precursor+of+the+cross-linked+envelope:+activation+of+the+cross-linking+by+calcium+ions.">Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions.</a> Cell. 18 (3), 681-694 (1979).</li><li>Eves, 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=Melanoma+invasion+in+reconstructed+human+skin+is+influenced+by+skin+cells+-+investigation+of+the+role+of+proteolytic+enzymes.">Melanoma invasion in reconstructed human skin is influenced by skin cells - investigation of the role of proteolytic enzymes.</a> Clin. Exp. Metastasis. 20 (8), 685-700 (2003).</li></ol>

作者什么都没有透露。

这个手稿描述适合用作实验平台，研究暴露于环境应激经食管上皮的影响的生物相关的人类食管粘膜模型的制备和表征。 对于成功制造人工食道粘膜模型的最重要的步骤是:确保多数上皮细胞保持增殖和尚未开始播种它们在支架上之前，分化;维持空气 - 液体界面的复合，以确保上皮的正确的成熟;保持无菌整个扩展文化时期。 由于人体组织的可用于细胞分...

食管粘膜包括分层，鳞状上皮以上的层结缔组织，固有层，并且是遇到摄取环境应激第一站点之一。暴露于饮食毒素牵涉于食管鳞状细胞癌的发展，而duodenogastro食管返流是在巴雷特上皮化生，这是与进展的风险增加至食管腺癌相关的发病机理的关键因素。食管癌是在英国男性和食道腺癌的第 8 次最常见的恶性肿瘤是在西方世界1迅速增加。此外，很少有改善疾病的预后，具有大约15％的总体5年存活率。因此，有必要对实验平台来调查对环境应激有关此食管上皮的开发板的曝光的影响和其潜在参与pment化生或肿瘤形成。 虽然永生化或肿瘤细胞系允许研究人员研究上皮细胞向这些压力在体外的反应，它们保持增殖和失败分化成的食管粘膜的最上层中发现的成熟上皮细胞。此外，那些已经经历了肿瘤细胞株可提供关于内上皮环境因素正常细胞的初始响应仅有限的信息;这是当用于治疗性干预的潜在可能是最高的阶段。最后，传统的细胞培养系统无法捕捉上皮细胞和间质细胞之间，并且这些细胞和周围基质在体内组织内发生之间的潜在的重要相互作用。 动物模型对于研究食管epitheli的答复提供更逼真的微环境微米和可以结合的人工诱导胃食管反流病2。然而，它可以是更具挑战来操纵这些模型的环境压力，他们可能不能完全代表人类食道内的响应。 其他实验人食管癌模型已开发了利用原代细胞，永生化细胞或肿瘤细胞系上的胶原蛋白，或结合胶原/基质胶，含有支架的成纤维细胞3,4。它是低劳动强度，以产生这些支架比本手稿中描述的脱细胞食管支架，这些器官模型提供了有用的工具，特别是在肿瘤侵袭5,6-，其中肿瘤细胞渗入胶原凝胶可以容易的研究观察。然而，这些胶原凝胶具有非天然的机械性能，并且缺乏原组织的某些特征，包括一个特定的基底膜和都是适宜E面形貌。这可以使用胶原凝胶支架7时影响导致细胞的行为，例如，上皮细胞和支架之间较差的粘附。作为结果，脱细胞猪食管支架被开发，与作为一个多种生物逼真脚手架，从而更适合用作实验平台的优点。还已经表明，最好是采用了初级细胞进入食管构建体比永生化食管上皮细胞系，如的Het-1A，因为这些细胞形成的多层上皮，但未能分层或分化4,7,8 。 因此，该协议已被改编自一个方法已经在麦克尼尔实验室做组织工程皮肤和口腔黏膜9,10使用，并采用了脱细胞化猪食管支架结合初级人食管上皮细胞和成纤维细胞。 THI规程产生一个成熟的，分层上皮，类似于正常人食道的具体表现为CK4，CK14，Ki67和外皮蛋白染色。由此产生的模型提供了一个实验平台，研究应对环境压力，并已被有效地用在食管上皮调查基因表达的变化响应于反流部件11。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Trypsin</td>
			<td>BD Biosciences</td>
			<td align="right">215240</td>
			<td>Prepare 0.1% w/v solution in PBS and filter sterilise. Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Labtech</td>
			<td>LM-D1112</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Labtech</td>
			<td>LM-H1236</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Foetal Calf Serum</td>
			<td>Labtech</td>
			<td>FB-1090</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>R+D Systems</td>
			<td>236-EG-200</td>
			<td>Prepare 200 &#181;g/ml stock solution in 10 mM acetic acid, 1% FCS</td>
		</tr>
		<tr>
			<td>Hydorcortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0396</td>
			<td>Prepare stock solution in PBS and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma-Aldrich</td>
			<td>A2786</td>
			<td>Prepare stock solution in PBS and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I2767</td>
			<td>Prepare 10 mg/ml solution in 0.01M HCl, dilute 1:10 in distilled water and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T2036</td>
			<td>Prepare stock solution in distilled water and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Triiodothyronine</td>
			<td>Sigma-Aldrich</td>
			<td>T2752</td>
			<td>Prepare stock solution in distilled water and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td>Prepare stock solution in water</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15290-026</td>
			<td>Brand name Fungizone</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Oxoid</td>
			<td>BR0014</td>
			<td>Dissolve 1 tablet in 100 ml water and autoclave to sterilise</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td align="right">10103578001</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine solution</td>
			<td>Ecolab</td>
			<td>10830E</td>
			<td>Brand name Videne</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td align="right">433209</td>
			<td>Prepare 1M solution and autoclave to sterilise before use (121 &#730;C for 15 min)</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G2025</td>
			<td>Autoclave to sterilise before use (121 &#730;C for 15 min)</td>
		</tr>
		<tr>
			<td>Chelex 100</td>
			<td>Sigma-Aldrich</td>
			<td>C7901</td>
			<td></td>
		</tr>
		<tr>
			<td>Newborn calf serum</td>
			<td>Gibco</td>
			<td align="right">26010074</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma-Aldrich</td>
			<td>P8783</td>
			<td>Prepare stock solution in DMEM and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>E9508</td>
			<td>Prepare stock solution in DMEM and filter sterilise before use</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>Prepare stock solution in DMEM and filter sterilise before use use</td>
		</tr>
		<tr>
			<td>O-phosphorylethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>P0503</td>
			<td>Prepare stock solution in DMEM and filter sterilise before use</td>
		</tr>
		<tr>
			<td>ITS (insulin, transferrin, selenium)</td>
			<td>Lonza</td>
			<td>17-838Z</td>
			<td>Used for composite media preparation</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T3924</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>EDTA 0.02% solution</td>
			<td>Sigma-Aldrich</td>
			<td>E8008</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>T75 culture flask</td>
			<td>VWR</td>
			<td>734-2313</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>Fisher</td>
			<td align="right">11819650</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml universal tube</td>
			<td>SLS</td>
			<td>SLS7504</td>
			<td></td>
		</tr>
		<tr>
			<td>180 ml pot</td>
			<td>VWR</td>
			<td>216-2603</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>SLS</td>
			<td align="right">150350</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>VWR</td>
			<td>734-2323</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel rings</td>
			<td></td>
			<td></td>
			<td>Manufactured in house - medical grade stainless steel, internal diameter 10 mm, external diameter 20 mm</td>
		</tr>
		<tr>
			<td>steel mesh grids</td>
			<td></td>
			<td></td>
			<td>Manufactured in house - sheets have 0.3 cm diameter holes, bent to produce grid 2cm (w) x2 cm (d) x 0.5 cm (h)</td>
		</tr>
		<tr>
			<td>ki67</td>
			<td>Novocastra</td>
			<td>KI67-MM1-L-CE</td>
			<td>Clone MM1 Use at 1:100</td>
		</tr>
		<tr>
			<td>CK4</td>
			<td>Abcam</td>
			<td>ab9004</td>
			<td>Clone 6B10 Use at 1:200</td>
		</tr>
		<tr>
			<td>CK14</td>
			<td>Novocastra</td>
			<td>LL002-L-CE</td>
			<td>Clone LL002 Use at 1:200</td>
		</tr>
		<tr>
			<td>Involucrin</td>
			<td>Novocastra</td>
			<td>INV</td>
			<td>Clone&#160; SY5 Use at 1:100</td>
		</tr>
		<tr>
			<td>OE21</td>
			<td>Sigma-Aldrich</td>
			<td align="right">96062201</td>
			<td></td>
		</tr>
		<tr>
			<td>OE33</td>
			<td>Sigma-Aldrich</td>
			<td align="right">96070808</td>
			<td></td>
		</tr>
		<tr>
			<td>Het-1A</td>
			<td>ATCC-LGC</td>
			<td>CRL-2692</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse 3T3 fibroblasts</td>
			<td>ATCC-LGC</td>
			<td>CRL-1658</td>
			<td>previously growth arrested by irradiation (60 Gy)</td>
		</tr>
	</tbody>
</table>

production, characterization and potential uses of a 3d tissue-engineered human esophageal mucosal model

两者食管腺癌和其前体，巴雷特化生的发生，正在迅速在西方世界上升。此外食管腺癌一般预后较差，在生存率几乎没有改善的排头兵。这些都是困难的条件下，研究，至今缺乏合适的实验平台，以探讨食管粘膜的疾病。 已经开发了麦克尼尔实验室，它不象常规的2D细胞培养系统，概括本体内的细胞-细胞和细胞-基质相互作用，并产生一个成熟的，分层类似于正常人上皮的人食管粘膜的模型食道。简要地说，该模型利用内的猪源性脱细胞食管支架生长的未转化的正常原代人食管成纤维细胞和上皮细胞。这种模式的表征免疫组化通过CK4，CK14，Ki67和外皮染色展示了人类正常食管黏膜的组织学再演合适。 这个模型提供了人类食管粘膜的稳健，生物相关的实验模型。它可以很容易被操纵以调查了若干研究问题，包括药理学试剂的有效性和暴露于环境因素，如醇，毒素，高温或胃食道反流部件的影响。该模型还有助于延长培养期间无法实现与传统的二维细胞培养，启用， 除其他外 ，一个成熟的上皮反复暴露于感兴趣的代理的影响为长达20天的研究。此外，各种细胞系，诸如那些自食管肿瘤或巴雷特化生衍生，可并入该模型来调查如肿瘤侵袭和药物responsivene流程SS在更生物学相关的环境。

这个手稿描述所需的过程，在图1以示意形式示出，以人类食管上皮成功培养3D模型。以确认模式的适用性作为实验平台的组织学和免疫组化研究已进行用正常人食管鳞状黏膜比较培养组织。 通过所描述的方法产生的上皮的组织学评估显示了成熟的，多层，层鳞状上皮（ 图2B），这是相媲美，与正常人食管（ 图2A）观察到，虽然稀释剂（5?...

Watch this Scientific Journal Video about Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model at JoVE.com

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

这份手稿描述了生产，表征和组织工程食管三维结构从正常原代人成纤维食管，并在脱细胞化猪接种支架鳞状上皮细胞制备的潜在用途。结果证明类似于正常人食道成熟复层上皮的形成。

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

production-characterization-potential-uses-3d-tissue-engineered-human

Research

JoVE Journal

Bioengineering

10.4K 次观看.  University of Sheffield. 这份手稿描述了生产，表征和组织工程食管三维结构从正常原代人成纤维食管，并在脱细胞化猪接种支架鳞状上皮细胞制备的潜在用途。结果证明类似于正常人食道成熟复层上皮的形成。 

视频: Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

科研

生物工程

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

人类乳腺上皮细胞的激素反应3D文化模式

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

一种混合的3D组织工程<em&gt;体外</em&gt; /<em&gt;在硅片</em在具体的突变背景预测药物的有效性&gt;肺肿瘤模型

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

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.

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

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., ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

一种新型的腱绦缝合技术，采用组织工程胶原蛋白移植物修复大肌腱缺损

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

小鼠模型中生物合成组织工程气管移植的育苗与植入

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...