This work was supported by the Biomedical Research Council, Singapore through core support to the Institute of Medical Biology and grant SPF2013/005. M.R. was supported by a NUS Faculty Research Committee Grant (Engineering in Medicine) (M.R.) R-397-000-081-112, and the NUS Tissue Engineering Program (NUSTEP). The authors would like to thank Professor Irene Leigh for providing the collagen VII antibody. Electron microscopy work was carried out at the National University of Singapore Electron Microscopy Unit.

1.大分子拥挤在2D皮肤细胞培养<ol><li>种子50,000个细胞（初级成纤维细胞或角质形成初级或初级成纤维细胞和角化细胞的共培养）每一个24孔板的孔。种子细胞在1ml对应的小区类型的生长介质。 </li><li>使细胞附着过夜，在5％CO 2 37℃培养箱中。 </li><li>废旧媒体和含大分子沟渠扫污和100μM抗坏血酸苯磺酸1毫升的新鲜介质代替。使用克劳德鸡尾酒组成的37.5毫克/毫升聚蔗糖70和25毫克/毫升的Ficoll 400使用成纤维细胞培养基（FM）的组成的DMEM补充有10％FBS和1％青霉素 - 链霉素抗生素。生长的角质形成细胞在无血清培养基，KSFM或CNT-57。保持共培养物中50％的FM和50％无血清培养基的混合物。 </li><li>孵育6天培养物在37℃培养箱中于5％的CO 2，与媒体的变化，每2天。 </li></ol>2.皮肤细胞培养免疫荧光染色<ol><li>洗细胞培养物两次用1×PBS。 </li><li>固定用甲醇或聚甲醛（取决于抗体规格）的细胞培养物。 <ol><li>甲醇固定<ol><li>等分500μl的冷甲醇到每个孔中，并在-20℃下进行10分钟孵育。吸固定剂和丢弃甲醇浪费。空气干燥在层通风柜。 </li></ol></li><li>对于多聚甲醛固定<ol><li>等分试样500微升的2％多聚甲醛溶液导入各孔中，并孵育在室温下进行15分钟。吸固定剂和丢弃多聚甲醛的浪费。 </li><li>用1X PBS洗涤，用于洗三次，每洗5分钟。 </li><li>等分试样1毫升1×PBS中的每个孔。 </li></ol></li></ol></li><li>免疫荧光染色使用抗体<ol><li>等分试样的3％牛血清白蛋白（在1×PBS稀释）到每个500微升良好。在室温下孵育30分钟。吸出封闭溶液并丢弃。 </li><li>一抗<ol><li>直接从印版成像:等分200μl的初级抗体（1:100稀释在10％山羊血清）溶液到每个孔中，并孵育在室温下90分钟。 </li><li>为盖玻片:分装50μl的初级抗体（1:100稀释在10％山羊血清）溶液在平坦的片封口膜。放置盖玻片上封口膜，但面向溶液单元侧（与细胞盖玻片的一部分）。覆盖整个单元侧，与无气泡。孵育在室温下90分钟。 </li></ol></li><li>吸去第一抗体溶液并丢弃。 </li><li>用1X PBS洗涤，用于洗三次，每洗5分钟。 </li><li>二抗<ol><li>直接从印版成像:等分200μl的第二抗体（1:400稀释度在10％山羊血清）溶液到每个孔中，并孵育30分钟，在室温下，覆盖板与铝FO<html>金正日。 </li><li>对于盖玻片:分装50微升二抗（1:400稀释10％山羊血清）解决方案到一块平坦的封口膜。放置盖玻片上封口膜，但面向溶液单元侧（与细胞盖玻片的一部分）。覆盖整个单元侧，与无气泡。孵育在室温下30分钟，覆盖用铝箔封口膜。 </li></ol></li><li>吸二级抗体溶液并丢弃。 </li><li>用1X PBS洗涤，用于洗三次，每洗5分钟。等分试样1毫升1×PBS中的每个孔。 </li><li>安装<ol><li>对于直接从印版成像:不安装。继续显微镜。 </li><li>对于盖玻片成像摩盖玻片，在安装媒体，玻片上。干通宵在覆盖着铝箔的容器。继续显微镜。 </li></ol></li></ol></li><li>获得使用激光共聚焦显微镜与40X / 1.30油浸物镜图像。 </li></ol>e"的皮肤细胞培养&gt; 3。免疫印迹<ol><li>用1×PBS洗涤细胞层两次。 </li><li>等份加入100μl裂解缓冲液（见材料列表）到每个孔（6孔板格式）。刮用细胞刮刀和转让溶液放入微量离心管中的细胞层。 </li><li>在15330×g离心30分钟，离心的溶液在4℃。将上清转移至另一微量离心管中，弃去沉淀。 </li><li>混合13微升样品缓冲液5微升和2μl还原剂的各样品的（见材料清单）。热样品在95℃下进行10分钟。 </li><li>在15330 XG 1分钟离心样品，收集冷凝水。负载样品成预制-PAGE凝胶上并在100V为约1小时运行。 </li><li>到分离的蛋白质转移至硝酸纤维素膜，夹着纸片和海绵（相同尺寸的）之间的凝胶和膜。确保有胶，膜，纸和海绵之间无气泡秒。关闭三明​​治盒，并在2小时运行在70 V输出用于传输。 </li><li>要检查是否转让完成后，用0.1％PBS吐温20，三次，每次洗10分钟，用10毫升丽春红S.洗膜孵育膜。 </li><li>用10毫升的5％的牛奶在室温下1小时阻断。用0.1％的PBS-Tween-20中，三次，每次洗涤10分钟洗膜。 </li><li>用90分钟10毫升初级抗体（1:1,000稀释在5％的牛奶的小鼠抗胶原VII，LH 7.2，1）孵育。用0.1％的PBS-Tween-20中，三次，每次洗涤10分钟洗膜。 </li><li>用30分钟10毫升二级抗体（:2,000稀释在5％的牛奶的山羊抗小鼠-HRP，1）孵育。用0.1％的PBS-Tween-20中，三次，每次洗涤10分钟洗膜。 </li><li>膜转移到磁带，加入1ml ECL检测试剂。将在膜清晰，薄塑料片。 </li><li>为了检测化学发光，将感光膜在塑料片材和关闭磁带。 2之后-5分钟，从盒取出胶片，并将其放置到一个显影剂。 </li></ol> 4.制作和使用细胞衍生矩阵<ol><li>种子50,000个细胞（初级成纤维细胞或角质形成初级或初级成纤维细胞和角化细胞的共培养）每一个24孔板的孔。 </li><li>使细胞附着过夜，在5％CO 2 37℃培养箱中。 </li><li>废旧媒体和含大分子沟渠扫污和100μM抗坏血酸新鲜培养基更换。的克劳德鸡尾酒包含37.5毫克/毫升聚蔗糖70和25毫克/毫升的Ficoll 400成纤维细胞培养基（FM），由DMEM补充有10％FBS和1％青霉素 - 链霉素的。角质形成细胞生长在无血清的培养基，KSFM或CNT-57。保持共培养物中50％的FM和50％KSFM或CNT-57的混合物。 </li><li>孵育6天培养物在37℃培养箱中于5％CO 2，改变介质每2天。 </li><li> Decellularize细胞层，以获得一个细胞衍生马特里X（F-垫 =成纤维细胞来源基质中，k-垫 =角质细胞衍生的基质， 共垫 =从成纤维细胞和角化细胞的共培养衍生的矩阵）。 <ol><li>在1×PBS洗涤细胞层两次。 </li><li>加入250微升0.5％去氧胆酸钠。孵育，在冰上进行10分钟。吸细胞裂解液。 </li><li>重复步骤4.5.2三次。 </li><li>在蒸馏H 2 O洗涤细胞衍生基质两次保持细胞衍生基质在1×PBS中。矩阵可以储存在4℃长达1周℃。 </li></ol></li><li>准备二次细胞悬液（例如，在顶部F-垫播种角质形成细胞）。 <ol><li>吸1X PBS。 </li><li>种子的细胞衍生的基质（F-MAT）的在前50000角质形成细胞。使细胞附着过夜，在5％CO 2 37℃培养箱中。 注:测定可直接在贴壁细胞来进行。 </li></ol></li></ol> 5.表征小区的衍生矩阵<ol><li>免疫荧光染色:参照协议2。 </li><li> 干扰反射显微镜 <ol><li>种子细胞上盖玻片，以下4.1-4.5协议。 </li><li>获得细胞来源的基质后，修复以下协议2.2样本。 注:抗体染色是可选的。如果需要的话，请参阅2.3协议。 </li><li>通过使用共聚焦激光扫描显微镜具有40X / 1.30油物镜进行图像采集。在74毫米设置针孔。 </li><li>使用LP 505为干扰重新挠度显微镜（IRM）通道和BP 575-615 IR为FL为荧光滤镜红色通道。使用NT 80/20的IRM通道和HFT 405/488/561，NFT 565，板为FL荧光的分光器红色通道。请使用以下激光器:DPSS 561-10（波长561纳米）以1.1％的电力为FL荧光红色通道，并在HeNe633为IRM通道5.0％的功率（波长633纳米）。 </li></ol></li></ol> 6. 3D器官皮肤共培养<ol><li>投出胶原蛋白凝胶，与成纤维细胞封装，在细胞培养插入（6孔板格式）。 <ol><li>分装10毫升鼠尾Ⅰ型胶原感冒烧杯中。 </li><li>加入1毫升冷的DMEM。将0.5ml的1M NaOH来中和酸性的胶原蛋白。加入0.5毫升（含120万成纤维细胞）成纤维细胞悬液。 </li><li>一个6孔板内转移该溶液中，在寒冷的吸移管，向细胞培养插入物。 12毫升总溶液的均匀分为6细胞培养插入。 </li><li>孵育在37℃培养箱的凝胶在5％的CO 2，1小时。 </li><li>凝胶已凝固后，淹没在调频的凝胶。 24小时后，丢弃旧媒体和新的媒体替代，含大分子沟渠扫污。 2毫升的细胞培养插管的内部和2ml的体积添加到细胞培养插管的外侧。 </li></ol></li><li> 24小时后，种子在骨胶原的顶部角质形成细胞。 <ol><li> Trypsinize角质用0.125％胰蛋白酶/螯合剂5分钟，在37℃。点击烧瓶两侧轻轻打跑细胞，并用含血清培养基中和胰蛋白酶。计数细胞并制备角质形成细胞悬浮液（每凝胶300000角化细胞并悬浮于200μl培养基）。 </li><li>吸旧媒体从细胞培养插入。添加在凝胶顶部角化细胞。 </li><li>孵育在37℃培养箱的凝胶在5％的CO 2，1小时。 </li><li>角化细胞有附着到凝胶表面后，淹没用2ml KSFM或凝胶CNT-57的细胞培养插管的内部和2ml调频到细胞培养插管的外侧。 </li></ol></li><li> 24小时后，丢弃旧媒体和新的媒体替代，含大分子沟渠扫污。 </li><li>经过7天浸没培养，提高培养至空气 - 液体界面。 <ol><li>转移细胞培养插件ERT到深孔板。 10毫升分层介质添加到细胞培养插管的外侧。保持细胞培养插管干的内部。 </li></ol></li><li>孵育在37℃培养箱培养在5％的CO 2和改变介质每3天，在接下来的14天。 注意:2周的空气 - 液体界面之后，皮肤等效的是成熟的，可以收获（或者快速冷冻或福尔马林固定的）。 </li></ol> 7.采收和器官的表征皮肤替代物<ol><li>吸分层介质。 </li><li>使用镊子，转移培养插入到纸巾和​​从插入件的基部轻拍远离剩余的介质。 </li><li>使用手术刀，沿着培养插管的内周切割。 </li><li> （与PET膜合）修复皮肤器官共培养。 <ol><li>捕捉冻结<ol><li>凝胶结构转移到cryomold。盖上华侨城cryomold的内部复合。放置cryomold到液氮中持续10秒。 </li><li>使用镊子，从液氮取出冷冻cryomold，包裹在铝箔和冷冻样品储存在-80℃冰箱中。 </li><li>部冷冻样品用低温恒温器，具有7μm的切片厚度。 </li></ol></li><li>福尔马林固定<ol><li>放置一个&#39;活检海绵垫&#39;到&#39;活检盒"。转移凝胶构建到海绵垫。轻轻地放在另一个海绵垫的凝胶结构的顶部。关闭录像带。 </li><li>完全浸没在10％的中性缓冲的福尔马林盒48小时，在室温下。取出磁带并丢弃福尔马林浪费。 </li><li>通过首先用乙醇系列脱水，然后逐渐渗透用蜡样品嵌入固定样品放入蜡块。部分中的蜡块用切片机。 </li></ol></li></ol></li><li>在皮肤替代物的表征<ol><li>免疫染色<ol><li>对于冰冻切片: <ol><li>孵育在10％山羊血清（在1×PBS中稀释的）切片在室温下1小时。在1X PBS洗净切片，以去除山羊血清。 </li><li>孵育初级抗体部分（1:在10％山羊血清100稀释）在室温下90分钟。洗切片在1×PBS中，三次，每次洗涤5分钟。 </li><li>孵育二级抗体部分（1:在10％山羊血清1:400稀释）在室温下30分钟后，在用铝箔覆盖的容器。洗切片在1×PBS中，三次，每次洗涤5分钟。 </li><li>山滑，带安装媒体，到一个玻璃罩。盖样品用铝箔并晾干过夜。 </li><li>图片使用共聚焦显微镜与40X / 1.30油浸物镜。 </li></ol></li><li>对于福尔马林固定的石蜡切片: <ol><li>脱蜡切片（和再水合）以递减的乙醇百分比，以水（二甲苯 - 100％的乙醇- 90％的乙醇 - 80％的乙醇 - 70％的乙醇 - 水）。在解决方案完全淹没样品。每个脱蜡步骤应该是3分钟。 </li><li>孵育在1％的过氧化氢的部分30分钟。洗切片在1×PBS中5分钟。 </li><li>孵育在120℃下在柠檬酸盐溶液的部分（10毫升柠檬酸盐溶液溶解于90ml水）20分钟。允许滑动冷却过夜，在室温下。洗切片在1×PBS中，三次，每次洗涤5分钟。 </li><li>孵育在10％山羊血清（在1×PBS中稀释的）切片在室温下1小时。在1X PBS洗净切片，以去除山羊血清。 </li><li>孵育初级抗体部分（1:在10％山羊血清100稀释）在室温下90分钟。洗切片在1×PBS中，三次，每次洗涤10分钟。 </li><li>孵育30分钟HRP标记的聚合物（未稀释），在室温下部分。洗切片用1×PBS，三次，每次洗涤10分钟。 </li><li>到微量离心管中，结合1毫升DAB底物（3,3&#39;-二氨基联苯胺）与色原的1滴。孵育部分用该溶液，持续15秒 - 5分钟（棕色=阳性信号）。 </li><li>停止反应，在流水中洗涤切片。 </li><li>对细胞核用苏木，5分钟。漂洗在自来水部分。 </li><li>孵育切片在酸醇溶液，1分钟。漂洗在自来水部分。 </li><li>孵育节在斯科特的自来水液，2分钟。漂洗在自来水部分。 </li><li>可选:孵育切片在曙红1分钟，在流水洗净。 </li><li>脱水在乙醇升序百分比二甲苯部分（70％的乙醇 - 80％的乙醇 - 90％的乙醇 - 100％乙醇 - 二甲苯）。确保样品完全浸没每阶段3分钟。 </li><li>山幻灯片，使用安装媒体，一个玻璃罩。空气干燥过夜。 </li><li>图片使用共聚焦显微镜与40X / 1.30油浸物镜。 </li></ol></li></ol></li><li>透射电子显微镜（形象化锚纤维） <ol><li>固定在2.5％戊二醛的样品72小时。 </li><li>洗涤样品在1×PBS中，三次，每次洗涤10分钟。 </li><li>切成小块（1 立方毫米）。修复交样品在1％的四氧化锇，pH为7.4，在室温下1小时。洗涤样品中的蒸馏水。 注意:四氧化锇是有毒和无毒和应在通风橱中小心处理。 </li><li> （ - 50％的乙醇 - 75％的乙醇 - 95％的乙醇 - 100％的乙醇 - 丙酮25％乙醇）对每步20分钟，通过一升乙醇系列脱水样品。 </li><li>通过浸泡在100％丙酮样品渗透:ARALDITE树脂（1:1）在室温下30分钟，然后在1:3 1小时，接着1:6过夜。浸泡样品在新鲜ARALDITE树脂在室温下2小时，随后新鲜ARALDITE树脂的1小时，在40℃的第二变化。离开三一新鲜在45℃下1小时raldite树脂变化和离开1小时第四新鲜ARALDITE树脂变更为50℃。 </li><li>在60℃孵育样品24小时，以允许聚合。 </li><li>用玻璃刀，部分样本以获得半薄切片，厚度为1微米。用亚甲蓝染色部分观察组织学。部分样品，以获得为70nm的超薄切片，并收集各部分在铜网格。 </li><li>染色切片用15分钟，随后进行10分钟的3％柠檬酸铅溶液5％乙酸双氧铀溶液。 </li><li>获得用透射电子显微镜（100千伏）的图像，在30,000X的放大倍数。 </li></ol></li></ol></li></ol>

<ol>
	<li>Breitkreutz, D., Mirancea, N., Nischt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membranes+in+skin:+unique+structures+with+diverse+functions.">Basement membranes in skin: unique structures with diverse functions.</a> Histochem Cell Biol. 132 (1), 1-10 (2009).</li><li>Lane, E. B., 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=mutation+in+the+conserved+helix+termination+peptide+of+keratin+5+in+hereditary+skin+blistering.">mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering.</a> Nature. 356 (6366), 244-246 (1992).</li><li>Fuchs, E., Raghavan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+under+the+skin+of+epidermal+morphogenesis.">Getting under the skin of epidermal morphogenesis.</a> Nat Rev Genet. 3 (3), 199-209 (2002).</li><li>Niessen, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junctions/adherens+junctions:+basic+structure+and+function.">Tight junctions/adherens junctions: basic structure and function.</a> J Invest Dermatol. 127 (11), 2525-2532 (2007).</li><li>Jarvelainen, H., Sainio, A., Koulu, M., Wight, T. N., Penttinen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+molecules:+potential+targets+in+pharmacotherapy.">Extracellular matrix molecules: potential targets in pharmacotherapy.</a> Pharmacol Rev. 61 (2), 198-223 (2009).</li><li>Schaefer, L., Schaefer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteoglycans:+from+structural+compounds+to+signaling+molecules.">Proteoglycans: from structural compounds to signaling molecules.</a> Cell Tissue Res. 339 (1), 237-246 (2010).</li><li>Frantz, C., Stewart, K. M., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+at+a+glance.">The extracellular matrix at a glance.</a> J Cell Sci. 123 (Pt 24), 4195-4200 (2010).</li><li>Ghalbzouri, A., Jonkman, M., Dijkman, R., Ponec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membrane+reconstruction+in+human+skin+equivalents+is+regulated+by+fibroblasts+and/or+exogenously+activated+keratinocytes.">Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes.</a> J Invest Dermatol. 124 (1), 79-86 (2005).</li><li>Lareu, R. R., Arsianti, I., Harve, K. S., Peng, Y. X., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Enhancement+of+Collagen+Matrix+Formation+and+Crosslinking+for+Applications+in+Tissue+Engineering--a+Preliminary+Study.">In Vitro Enhancement of Collagen Matrix Formation and Crosslinking for Applications in Tissue Engineering--a Preliminary Study.</a> Tiss Eng. 13 (2), 385-391 (2007).</li><li>Lareu, R. 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=Collagen+matrix+deposition+is+dramatically+enhanced+in+vitro+when+crowded+with+charged+macromolecules:+the+biological+relevance+of+the+excluded+volume+effect.">Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect.</a> FEBS Lett. 581 (14), 2709-2714 (2007).</li><li>Lareu, R. R., Harve, K. S., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emulating+a+crowded+intracellular+environment+in+vitro.+dramatically+improves+RT-PCR+performance.">Emulating a crowded intracellular environment in vitro. dramatically improves RT-PCR performance.</a> Biophys Biochem Res Comm. 363 (1), 171-177 (2007).</li><li>Harve, K. S., Ramakrishnan, V., Rajagopalan, R., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+in+vitro+as+means+of+emulating+cellular+interiors:+when+less+might+be+more.">Macromolecular crowding in vitro as means of emulating cellular interiors: when less might be more.</a> Proc Natl Acad USA Sci. 105 (51), E119 (2008).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Scar-in-a-Jar:+Studying+antifibrotic+lead+compounds+from+the+epigenetic+to+extracellular+level+in+a+single+well.">The Scar-in-a-Jar: Studying antifibrotic lead compounds from the epigenetic to extracellular level in a single well.</a> Br J Pharmacol. 158 (5), 1196-1209 (2009).</li><li>Harve, K. S., Lareu, R., Rajagopalan, R., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+how+the+crowded+interior+of+cells+stabilizes+DNA/DNA+and+DNA/RNA+hybrids-in+silico+predictions+and+in+vitro+evidence.">Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids-in silico predictions and in vitro evidence.</a> Nucleic Acids Res. 38 (1), 172-181 (2009).</li><li>Satyam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+meets+tissue+engineering+by+self-assembly:+A+paradigm+shift+in+regenerative+medicine.">Macromolecular crowding meets tissue engineering by self-assembly: A paradigm shift in regenerative medicine.</a> Adv Mat. 26 (19), 3024-3034 (2014).</li><li>Chen, C. Z. C., Loe, F., Blocki, A., Peng, Y., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+macromolecular+crowding+to+enhance+extracellular+matrix+deposition+and+its+remodeling+in+vitro+for+tissue+engineering+and+cell-based+therapies.">Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies.</a> Adv Drug Deliv Rev. 63 (4-5), 277-290 (2011).</li><li>Rashid, 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=Novel+use+for+Polyvinylpyrrolidone+as+a+Macromolecular+Crowder+for+Enhanced+Extracellular+Matrix+Deposition+and+Cell+Proliferation.">Novel use for Polyvinylpyrrolidone as a Macromolecular Crowder for Enhanced Extracellular Matrix Deposition and Cell Proliferation.</a> Tiss Eng C. , (2014).</li><li>Dewavrin, J. Y., Hamzavi, N., Shim, V. P. W., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+Architecture+of+3D+Collagen+Hydrogels+by+Physiological+Macromolecular+Crowding.">Tuning the Architecture of 3D Collagen Hydrogels by Physiological Macromolecular Crowding.</a> Acta Biomaterialia. 10 (10), 4351-4359 (2014).</li><li>Dewavrin, J. Y., 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=Synergistic+Rate+Boosting+of+Collagen+Fibrillogenesis+in+Heterogeneous+Mixtures+of+Crowding+Agents.">Synergistic Rate Boosting of Collagen Fibrillogenesis in Heterogeneous Mixtures of Crowding Agents.</a> J Phys Chem B. 119 (12), 4350-4358 (2015).</li><li>Auger, F. A., Berthod, F., Moulin, V., Pouliot, R., Germain, L. <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+skin+substitutes:+from+in+vitro+constructs+to+in+vivo+applications.">Tissue engineered skin substitutes: from in vitro constructs to in vivo applications.</a> Biotechnol Appl Biochem. 39, 263 (2004).</li><li>Chen, B., 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=Macromolecular+crowding+effect+on+cartilaginous+matrix+production:+a+comparison+of+two-dimensional+and+three-dimensional+models.">Macromolecular crowding effect on cartilaginous matrix production: a comparison of two-dimensional and three-dimensional models.</a> Tiss Eng Part C Methods. 19 (8), 586-595 (2013).</li><li>Zeiger, A. S., Loe, F. C., Li, R., Raghunath, M., van Vliet, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+directs+extracellular+matrix+organization+and+mesenchymal+stem+cell+behavior.">Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior.</a> PLOS One. 7 (5), e37904 (2012).</li><li>Kumar, 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=Macromolecularly+crowded+in+vitro+microenvironments+accelerate+the+production+of+extracellular+matrix-rich+supramolecular+assemblies.">Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.</a> Scientific Reports. 5, 8729 (2015).</li><li>Ang, X. 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=Macromolecular+crowding+amplifies+adipogenesis+of+human+bone+marrow-derived+MSCs+by+enhancing+the+pro-adipogenic+microenvironment.">Macromolecular crowding amplifies adipogenesis of human bone marrow-derived MSCs by enhancing the pro-adipogenic microenvironment.</a> Tiss Eng Part A. 20 (5-6), 966-981 (2014).</li><li>Peng, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Fibroblast+Matrices+Bioassembled+Under+Macromolecular+Crowding+Support+Stable+Propagation+Of+Human+Embryonic+Stem+Cells.">Human Fibroblast Matrices Bioassembled Under Macromolecular Crowding Support Stable Propagation Of Human Embryonic Stem Cells.</a> J Tiss Eng Regen Med. 6 (10), e74-e84 (2012).</li><li>Benny, P., Badowski, C., Lane, E. B., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+More+Matrix:+Enhancing+the+deposition+of+dermal-epidermal+junction+components+in+vitro+and+accelerating+organotypic+skin+culture+development,+using+macromolecular+crowding.">Making More Matrix: Enhancing the deposition of dermal-epidermal junction components in vitro and accelerating organotypic skin culture development, using macromolecular crowding.</a> Tiss Eng A. 21 (1-2), 182-192 (2015).</li></ol>

The authors have no conflicting interests to disclose.

Enhanced extracellular matrix is obtained upon introduction of macromolecular crowders to cell culture, owing to the excluded volume effect which increases the propeptide cleavage by proteinases. This results in extracellular matrix, in particular collagen, to be processed faster and deposited on the culture surface. While other groups have obtained thick fibroblast cell layers, it involved a culture time of several weeks20. In contrast, MMC described in this Protocol, dramatically shortens the culture time wh...

皮肤形成通过防止水分流失和病原体进入的保护性屏障。它是由三大部分组成;基质丰富的真皮1，分层表皮2,3,4在它的上面，并在5,6之间的真皮表皮交界处。真皮是由主要的胶原蛋白和弹力纤维，并用稀疏的7成纤维细胞填充。与此相反，细胞丰富表皮是由角化细胞的多层组成。最内层的角质形成细胞是增殖性并提供新的基底细胞，其更新和替换末端分化的角质形成细胞是不断移动到皮肤的最外层，并失去了核和细胞质材料，导致其经历角化层脱屑。 真皮 - 表皮交界，特定类型的基底膜，是互连矩阵分子，其tethe组成的复杂结构RS表皮到真皮。真皮的胶原蛋白I的纤维交织胶原VII锚定它们锚定到IV型胶原丰富致密板纤维。反过来锚固丝（5层粘连蛋白，胶原十七和整合）与基底角朊细胞的半桥粒连接致密板。基底角质形成细胞（基底层）有能力的增殖和更新​​，以及分化和分层，以形成基底层层;棘层，颗粒层，以及最后的角质层，它代表了皮肤的接触面与环境。从基底层的角化层的途中 ，角化切换角蛋白的表达模式和最后经历细胞凋亡和包住自己在角化信封，共价由转谷氨酰胺酶活性的交联的特定蛋白质的船体。 重建皮肤及其体外层，包括的复杂结构<html>真皮 - 表皮连接和真皮细胞外基质，并以模拟角化过程中，具有长好奇科学家和生物工程作为一个具有挑战性的任务。已经出现了皮肤组织工程显著的进步，例如，皮肤细胞从病人活检成功提取和皮肤器官培养物使用源自患者的皮肤细胞8的生成。然而，未解决的问题保持由皮肤细胞有关的细胞外基质蛋白的分泌差本身并导致次优的皮肤模型。此外，所需要的时间，以产生使用当前的协议三维器官皮肤共培养四到八周之间变化，这可能潜在地与大分子沟渠扫污机的掺入缩短一个时间段。减少培养时间可以节省试剂成本，减少细胞衰老的发生率，降低了患者的等待时间应该产品在临床中使用。 t"的&gt;高分子拥挤（MMC）涉及引入特定大分子到培养基中，以产生排除体积效应。这些影响酶促反应速率，包括原胶原的蛋白水解切割这是标准的水性培养条件9-13下缓慢。在MMC中，酶反应被加速，而不相比不拥挤的对照10增加试剂导致14,15的量，以原胶原裂解，I型胶原在拥挤的培养物分子的量增加的情况下，由于原胶原胶原的转换允许胶原蛋白的形成组件，具有MMC 48小时培养成纤维细胞产生显著更多的胶原蛋白我相比，监测四个星期11,16,17拥挤的成纤维细胞培养的。除了对影响ECM的形成，稳定和重塑酶活性，MMC还效应已显示直接提升和调制胶原纤维的形成18,19。 我们在这里提出的方法，以增强由皮肤细胞的胞外基质（ECM）的生产，特别是真皮成纤维细胞和表皮角质形成细胞。此外，我们表明，在单层培养下的MMC中产生的富集的ECM可脱细胞并用作纯细胞衍生的基质（CDM）的。 我们使用非传统的方式来可视化和充分理解皮肤细胞培养MMC沉积在细胞外基质。干扰反射镜通常用于研究细胞 - 基质相互作用或细胞至玻璃的接触点。这种技术是在我们的系统中使用，以查看基质的沉积在玻璃表面上的总金额。干扰反射显微镜加上荧光免疫染色，以获得在细胞外基质组合物和图案方面最量的信息，在存在和不存在的MMC。 Organotypic皮共培养是在皮肤上的体外在三维上下文建模经典方法。而二维共培养可能提供显著的信息，它转换该数据和应用回体内环境，这是固有的三维结构时是有限的。皮肤角质形成细胞，尤其是偏振和含有心尖和基底段这对于稳态和细胞粘附是必不可少的。此外，典型的基底层蛋白在角化细胞的基底层之上，如角蛋白1，角蛋白10和聚丝蛋白的表达仅在分层和角化细胞的终末分化的过程中存在。由于终末分化是典型的单层培养几乎不存在，基底层蛋白的表达通常不会在这个培养体系来实现的。因此，器官型培养开始淹没在培养基中，但随后提升到空气 - 液体界面驱动keratinocytË分化。这导致分层的标记，甚至角化和表皮生理学的总体上更好的反射的表达。虽然其他团体先前生成的器官皮肤共培养成功，建立了功能真皮表皮交界地带一直是一个问题。这里，我们提出用于培养器官皮肤共培养具有增强的基底膜，在稠时间帧，在不损害这些构建体的成熟的新方法。这将提供皮肤类似物在体外模型，皮肤生物学研究和筛选试验的品种。

<table border="0" cellpadding="0" cellspacing="0" width="1154">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ascorbic acid</td>
			<td style="width:181px;">Wako</td>
			<td style="width:175px;">013-12061</td>
			<td style="width:499px;">Cell culture media additive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture inserts&#160; (6-well format)</td>
			<td style="width:181px;">Greiner Bio-One</td>
			<td style="width:175px;">657610</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citrate solution</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">S2369</td>
			<td style="width:499px;">DakoCytomation Target Retrieval Solution Citrate, pH 6 (x10)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Collagen (rat tail)</td>
			<td style="width:181px;">Corning</td>
			<td style="width:175px;">354236</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">CnT-57&#160;</td>
			<td style="width:181px;">CELLnTEC</td>
			<td style="width:175px;">CnT-57</td>
			<td>Cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DAB Substrate + chromogen kit</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">K3468</td>
			<td>Histology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Deep-well plate (6-well format)</td>
			<td style="width:181px;">Corning</td>
			<td style="width:175px;">355467</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">ECL detection reagent</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">RPN2106</td>
			<td>Amersham ECL Western Blotting Detection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Electron microscope (TEM)</td>
			<td>&#160;JEOL&#160;</td>
			<td>&#160;JEM-1010 (100kV)</td>
			<td>Transmission electron microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fibroblast media (FM)</td>
			<td></td>
			<td></td>
			<td>High Glucose-DMEM + 10% Fetal Bovine Serum + 1% Penicillin Streptomycin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Ficoll PM70</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">17-0310-05</td>
			<td>Macromolecular crowder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Ficoll PM400</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">17-0300-05</td>
			<td>Macromolecular crowder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Keratinocyte serum-free media (KSFM)</td>
			<td style="width:181px;">Life Technologies</td>
			<td style="width:175px;">17005-042</td>
			<td>Cell culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Lysis buffer</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">89900</td>
			<td style="width:499px;">Lysis buffer for protein extraction. A protease inhibitor (Roche, #11836170001) was added to this lysis buffer.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;">LSM510</td>
			<td style="width:499px;">Red, green and blue channels to visualize AF594, AF488 and DAPI fluorescent immunostainings (40X mag).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mounting media (Hydromount)</td>
			<td style="width:181px;">National Diagnostics</td>
			<td style="width:175px;">HS-106</td>
			<td>Fluorescent staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mounting media (Cytoseal)</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">8310-16</td>
			<td>Histology (HRP)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">OCT compound (Tissue Tek)</td>
			<td style="width:181px;">Sakura</td>
			<td style="width:175px;">4583</td>
			<td>Embedding for cryotomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-streptomycin antibiotics</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:175px;">A5955</td>
			<td>Cell culture media additive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Primary antibodies</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Collagen I antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:175px;">#ab6308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Collagen Type IV</td>
			<td style="width:181px;">Novocastra</td>
			<td style="width:175px;">#NCL-COLL-IV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-collagen VII</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>LH7.2 (in house)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Fibronectin antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:175px;">#ab2413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Reducing agent&#160;</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">NP0009</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sample buffer</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">NP0008</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Secondary antibodies (Immunostaining)</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:175px;">#D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 594 goat-anti-rabbit</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-11037&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 594 goat-anti-mouse</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">&#160;#A-11005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 488 chicken-anti-rabbit</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-21441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 488 goat-anti-mouse</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-11001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">Secondary antibodies (HRP)</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">Envision + System - HRP Labelled Polymer Anti-Rabbit and Anti-Mouse</td>
			<td>undiluted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Sodium deoxycholate</td>
			<td style="width:181px;">Prodotti Chimicie Alimentari</td>
			<td style="width:175px;"></td>
			<td>Decellularization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Stratification media</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dulbecco&#39;s Modified Eagle&#39;s Medium&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ham&#8217;s F12&#160;&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">10% fetal bovine serum&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">100U/mL penicillin and 100U/mL streptomycin&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">0.4mg/mL hydrocortisone&#160;&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5mg/mL insulin</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">1.8&#183;10-4 M adenine&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5mg/mL transferrin&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">2&#183;10- 11 M triiodothyronine&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Trypsin</td>
			<td style="width:181px;">Biopolis Shared Facilities</td>
			<td style="width:175px;">0.125% Trypsin/Versene&#160;&#160; pH 7.0 + 0.3&#160;</td>
			<td>Used to trypsinize cells</td>
		</tr>
	</tbody>
</table>

improving 2d and 3d skin in vitro models using macromolecular crowding

胶原的糖蛋白家族代表在人体内的主要结构蛋白，并且是在现代组织工程中使用的生物材料的重要组成部分。一个技术瓶颈是胶原的体外沉积，因为它是相当慢，导致结缔组织和随后的组织凝聚力亚最佳的形成，特别是在皮肤的模型。在这里，我们描述了包括加入差分大小蔗糖共聚物的皮肤培养物，以产生高分子拥挤（MMC），其导致胶原沉积的急剧增强的方法。具体地，真皮成纤维细胞相比，控制沉积下的MMC胶原I / IV / VII和纤连蛋白的一个显著量。 该协议还描述了一种方法，以decellularize拥挤的细胞层，暴露显著量细胞外基质（ECM）的该被保留的培养表面上由immunocy证明组化。使用干扰反射显微镜研究总矩阵的质量和分布格局。变化的组成和形态的有趣的是，成纤维细胞，角质形成细胞和共培养生产细胞衍生的基质（CDM）的。 CDM可作为"生物支架"进行二次细胞接种，其中当前使用的涂层或支架的，典型地从异种动物来源的，是可以避免的，由此实现更临床相关的应用程序移动。 此外，该协议描述中一个三维器官皮肤共培养模型这是足以增强的ECM沉积于真皮 - 表皮交界处（DEJ），特别是，胶原VII，主要部件的浸没阶段MMC卡的应用锚固纤维。电子显微镜确认为与对照相比锚固在使用MMC的发展，培养原纤维的存在。这是因为显著锚原纤维系绳真皮表皮，因此，具有预成形的成熟DEJ可以以接枝稳定性和整体的伤口愈合方面受益皮肤移植接受者。此外，培养时间第5周冷凝至3周，以获得成熟的构建物，使用MMC时，降低了成本。

大分子拥挤能够提高ECM的沉积，特别是成纤维细胞沉积的更多的胶原蛋白I，IV和纤连蛋白相比，对照培养物（ 图1，细胞层; 1A，I型胶原; 1B，IV型胶原; 1C，纤连蛋白）。在脱细胞，这是明显的，因为相比于角质形成细胞（ 图1，基质），该成纤维细胞胶原I，IV和纤连蛋白的主要存。 <p class="jove_content" fo:keep-t...

Watch this Scientific Journal Video about Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding at JoVE.com

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

我们提出了一个协议，以获得富含胞外基质蛋白的细胞来源的基质，使用大分子沟渠扫污机（MMC）。此外，我们提出该结合MMC在三维器官皮肤共培养产生，从而降低了培养时间，同时保持结构的成熟的协议。

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

improving-2d-and-3d-skin-in-vitro-models-using-macromolecular-crowding

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

12.4K 次观看.  National University of Singapore. 我们提出了一个协议，以获得富含胞外基质蛋白的细胞来源的基质，使用大分子沟渠扫污机（MMC）。此外，我们提出该结合MMC在三维器官皮肤共培养产生，从而降低了培养时间，同时保持结构的成熟的协议。 

视频: Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

负染色的EM可视化蛋白质和大分子复合物:从电网的制备图像采集

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

科研

生物工程

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

细胞外基质刚性三维肿瘤模型的纵向测量用粒子跟踪Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

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

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

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

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

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

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

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

2D和3D矩阵研究线性侵袭体形成和活性

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

利用大分子挤出的人类食性增大疤痕的体外模型

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

以自动化和标准化方式制造基于水凝胶的3D培养模型的开源技术平台

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

提高2D和3D皮肤

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