我们感谢Wistar研究所的动物设施，显微镜基金，Histotechnology基金和研究供应中心。这项研究是由国家卫生CA 076674研究院，CA 098101，025874和CA的CA 10815的资助部分经费。

1。三维人体皮肤重建模型： <ol><li>脱细胞层的制备：混合在50毫升管上冰的下列试剂：0.59 mL的10倍EMEM，50μL200mm的L -谷氨酰胺，0.6毫升胎牛血清，120μL7.5％碳酸氢钠和4.6 mL的牛胶原蛋白一加1毫升插入到每个组织培养托盘的混合物。孵育在室温下30分钟，让凝胶巩固。混合物的颜色应该是从稻草黄色粉色。 </li><li> Trypsinize人成纤维细胞培养瓶中，用0.25％胰蛋白酶/ EDTA，加入DMEM培养液含10％胎牛血清，以抵销。收集细胞于1.5 mL的DMEM含10％胎牛血清，离心，重悬0.45 × 10 6细胞。皮肤干细胞，攻烧瓶中分离的领域，离心收集6600（皮肤干细胞的干细胞的培养基中生长时，它们形成神经球以同样的方式来球）真皮领域。 </li><li>细胞层的制备方法：上冰混合以下，在50毫升管：1.65毫升的10倍EMEM，150μL200毫米L -谷氨酰胺，1.85毫升胎牛血清，350μL7.5％碳酸氢钠14毫升，牛胶原蛋白I和1.5毫升的成纤维细胞悬浮从第2步（另外，使用在皮肤1.5毫升0.45 × 10 6的成纤维细胞和6600真皮领域相结合，重建培养基皮肤干细胞重建我） ，拌匀。加入3毫升的混合液，每脱细胞层涂层插入。孵育45分钟，在37℃，5％的CO 2组织培养孵化器。混合物的颜色应该是淡黄色至粉红色。凝胶固化后，加入DMEM培养液含10％胎牛血清（2毫升内，10毫升的插入皮肤重建托盘以及在每个外）。孵育4天，并确保凝胶合同。 </li><li>吸从每个插入的内部和外部的介质。添加洗涤液（1％透析胎牛血清的HBSS）内2毫升和10毫升之外插入以定期血清洗掉。 1小时在37 ° C。 </li><li>皮肤准备重建介质： <ol><li>对于正常的黑素细胞和皮肤干细胞重建：为了使基本培养基（500毫升），混合下列试剂：490毫升角质细胞无血清培养液，1.8毫升牛垂体提取物，10毫升透析胎牛血清，10微克/ 500μL毫升SCF，562.5微升4μg/ mL的bFGF和500μL的264微克/毫升的ET - 3。中等我：100微克/毫升至100毫升的基本培养基，添加10μL的EGF。中期（二）：100毫升基本培养基加入2μL的EGF。中等三：到300毫升的基本培养基，添加720μL的氯化钙，1米2。 </li><li>对于黑色素瘤皮肤重建：要中等我（100毫升），混合下列试剂：72.5毫升的DMEM，24，F12毫升（火腿的），2迷你大号-谷氨酰胺200毫米，200 269克/毫升，200μL氢化可的松500X，200μLØ - phosphorylethanolamine 0.05米，200μL腺嘌呤90毫米，200μL孕酮2 nm的，240μL氯化钙2 1米，200μL10纳米和100μLchelexed新生小牛血清甲状腺素μLITES （3 Chelex 100克溶于100毫升血清中，搅拌1.5小时，在4 ° C，过滤消毒）。为了使中等二（100毫升），混合下列试剂：72.5毫升的DMEM，24，F12毫升（火腿的），2毫升大号-谷氨酰胺200毫米，200 269克/毫升，200，500XμLITESμL氢化可的松， 200μLØ - phosphorylethanolamine 0.05米，200μL腺嘌呤90毫米，200μL孕酮2 nm的，240μL1米氯化钙，200μL10 nm和100μL新生小牛血清甲状腺素。为了使中等三（300毫升），混合下列试剂：142.5毫升的DMEM，142.5的F12毫升（火腿的），6 200毫米大号-谷氨酰胺毫升，600μL氢化可的松269克/毫升，600，500XμLITES 600μLO型phosphorylethanolamine 0.05米，600μL腺嘌呤90毫米，600μL孕酮2 nm的，720μL氯化钙1 2米 ，600μL的10纳米和6 mL新生小牛血清甲状腺素。 </li></ol></li><li> Trypsinize人角质形成细胞从培养瓶中，用0.05％胰蛋白酶/ EDTA，中胰蛋白酶大豆胰蛋白酶抑制剂（250毫克/大号1X磷酸盐缓冲液），自旋向下，细胞沉淀重悬在4.17 × 10 6细胞/ mL重建皮肤中等一</li><li> Trypsinize人类黑色素细胞或黑色素细胞培养瓶中，用0.05％胰蛋白酶/ EDTA，胰蛋白酶抑制大豆胰蛋白酶抑制剂，降速，一个细胞沉淀重悬在0.83 × 10 6细胞/毫升在皮肤重建中型一</li><li>删除洗衣机从每个插入的内部和外部的介质。 </li><li>皮肤重建中等我（1.5毫升内和10毫升每个插入的外）。 </li><li>角质形成细胞和黑色素细胞或黑色素细胞600μL细胞悬液600μL细胞悬液，拌匀。分配由每个插入内下降到200μL混合细胞悬液下降。皮肤干细胞的重建，使用100μL只角质形成细胞悬液。 2天在37 ° C。 </li><li>吸皮肤重建中我从每个插入内和外。皮肤重建中II（2毫升内和10毫升外）。再过2天在37 ° C。 </li><li>吸皮肤由内而外重建中等II和每个插入的外，添加7.5毫升的皮肤重建中型三只外。从这一点来说，表面重建开始暴露在空气中。更改中等三隔日直到第18天。 </li><li>在第18天收获皮肤重建： <ol><li>从内部和外插入吸媒体。 </li><li>从托盘与钳插入。 </li><li>通过跟踪一个圆圈靠近边缘，用手术刀的刀片切出的重建（包括聚碳酸酯过滤器）。 </li><li>剪切重建的一半，在坚硬的表面滤波器。 </li></ol></li><li>对于石蜡切片：将一半的重建组织学之间的2个黑色的TBS活检论文的卡带和10％的福尔马林浸泡在4个多小时的整个磁带。然后放入70％乙醇的录像带，并存储在4 ° C，直到您准备过程中重建石蜡包埋。 </li><li>对于冰冻切片： <ol><li>广场的另一半重建在4 ° 50％的蔗糖为1-2小时，然后更改为2M的蔗糖，并保存在4 ° C，另外1-2个小时。 </li><li>免除成一次性底模的最佳切削温度冻结媒体技术（OCT），以便它填充船约​​50％。在OCT避免任何气泡。 </li><li>抓住边缘的重建与钳和删除重建蔗糖。广场上Kimwipes直到Kimwipes吸收的蔗糖。 </li><li>传输改建为底模在OCT上，使用产钳和抹刀。盖的重建更华侨城的顶部，直到基模是完全充分的。确保你没有任何气泡在华侨城，这将会使切削困难。 </li><li>将底模上均匀地粉碎的干冰，使华侨城完全冻结。 </li><li>裹在锡纸底模，并储存于-70 ° C，直到您准备切断与低温恒温器。 </li></ol></li><li>移植皮肤重建鼠标： <ol><li>准备用5毫升的DMEM（冻结和解冻的小鼠皮肤）50毫升猎鹰管。 </li><li>使用约6个星期，一个女的S​​CID无毛远交鼠标。 </li><li>鼠标与异氟醚（EZ麻醉系统）麻醉：最初的麻醉是4％异氟醚（鼠标移动到手术床上之前）和维持麻醉诱导室通过在程序的头锥喘息（异氟醚：1.5-2％ ）。热支持一个加热垫，以防止低温和应用无菌眼科润滑剂，以防止在麻醉过程中的眼外伤。 </li><li> 600毫升的水放入一个烧杯中，并留下一个热点板块之上，水温保持在40 - 60 ° C </li><li>复方安息香酊和酒精准备棉签清洁老鼠的皮肤。 </li><li>标记鼠标背部上的线缝合后保持相同的位置。 </li><li>切鼠标上部回用虹膜剪刀和镊子直径约1.2厘米的圆形伤口床。封面用无菌纱布海绵伤口床。把5毫升的DMEM一轮的老鼠的皮肤，然后离开在液态氮容器中，直到它完全冻结。解冻的一个热点板块之上，在热水管，直到完全解冻。重复3次。 </li><li>取下皮肤重建使用手术刀片Transwell小，非常仔细地取出膜，转移重建皮肤上的伤口床的上方（表皮朝上）。 </li><li>皮肤重建的顶部放置在老鼠的皮肤（表皮朝上）。 </li><li>设为缝合对先前标记的标记第一，第二个在底部，然后对双方缝合修复小鼠皮肤。 </li><li>嫁接后的清洁老鼠的皮肤。取出头锥，并保持直到清醒和门诊加热垫鼠标。 </li><li>放入一个新的笼子鼠标。 </li><li>皮下注射后立即手术，手术后的24小时和48小时，手术后镇痛：美洛昔康（1毫克/公斤）。 </li></ol></li></ol> 2。代表性的成果： 皮肤重建与正常黑色素细胞和皮肤干细胞 皮肤重建是在一个特殊的6孔与插入托盘的培养。真皮车厢是培养含10％胎牛血清的DMEM的前四天（图1A）。皮肤重建我是角质细胞与黑色素细胞或黑色素细胞（图1B）接种时使用的介质。表皮是暴露在空气中，在第9天，这使角质形成细胞分化（图1C）。在18天，皮肤表皮重建分层的角质细胞层组成。无差别的基底层和顺序区别层垂直方向（图1D）。污点ING的重建与黑素细胞标记S100节表明，黑色素细胞是在表皮和沟通，通过多个枝蔓扩展（图1E）角质形成细胞的基底层对齐。真皮车厢包含嵌入在I型胶原基质的成纤维细胞。敷IV型胶原表示基底膜，分离表皮从真皮（图1F）。当皮肤干细胞（与绿色荧光蛋白的慢病毒载体标记）与成纤维细胞在Ⅰ型胶原基质中，他们迁移到表皮和分化成黑素细胞（1），（图2）。在表皮角质形成细胞的多层次开发。 黑色素瘤皮肤重建 临床研究表明，黑色素瘤：常见的后天性痣，发育不良痣，RGP（径向生长阶段），黑色素瘤，VGP（垂直生长阶段）黑素瘤和转移性黑色素瘤（7）以逐步的方式进展。黑色素瘤细胞株的不同阶段的形态相似，在对方的2 - D文化（图3A - D），但是当他们在皮肤重建中，细胞的行为反映了他们体内的特点。严格控制在皮肤重建（图3E）的位置和正常黑素细胞的生长率。 RGP的原发性黑色素瘤WM35增殖主要在表皮（图3F），而VGP黑色素瘤WM793增长侵入真皮（图3G）。转移性黑色素瘤1205Lu积极深入到真皮（图3H）侵入。 <img alt="图1" src="/files/ftp_upload/2937/2937fig1.jpg" /> 图1。皮肤重建与正常黑色素细胞 ，皮肤总值的外观重建是在AC 。 A.与胶原混合成纤维细胞生长的DMEM含10％FBS和形式真皮车厢。 B.角质形成细胞和黑素细胞接种于真皮层的顶部和成长在皮肤重建介质。 C.表皮暴露于空气中，在第9天。 D. H＆E -染色的皮肤重建提出了包括垂直的表皮，面向基底层，并分层连续分化的细胞层。真皮包含嵌入在I型胶原基质的成纤维细胞。 E. S100 -积极的黑色素细胞（黑色箭头）对准多个角质形成细胞基底膜和沟通。 F. IV型胶原染色表明，分离表皮从真皮的基底膜。在DF福尔马林固定，石蜡包埋切片染色法。 <img alt="图2" src="/files/ftp_upload/2937/2937fig2.jpg" /> 图2。在皮肤的真皮干细胞重建迁移到表皮黑色素细胞分化成。第5天播种后角质形成细胞，单GFP阳性细胞（绿色）开始迁移领域。表皮仍然是一个单独的层组成。在第8天，少数细胞到达表皮，真皮​​层的接口。第10天，GFP阳性细胞紧密排列在基底膜的位置。迁移的GFP阳性细胞表达的表皮黑色素细胞HMB45标记（红色，白色箭头表示）。细胞核用DAPI染色（蓝色）。表皮是作为多层次开发。基底膜是白色虚线表示。 <img alt="图3" src="/files/ftp_upload/2937/2937fig3.jpg" /> 图3。皮肤黑色素瘤的不同阶段重建：公元。正常黑色素细胞和黑色素瘤细胞生长的2D文化。 A.从包皮的黑素细胞。 B. RGP的WM35细胞。 C. VGP WM793细胞。 D.转移性黑色素瘤1205Lu细胞。 E.正常黑色素细胞位于基底膜。 F. RGP的黑色素瘤WM35细胞生长在表皮的细胞团。 G. VGP黑色素瘤WM793细胞真皮基底膜侵入到通过。 H.转移性黑色素瘤1205Lu细胞大举入侵到真皮深。 故障排除<table border="1"><tbody><tr><td> 问题 </td><td> 故障排除 </td></tr><tr><td>胶原蛋白的混合物并没有巩固</td><td>胶原蛋白的混合物的颜色应为稻草黄色为粉红色，否则pH值是错误的，胶原蛋白可能不是凝胶。如果颜色是明亮的黄色，应增加更多的碳酸氢钠一滴一滴。 </td></tr><tr><td>胶原过早沉淀在mixuture </td><td>所有元件置于冰上，直到胶原蛋白的混合物放置到插入</td></tr><tr><td>签约胶原甚至没有（一侧比另一侧厚） </td><td>校准孵化器的架子</td></tr><tr><td>表皮是成立不到三年的角质细胞层。 </td><td>在较低的通道上使用的未分化的角质形成细胞</td></tr></tbody></table>

<ol>
	<li>Sun, T., Jackson, S., Haycock, J. W., 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=Culture+of+skin+cells+in+3D+rather+than+2D+improves+their+ability+to+survive+exposure+to+cytotoxic+agents.">Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents.</a> J Biotechnol. 122, 372-381 (2006).</li><li>Smalley, K. S., Lioni, M., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+isn't+flat:+taking+cancer+biology+to+the+next+dimension.">Life isn't flat: taking cancer biology to the next dimension.</a> In Vitro Cell Dev Biol Anim. 42, 242-247 (2006).</li><li>Sun, T., Norton, D., McKean, R. J., Haycock, J. W., Ryan, A. J., 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=Development+of+a+3D+cell+culture+system+for+investigating+cell+interactions+with+electrospun+fibers.">Development of a 3D cell culture system for investigating cell interactions with electrospun fibers.</a> Biotechnol Bioeng. 97, 1318-1328 (2007).</li><li>Kremer, M., Lang, E., Berger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypical+engineering+of+differentiated+composite-skin+equivalents+of+human+keratinocytes+in+a+collagen-GAG+matrix+(INTEGRA+Artificial+Skin)+in+a+perfusion+culture+system.">Organotypical engineering of differentiated composite-skin equivalents of human keratinocytes in a collagen-GAG matrix (INTEGRA Artificial Skin) in a perfusion culture system.</a> Langenbecks Arch Surg. 386, 357-363 (2001).</li><li>Esc&#225;mez, M. J., Garc&#237;, M., Larcher, F., Meana, A., Mu&#241;oz, E., Jorcano, J. L., Del R&#237;o, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+model+of+wound+healing+in+genetically+modified+skin-humanized+mice.">An in vivo model of wound healing in genetically modified skin-humanized mice.</a> J Invest Dermatol. 123, 1182-1191 (2004).</li><li>Li, L., Fukunaga-Kalabis, M., Yu, H., Xu, X., Kong, J., Lee, J. T., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dermal+stem+cells+differentiate+into+functional+epidermal+melanocytes.">Human dermal stem cells differentiate into functional epidermal melanocytes.</a> J Cell Sci. 123, 853-860 (2010).</li><li>Meier, F., Nesbit, M., Hsu, M. Y., Martin, B., Van Belle, P., Elder, D. E., Schaumburg-Lever, G., Garbe, C., Walz, T. M., Donatien, P., Crombleholme, T. M., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanoma+progression+in+skin+reconstructs:+biological+significance+of+bFGF.">Human melanoma progression in skin reconstructs: biological significance of bFGF.</a> Am J Pathol. 156, 193-200 (2000).</li><li>Yu, H., McDaid, R., Lee, J., Li, L., Kumar, S. M., Elder, D. E., Van Belle, P., Gimotty, P., Guerra, M., Hammond, R., Nathanson, K. L., Dalla Palma, M., Herlyn, M., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+BRAF+mutation+and+p53+inactivation+during+transformation+of+a+subpopulation+of+primary+human+melanocytes.">The role of BRAF mutation and p53 inactivation during transformation of a subpopulation of primary human melanocytes.</a> Am J Pathol. 174, 2367-2377 (2009).</li><li>Lee, J. T., Li, L., Brafford, P. A., van den Eijnden, M., Halloran, M. B., Sproesser, K., Haass, N. K., Smalley, K. S., Tsai, J., Bollag, G., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLX4032,+a+potent+inhibitor+of+the+B-Raf+V600E+oncogene,+selectively+inhibits+V600E-positive+melanomas.">PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas.</a> Pigment Cell Melanoma Res. 23, 820-827 (2010).</li><li>Tsai, J., Lee, J. T., Wang, W., Zhang, J., Cho, H., Mamo, S., Bremer, R., Gillette, S., Kong, J., Haass, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+selective+inhibitor+of+oncogenic+B-Raf+kinase+with+potent+antimelanoma+activity.">Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.</a> Proc Natl Acad Sci U S A. 105, 3041-3046 (2008).</li></ol>

没有利益冲突的声明。

我们已经描述了正常的人类黑色素细胞，皮肤干细胞和黑色素细胞生成3D的皮肤与重建。在单层培养生长时，黑色素细胞中存在类似的形态（扁平，梭形或更多的树突状），不论其原产地的临床阶段。相比之下，3D皮肤重建复述黑色素瘤细胞的阶段特定的属性。正常人体黑色素细胞驻留在单细胞的表皮和真皮层之间的基底膜。 RGP的原发性黑色素瘤细胞沿基底膜成长为小群，而更积极的VGP黑色素瘤细胞突破基底膜进入真皮的大型集群成长。转移性黑色素瘤细胞生长的各个方向，并侵入到真皮单细胞或集群深。在此模型上，通过测量深度的入侵和扩散程度，可以进行定量分析。除了 ​​正常的和恶性黑色素细胞的特性，利用该模型，我们可以直接诱发的皮肤干细胞分化为成熟黑色素细胞，表皮基底层，并建立与角质形成细胞通过上调E - cadherin的善意沟通（ 6）。 使用病毒载体，我们能够激活或灭活基因的功能，一个更好地了解由不同的细胞类型表达的基因在皮肤的动态和功能意义重构，它有望成为一个有效的模型来研究不仅黑色素细胞的转化机制，而且进展的黑色素瘤（8）。通过嫁接皮肤重建免疫缺陷动物的长期观察细胞表型已成为可能。这种人类皮肤鼠嵌合体是一个很好的研究工具，研究melanomagenesis和黑色素瘤的转移。 皮肤重建也可以是一个有用的平台，药物评估。往往有许多药物消灭癌细胞在二维培养条件的影响不大，在实验和临床应用。看出，在体内肿瘤，黑色素瘤细胞的三维文化往往是2D文化细胞的药物具有耐药性，表明微环境调节信号转导通路在黑色素瘤。成功的药物发现，3D皮肤重建模型是一个理想的临床前的工具来预测化合物在体内（9,10）的影响。 总之，皮肤重建模型桥在体外和体内研究之间的差距。他们会导致更好地了解哪些基因参与改造和干细胞如何促进这一转变。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> 10X EMEM </td><td>生物惠特克</td><td> 12 - 684F </td></tr><tr><td> L -谷氨酰胺</td><td> Gibco公司</td><td> 25030-081 </td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH - 30071.02 </td></tr><tr><td>牛腱酸提取的胶原</td><td>器官</td><td> 200/50 </td></tr><tr><td>组织培养6托盘插入</td><td>器官</td><td> 9285 </td></tr><tr><td>角质细胞血清培养基</td><td> Gibco公司</td><td> 17005042 </td></tr><tr><td>透析胎儿小牛血清</td><td> Hyclone公司</td><td> SH - 30079.03 </td></tr><tr><td>重组人干细胞因子</td><td>杰拉德工业</td><td> RDI - 307 - 255X </td></tr><tr><td>基本的FGF </td><td>杰拉德工业</td><td> 30R - AF015 </td></tr><tr><td>内皮素- 3，人类</td><td>美国多肽有限公司</td><td> 88-5-10 </td></tr><tr><td>氯化钙</td><td>西格玛</td><td> C - 7902 </td></tr><tr><td> DMEM培养液</td><td> JRH Biosciences公司</td><td> 56430 - 10L </td></tr><tr><td> F12（火腿的） </td><td> Gibco公司</td><td> 11765-54 </td></tr><tr><td>氢化可的松</td><td>西格玛</td><td> H - 0888 </td></tr><tr><td>胰岛素，转铁蛋白，硒，乙醇胺（ITES） </td><td> BioWhittaker </td><td> 17839Z </td></tr><tr><td> O型Phosphorylethanolamine </td><td>西格玛</td><td> P0503 </td></tr><tr><td>腺嘌呤</td><td>西格玛</td><td> A9795 </td></tr><tr><td>孕酮</td><td>西格玛</td><td> P - 8783 </td></tr><tr><td>甲状腺素</td><td>西格玛</td><td> T - 5516 </td></tr><tr><td>新生小牛血清</td><td> Hyclone公司</td><td> SH3011802 </td></tr><tr><td> 10％缓冲福尔马林</td><td> Surgipath </td><td> 00600 </td></tr><tr><td>最佳切削温度冻结媒体（华侨城） </td><td>樱花</td><td> 4583 </td></tr><tr><td>多磁带</td><td> Surgipath </td><td> 02293 - BX </td></tr><tr><td> TBS的活检论文</td><td>三角生物医学科学</td><td> BP - B </td></tr><tr><td> TBS的活检论文</td><td>三角生物医学科学</td><td> BP - B </td></tr><tr><td>一次性基地模具</td><td>费舍尔</td><td> 15 - 182 - 501C </td></tr><tr><td>复方安息香酊</td><td>专业耗材公司， </td><td> S42450 </td></tr><tr><td>酒精准备棉签</td><td> CVS的药店</td><td></td></tr><tr><td>丝绸黑色编织5-0 </td><td>爱惜康</td><td> 682G </td></tr><tr><td>纱布海绵（无菌） </td><td> CVS的药店</td><td></td></tr><tr><td>镊子</td><td> Roboz </td><td> RS5070 </td></tr><tr><td>虹膜剪刀</td><td> Roboz </td><td> RS5913 </td></tr><tr><td>手术刀</td><td>羽毛</td><td> 2976 </td></tr><tr><td>简易麻醉</td><td> Euthanex公司</td><td></td></tr><tr><td> SCID的无毛远交鼠标</td><td>查尔斯河</td><td></td></tr></tbody></table>

the three-dimensional human skin reconstruct model: a tool to study normal  skin and melanoma progression

在体外实验皮肤生物学研究中，大部分已经完成2维（2D）的单一种植，而越来越多的证据表明细胞有不同的行为时，他们都生长在一个三维的细胞外基质，并与其他细胞相互作用（1-5） 。小鼠模型已被广泛用来研究在体内的组织形态发生。然而，老鼠和人类的皮肤细胞结构和生理，这使得它难以推断小鼠研究人类的显着性差异。由于在老鼠皮肤中的黑色素细胞在毛囊大多是本地化，他们有不同的生物学特性，从人类，它主要定位于表皮的基底层。最近开发三维人体皮肤重建模型，使实地调查不同类型的细胞之间的细胞 - 基质和细胞间的相互作用。包括一个嵌入式成纤维细​​胞在Ⅰ型胶原基质，“表皮”，这是分层，差异化的角质形成细胞和基底膜功能，分离表皮从真皮层组成的“真皮”的重建。胶原蛋白提供了脚手架，营养输送，和细胞与细胞之间的相互作用的潜力。皮肤三维模型纳入复述黑色素细胞在人体皮肤的黑素细胞的动态平衡和黑色素瘤进展的自然特征。 在体内 ，重建皮肤中的黑色素细胞在基底膜与基底层角质穿插进行本地化。黑色素瘤细胞表现出相同的特征，反映原发肿瘤阶段（rgp，VGP和转移性黑素瘤细胞）在体内。近日，皮肤干细胞已经确定在人类真皮（6）。这些多有力的干细胞可以迁移到表皮分化为黑素细胞。

Watch this Scientific Journal Video about The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression at JoVE.com

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

在这份报告中，我们描述了皮肤三维重建模型模仿人体皮肤结构和组成。使用皮肤重建模型被查处的黑素细胞的生理，黑色素瘤的进展和皮肤干细胞的命运。该模型也可作为临床前药物评估工具。

三维人体皮肤重建模型：一种工具来研究正常皮肤恶性黑色素瘤进展

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

the-three-dimensional-human-skin-reconstruct-model-tool-to-study

Research

JoVE Journal

Bioengineering

49.2K 次观看.  The Wistar Institute. 在这份报告中，我们描述了皮肤三维重建模型模仿人体皮肤结构和组成。使用皮肤重建模型被查处的黑素细胞的生理，黑色素瘤的进展和皮肤干细胞的命运。该模型也可作为临床前药物评估工具。

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

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

科研

生物工程

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

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

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

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

研究模型声乐折叠聚合物引起的三维流量分离

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

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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

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

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

三维全层皮肤的生成等效和自动伤人

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

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., ...

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 ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

大规模培育三维重建人类表皮

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

自组装血管化人体皮肤等价物的生成

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...