This work has been supported by the German Cancer Research Center (DKFZ), the EU-consortium &#8220;Tolerage&#8221;, the Deutsche Forschungsgemeinschaft (SFB 938) and the Landesstiftung Baden-W&#252;rttemberg.

这项研究已批准Regierungspräsidium卡尔斯鲁厄的伦理委员会。所有动物饲养无特定病原体条件下在德国癌症研究中心（DKFZ）下。对于所有的培养实验小鼠幼仔范围从1岁到7天被使用。 从胸腺1.隔离mTECs的注:如无菌条件下先前1中记载有一些修改如下进行以下的消化步骤。 <ol><li>斩首的乳鼠，取出胸腺。放置在冰上thymi在含有RPMI 1640培养基（含5％FCS）培养皿。 </li><li>切thymi成细，小块并置于圆底管〜5-30毫升RPMI培养基和轻轻搅拌用磁铁在室温10分钟。 </li><li>此后，滗主要含有胸腺细胞的上清液并与一个圆collagena的消化组织顺序的剩余本身IV型（0.2毫克/毫升和57 U / ml的最终concentartion）15分钟每个在37℃，随后通过胶原酶/分散酶（0.2毫克/毫升和1.2 U / ml的终浓度）为每25分钟，在37°下在水浴磁力搅拌下，直到thymi被完全消化。使用1ml的酶每两到三个thymi。 </li><li>搅动组织每隔7-10分钟用巴氏吸管。池胶原酶/分散酶组分，并通过一个70微米的纱布过滤。 </li><li>通过磁性细胞分选（MACS）充实mTECs。通过磁性细胞如先前11描述的，和在图1所示的排序执行mTECs的纯化。 注:对于mTECs的磁分选，我们使用了下列抗体:抗CD80-PE（16-10A1，使用以1:100稀释）和抗EpCAM-生物（G8.8，使用以1:100稀释）12。使用MACS未成熟和成熟mTECs被定义为:CD45 - EpCAM的+ CD80 -和CD45 - CD80 + respectiv伊利。 </li><li> MACS纯化（未成熟mTECs的纯度= 83.1±6.3％和成熟mTECs = 79.23±3.42％）后，种子mTECs到器官文化，如下所述（第2.3节）。 </li><li>可替换地，排序mTECs通过FACS（使用100微米的喷嘴）的CD45磁珠耗尽后使用下列抗体:抗CD45-PerCP（30-F11，使用以1:100稀释），抗Ly51-FITC（6C3，使用在1 :100稀释），抗EpCAM-Alexa647（G8.8，使用以1:500稀释），并在1抗CD80-PE（16-10A1，用途:100稀释度）。排除使用碘化丙啶（1:5000）死细胞（4天）。使用流式细胞仪未成熟和成熟mTECs被定义为:PI - CD45 - Ly51 - EpCAM的+ CD80 -和PI - CD45 - Ly51 - EpCAM的+ CD80 +，分别为。 </li></ol> 2. 3D器官联合培养（OTCS） 注:3D-真皮构造的器官培养keratinocyt的西文被如先前9,13所述制备。在所有步骤的细胞在37℃和5％的CO 2。使用mTECs的OTCS制备稍作修改如下。 <ol><li>人成纤维细胞的制备 注意:人皮肤成纤维细胞从脱epidermised真皮外植体培养物如先前9中所述获得。 <ol><li>简言之，切开人体皮肤（〜5cm长）的带和治疗与嗜热菌蛋白酶（0.5mg / ml的盐水中，用10毫摩尔的Hepes pH7.4）中O / N在4℃。 </li><li>其后，从利用镊子真皮分离表皮。 </li><li>细切真皮成小块，放置在10cm的培养皿，并允许下一个无菌气流中干燥1-2小时。用DMEM含20％FBS定期补充外植体。 </li><li>分裂出生长的成纤维细胞时汇合（通常大约3周）用0.1％胰蛋​​白酶确保植继续坚持该板用于进一步CON组secutive轮生长的。 </li><li>扩大从同一外植体成纤维细胞为最多3次在含10％FBS和低温保存它们14。使用相同批次的成纤维细胞的每个实验系列。 注:成纤维细胞不照射为集合式真皮等同物。 </li></ol></li><li>脚手架的制备<ol><li>削减0.4-0.6毫米厚的粘胶，非织造纤维材料（在支持Excel表单产品的详细信息）以及成圈划定，用锋利的直径11mm的金属冲床恰好适合12良好的滤芯。然后将其放入12井滤芯（聚酯毛细管孔膜，3微米孔径）作为支架。放置的完整的过滤器安装到无菌的12孔板中。 </li><li>准备使用纤维蛋白胶，试剂盒进行手术，包括纤维蛋白原和凝血酶的结合的血纤维蛋白凝胶。预稀释fibrinogen-以及试剂盒的凝血酶组分至8毫克/毫升和10单位/毫升。对于一个12孔板的单个孔的步骤如下。 <ol><li>稀释100μl的纤维蛋白原（8毫克/毫升）用100μl磷酸盐缓冲盐水（PBS）不含Ca 2+和Mg 2+，pH值7.0。稀释100μl的凝血酶（10单位/ ml）与含有270000成纤维细胞加入100μlFCS中。 </li><li>分配200μl的含有成纤维细胞的混合到该支架的凝血酶，向其中加入200微升血纤维蛋白原（1:1的混合物），产生的2毫克/毫升的2.5单位/ ml凝血酶和纤维蛋白原的终浓度。拌匀，均匀地分布在支架轻柔吹打（第1天）的整个区域。 注:30分钟，在37℃后凝块包封的成纤维细胞会形成，尽显无遗支架的内部空间，并形成一个光滑的上表面。 </li></ol></li></ol></li><li>共培养mTECs <ol><li>用于预培养，浸没在器官培养含10％FBS，50μg/ ml的L-抗坏血酸和1纳克/毫升的培养基改变TGF-β1的隔日4-5天。 </li><li>上MTEC播种当天，由RFAD介质更换介质（1:1的DMEM + DMEM / F12）有10％FBS，10 -10 M霍乱毒素，0.4毫克/ ml氢化可的松，50μg/ ml的L-抗坏血酸， RANK配体（0.1微克/毫升）和500单位/ ml抑肽酶，从而防止早熟纤溶由成纤维细胞分泌的丝氨酸蛋白酶。 </li><li> 7-8小时后，建立共培养播种250000 mTECs（无论是完整的mTECs，或CD80 Io和 CD80 喜子集），在每孔100μl的成纤维细胞支架的顶端的体积。算使用Neubauer室（4天）的细胞。 注:在任何时候胸腺器官3D文化是浸没在媒体不像皮肤OTCS这是空气解除。 </li><li>经过24小时培养，提供如上述（步骤2.3.2）与介质的培养物（总培养基交换）现在containiNG减少抑肽酶，250单位/毫升（5日）的金额。 </li><li>为了评估mTECs的增殖活性，终止培养物之前的EdU（6.7μM/ ml时， 即，10μM/孔）添加到OTCS 4小时。在每一种文化标本或流两节这些EDU成像套件结合共染色角蛋白14的描述确定mTECs的增殖指数，方法是计数K14 + +的EdU细胞进行的OTC冷冻切片染色术（EDU流式细胞术，第代替Ly51-FITC 1.7，但是使用的CDR1-PB 15在1:100稀释和2.3.6.4）。 </li><li>下列4-7天共培养的，终止OTCS和方法RNA分离，冷冻切片或FACS分析（8-11天）。 <ol><li>使用镊子，分离的支架/真皮当量从井刀片的过滤终止培养。 </li><li>对于冷冻切片，嵌入整个OTC华侨城化合物，冻结李冷冻切片之前，英镑氮蒸气。准备使用低温恒温器和存储5-7微米厚的非处方药部分在-20℃下直到使用。对于免疫组化的冷冻切片OTCS使用抗角蛋白14（AF64，使用以1:1000稀释）和抗波形蛋白（GP53，使用以1:100稀释）的抗体。执行使用各自的二级抗体的间接免疫组织化学染色。 </li><li>对于RNA分离，加入1 ml变性溶液（含有苯酚和异硫氰酸胍）在2 ml无RNase管的螺旋盖。切断整个场外成用手术刀片，并添加到含有变性溶液的管中。机械地以6.0的速度切丝与FastPrep仪OTCS两次30秒，放在冰上间为2分钟（样品可以储存在-80℃这点之后）。如果在-80℃下冻结，解冻在冰上。离心管在11,500-13,000转速在4℃下10分钟。将上清液转移到新的管中，孵育5分钟，在RT和f使用酸性硫氰酸胍 - 酚 - 氯仿提取如制造商所述ollow RNA分离协议。 </li><li>用于FACS分析，除去该脚手架和分离的膜与纤维蛋白/纤维细胞/ MTEC凝胶。切细的凝胶用手术刀和消化它在在FACS管中〜20分钟，或直至完全在水浴磁力搅拌下用2ml胶原酶/分散酶的消化在37℃。搅拌用巴斯德吸管每5分钟的酶溶液。完全消化后，通过过滤70微米的过滤器细胞悬浮液;染色使用如1.7所描述并通过流式细胞术分析抗体的单细胞悬浮液。 </li></ol></li></ol></li></ol>

<ol>
	<li>Derbinski, J., Schulte, A., Kyewski, B., Klein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promiscuous+gene+expression+in+medullary+thymic+epithelial+cells+mirrors+the+peripheral+self.">Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self.</a> Nat Immunol. 2, 1032-1039 (2001).</li><li>Kyewski, B., Klein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+central+role+for+central+tolerance.">A central role for central tolerance.</a> Annual review of immunology. 24, 571-606 (2006).</li><li>Bonfanti, 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=Microenvironmental+reprogramming+of+thymic+epithelial+cells+to+skin+multipotent+stem+cells.">Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells.</a> Nature. 466, 978-982 (2010).</li><li>Kont, V., 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=Modulation+of+Aire+regulates+the+expression+of+tissue-restricted+antigens.">Modulation of Aire regulates the expression of tissue-restricted antigens.</a> Molecular Immunology. 45, 25-33 (2008).</li><li>Mohtashami, M., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+architecture+of+the+thymus+is+required+to+maintain+delta-like+expression+necessary+for+inducing+T+cell+development.">Three-dimensional architecture of the thymus is required to maintain delta-like expression necessary for inducing T cell development.</a> J Immunol. 176, 730-734 (2006).</li><li>Palumbo, M. O., Levi, D., Chentoufi, A. A., Polychronakos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+proinsulin-producing+medullary+thymic+epithelial+cell+clones.">Isolation and characterization of proinsulin-producing medullary thymic epithelial cell clones.</a> Diabetes. 55, 2595-2601 (2006).</li><li>White, A., Jenkinson, E., Anderson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaggregate+thymus+cultures.">Reaggregate thymus cultures.</a> Journal of visualized experiments : JoVE. , (2008).</li><li>Stark, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+homeostasis+in+long-term+scaffold-enforced+skin+equivalents.">Epidermal homeostasis in long-term scaffold-enforced skin equivalents.</a> J Investig Dermatol Symp Proc. 11, 93-105 (2006).</li><li>Boehnke, K., 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=Effects+of+fibroblasts+and+microenvironment+on+epidermal+regeneration+and+tissue+function+in+long-term+skin+equivalents.">Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents.</a> Eur J Cell Biol. 86, 731-746 (2007).</li><li>Pinto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+organotypic+coculture+model+supporting+proliferation+and+differentiation+of+medullary+thymic+epithelial+cells+and+promiscuous+gene+expression.">An organotypic coculture model supporting proliferation and differentiation of medullary thymic epithelial cells and promiscuous gene expression.</a> J Immunol. 190, 1085-1093 (2013).</li><li>Gabler, J., Arnold, J., Kyewski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promiscuous+gene+expression+and+the+developmental+dynamics+of+medullary+thymic+epithelial+cells.">Promiscuous gene expression and the developmental dynamics of medullary thymic epithelial cells.</a> Eur J Immunol. 37, 3363-3372 (2007).</li><li>Farr, A., Nelson, A., Truex, J., Hosier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+heterogeneity+in+the+murine+thymus:+a+cell+surface+glycoprotein+expressed+by+subcapsular+and+medullary+epithelium.">Epithelial heterogeneity in the murine thymus: a cell surface glycoprotein expressed by subcapsular and medullary epithelium.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 39, 645-653 (1991).</li><li>Stark, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Authentic+fibroblast+matrix+in+dermal+equivalents+normalises+epidermal+histogenesis+and+dermoepidermal+junction+in+organotypic+co-culture.">Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermoepidermal junction in organotypic co-culture.</a> Eur J Cell Biol. 83, 631-645 (2004).</li><li>Schoop, V. M., Mirancea, N., Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+organization+and+differentiation+of+HaCaT+keratinocytes+in+organotypic+coculture+with+human+dermal+fibroblasts.">Epidermal organization and differentiation of HaCaT keratinocytes in organotypic coculture with human dermal fibroblasts.</a> J Invest Dermatol. 112, 343-353 (1999).</li><li>Rouse, R. V., Bolin, L. M., Bender, J. R., Kyewski, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+reactive+with+subsets+of+mouse+and+human+thymic+epithelial+cells.">Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells.</a> The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 36, 1511-1517 (1988).</li></ol>

作者宣称没有任何财务或商业利益冲突。

沿着RTOCs，三维OTCS已经于TEC分化和PGE维护/感应（ 表1）相对于其它（ⅰ）"简化3D培养&#39;使用的术语是由远优于-的成纤维细胞单独无脚手架; （ⅱ）二维系统使用-成纤维细胞/饲养细胞共培养的TECs 10，（ⅲ）3T3-J2细胞，其中的TEC克隆发展，但PGE丢失，（ⅳ）基质胶或（v）ECM组分（未发表数据）。 PGE下保持7天，在3D OTCS 4天为最佳时间点之后，PGE开始下降。其他形态的TEC特征被?...

显影胸腺细胞约占98％，胸腺，而其余的2％由多种细胞，它们共同构成了胸腺基质（ 即，上皮细胞，树突细胞，巨噬细胞，B细胞，成纤维细胞，内皮细胞）。外皮质上皮细胞（cTECs）促亲T细胞的移民从骨髓，T细胞谱系的诱导多能前T细胞和阳性选择自我的MHC限制性不成熟的胸腺细胞。所述内髓胸腺上皮细胞（mTECs）参与耐受诱导那些胸腺细胞具有高亲和性TCR为自身肽/ MHC复合物由两种诱导阴性选择或它们的偏差成T调节细胞谱系。在中央耐受诱导的上下文中，mTECs是因为它们表达了广泛的组织限制性自身抗原（的TRAs）因而镜像的周自谱是唯一的。这种现象被称为混杂基因表达（PGE）1,2。 关于这个迷人的细胞类型的最新研究依赖于体外分离的细胞，如各种短期二维培养系统总是导致PGE的损失和关键调节分子如MHC II类，FoxN1和艾尔内的第2天3-6 。它仍然不清楚然而，这尤其是元器件和胸腺的完整3D小梁的特点在2D模式不翼而飞。的再聚集胸腺器官培养（RTOC）一直迄今唯一的三维系统，使T细胞发育的研究中，一方面，与基质细胞生物学，另一方面，在一个完整的胸腺微环境7。然而，RTOCs有一定的局限性， 即，它们已经含有细胞的复杂混合物，需要胚胎基质细胞的输入和忍受的5到10天的最大培养期。 还原的体外培养体系的缺乏阻碍的研究T细胞发育和胸腺器官几个方面PGE的不仅仅是分子调控及其对mTECs的发育生物学关系。 由于紧密关联的皮肤和胸腺上皮细胞的结构化组织，我们选择了已被开发的最初仿效体外角质细胞的分化，从而创造一个真皮当量的三维器官培养（OTC）系统。场外交易系统由覆盖与被困在纤维蛋白胶，其上的角质形成细胞接种8,9真皮成纤维细胞惰性支架矩阵。在这里，我们代替角质形成细胞纯化mTECs。在保持这一模式的基本特征，我们优化的某些参数。 在通过场外交易模式mTECs增殖，进行终末分化和维护MTEC身份和铂族元素， 在体内 mTECs的发展密切相关，从而模仿<sup&gt; 10。本技术说明提供了一个详细的协议，允许逐步建立胸腺OTCS的。

<table><tbody><tr><td>Pregnant C57BL/6 mice&amp;nbsp;</td><td>Charles River WIGA</td><td></td><td></td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MS columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>CD45 Microbeads, mouse</td><td>Miltenyi Biotec</td><td>130-052-301</td><td></td></tr><tr><td>Anti-PE Microbeads</td><td>Miltenyi Biotec</td><td>130-048-801</td><td></td></tr><tr><td>Streptavidin Microbeads</td><td>Miltenyi Biotec</td><td>130-048-101</td><td></td></tr><tr><td>EpCAM (G8.8 -Alexa 647 and -biotin)</td><td>Ref. 12</td><td></td><td></td></tr><tr><td>CD80-PE antibody</td><td>BD Pharmingen</td><td>553769</td><td></td></tr><tr><td>CD45-PerCP antibody</td><td>BD Pharmingen</td><td>557235</td><td></td></tr><tr><td>Ly51-FITC antibody</td><td>BD Pharmingen</td><td>553160</td><td></td></tr><tr><td>CDR1-Pacific Blue</td><td>Ref. 15</td><td></td><td></td></tr><tr><td>Keratin 14 antibody</td><td>Covance</td><td>PRB-155P</td><td></td></tr><tr><td>Vimentin antibody</td><td>Progen</td><td>GP58</td><td></td></tr><tr><td>Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>111-165-003</td><td></td></tr><tr><td>Alexa 488-conjugated AffiniPure F(ab&#039;)2 Fragment Goat anti-Guinea Pig IgG (H+L)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>106-546-003</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Molecular Probes (Invitrogen GmbH)</td><td>A-11008</td><td></td></tr><tr><td>Click-iT EdU Alexa Fluor 594 Imaging Kit</td><td>Invitrogen</td><td>C10339</td><td></td></tr><tr><td>Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit</td><td>Invitrogen</td><td>C10425</td><td></td></tr><tr><td>12-well filter inserts (thincerts)</td><td>Greiner bio-one</td><td>657631</td><td></td></tr><tr><td>12-well plate</td><td>Greiner</td><td>665180-01</td><td></td></tr><tr><td>Jettex 2005/45</td><td>ORSA, Giorla Minore, Italy</td><td></td><td></td></tr><tr><td>Fibrinogen TISSUECOL-Kit Immuno</td><td>Baxter</td><td></td><td></td></tr><tr><td>Thrombin TISSUECOL-Kit Immuno</td><td>Baxter</td><td></td><td></td></tr><tr><td>PBS</td><td>Serva</td><td>47302.03</td><td></td></tr><tr><td>DMEM</td><td>Lonza</td><td>BE12-604F</td><td></td></tr><tr><td>DMEM/F12</td><td>Lonza</td><td>BE12-719F</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-049&amp;nbsp;</td><td></td></tr><tr><td>FBS Gold</td><td>GE Healthcare</td><td>A11-151</td><td></td></tr><tr><td>Aprotinin (Trasylol)</td><td>Bayer</td><td>4032037</td><td></td></tr><tr><td>Cholera toxin</td><td>Biomol</td><td>G117</td><td></td></tr><tr><td>Hydrocortisone</td><td>Seromed (Biochrom)</td><td>K3520</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>A4034</td><td></td></tr><tr><td>TGF-&amp;szlig;1</td><td>Invitrogen</td><td>PHG9214</td><td></td></tr><tr><td>RANKL</td><td>R&amp;amp;D systems</td><td>462-TR-010</td><td></td></tr><tr><td>Thermolysin</td><td>Sigma Aldrich</td><td>&amp;nbsp;T-7902</td><td></td></tr><tr><td>OCT Compound</td><td>TissueTek</td><td>4583</td><td></td></tr><tr><td>Trizol (aka. Denaturing solution - Acid guanidinium thiocyanate-phenol-chloroform extraction)</td><td>Invitrogen</td><td>10296028</td><td></td></tr><tr><td>FastPrep FP120</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Collagenase Type IV&amp;nbsp;</td><td>CellSystems</td><td>LS004189</td><td>0.2 mg/ml and 57U/ml final conc.</td></tr><tr><td>Neutrale Protease (Dispase)</td><td>CellSystems</td><td>LS002104</td><td>0.2 mg/ml and 1.2U/ml final conc.</td></tr><tr><td>DNase I&amp;nbsp;</td><td>Roche</td><td>11 284 932 001</td><td>25 &amp;micro;g/ml final conc.</td></tr></tbody></table>

3d organotypic co-culture model supporting medullary thymic epithelial cell proliferation, differentiation and promiscuous gene expression

帧内胸腺T细胞的发展需要一个复杂的三维网状组织的各种基质细胞， 即非T细胞组成。胸腺遍历这个支架在一个高度协调的时间和空间秩序，同时顺序通过强制性的检查点， 即 T细胞谱系的承诺，其次是T细胞受体库的生成和选择之前，其出口到周边。形成该支架的两个主要居民小区类型是皮质（cTECs）和髓胸腺上皮细胞（mTECs）。 mTECs的一个关键特征是众多组织限制性抗原所谓混杂的表达。这些组织限制性抗原呈现，以直接或间接的不成熟的胸腺细胞通过mTECs或胸腺树突状细胞，分别导致自身耐受。 合适的体外模型模拟cTECs和mTECs的发展途径和功能在当前LAC国王。这种缺乏足够的实验模型已例如阻碍混杂基因的表达，这仍然​​知之甚少在细胞和分子水平上的分析。我们适应了3D器官联合培养模式，培养体外分离mTECs。最初设计该模型培养的角质形成细胞在这样的方式来产生皮肤等效体外 。 3D模型保存MTEC生物学重点功能特点:（I）的增殖和CD80 LO，艾尔阴性终末分化成CD80 喜 ，艾尔阳性mTECs，（二）反映RANKL，并FoxN1（三）持续表达， AIRE和CD80 喜 mTECs组织限制性基因。

我们采用了3D器官联合培养模型（3D OTC）已被最初开发的角质9 体外长期培养。 MACS富集mTECs（见MACS富集方案图1）接种到含有纤维蛋白胶包裹和成纤维细胞的支架。成纤维细胞提供必要的细胞外基质（ECM） 体外支承mTECs。 MTECs培养在OTCS为4-14天在RANKL的存在下，在浸没培养不像角化细胞，这是空气暴露模仿它们在体内环境（参见场外的建立方案图2）。<...

Watch this Scientific Journal Video about 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression at JoVE.com

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

3d-organotypic-co-culture-model-supporting-medullary-thymic

Research

JoVE Journal

Developmental Biology

10.7K 次观看.  German Cancer Research Center (DKFZ). Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression. 

视频: 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

科研

癌症研究

Reaggregate Thymus Cultures

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

In this video the preparation of 2-dGuo-treated reaggregate thymus cultures is demonstrated.

Reaggregate胸腺文化

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and ...

生物学

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 dGuo治疗胸腺器官培养的制备

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

生物工程

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

基于细胞基和细胞外矩阵的Murine睾丸器官生成

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

发育生物学

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

制备及胸腺器官切片文化中的应用

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

免疫与感染

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

<meta charset="utf8"></head><body>使用 iPSC 来源的类器官重建胸腺微环境

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

<meta charset="utf8"></head><body>作者聚焦：利用人胸腺类器官推进胸腺上皮细胞和 T 细胞研究

Here, we describe a protocol for the formation of human iPSC-derived thymus organoids grown in 3D fibrin hydrogels aiming to support thymic epithelial cell (TEC) maturation and prolonged maintenance, as well as thymopoiesis in vitro.

在三维纤维蛋白水凝胶中形成人胸腺类器官

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

摘要

探索更多视频

此视频中的章节

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

Reaggregate胸腺文化

2 dGuo治疗胸腺器官培养的制备

工程三维上皮组织细胞外基质中嵌入

基于细胞基和细胞外矩阵的Murine睾丸器官生成

制备及胸腺器官切片文化中的应用

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

在三维纤维蛋白水凝胶中形成人胸腺类器官

在三维纤维蛋白水凝胶中形成人胸腺类器官

在三维纤维蛋白水凝胶中形成人胸腺类器官