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
Summary
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.
Abstract
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。
Introduction
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> 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。
Protocol
Representative Results
Discussion
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'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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work has been supported by the German Cancer Research Center (DKFZ), the EU-consortium “Tolerage”, the Deutsche Forschungsgemeinschaft (SFB 938) and the Landesstiftung Baden-Württemberg.
Materials
| Pregnant C57BL/6 mice | Charles River WIGA | ||
| LS columns | Miltenyi Biotec | 130-042-401 | |
| MS columns | Miltenyi Biotec | 130-042-201 | |
| CD45 Microbeads, mouse | Miltenyi Biotec | 130-052-301 | |
| Anti-PE Microbeads | Miltenyi Biotec | 130-048-801 | |
| Streptavidin Microbeads | Miltenyi Biotec | 130-048-101 | |
| EpCAM (G8.8 -Alexa 647 and -biotin) | Ref. 12 | ||
| CD80-PE antibody | BD Pharmingen | 553769 | |
| CD45-PerCP antibody | BD Pharmingen | 557235 | |
| Ly51-FITC antibody | BD Pharmingen | 553160 | |
| CDR1-Pacific Blue | Ref. 15 | ||
| Keratin 14 antibody | Covance | PRB-155P | |
| Vimentin antibody | Progen | GP58 | |
| Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 111-165-003 | |
| Alexa 488-conjugated AffiniPure F(ab')2 Fragment Goat anti-Guinea Pig IgG (H+L) | Jackson ImmunoResearch | 106-546-003 | |
| Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate | Molecular Probes (Invitrogen GmbH) | A-11008 | |
| Click-iT EdU Alexa Fluor 594 Imaging Kit | Invitrogen | C10339 | |
| Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit | Invitrogen | C10425 | |
| 12-well filter inserts (thincerts) | Greiner bio-one | 657631 | |
| 12-well plate | Greiner | 665180-01 | |
| Jettex 2005/45 | ORSA, Giorla Minore, Italy | ||
| Fibrinogen TISSUECOL-Kit Immuno | Baxter | ||
| Thrombin TISSUECOL-Kit Immuno | Baxter | ||
| PBS | Serva | 47302.03 | |
| DMEM | Lonza | BE12-604F | |
| DMEM/F12 | Lonza | BE12-719F | |
| HEPES | Gibco | 15630-049 | |
| FBS Gold | GE Healthcare | A11-151 | |
| Aprotinin (Trasylol) | Bayer | 4032037 | |
| Cholera toxin | Biomol | G117 | |
| Hydrocortisone | Seromed (Biochrom) | K3520 | |
| L-ascorbic acid | Sigma | A4034 | |
| TGF-ß1 | Invitrogen | PHG9214 | |
| RANKL | R&D systems | 462-TR-010 | |
| Thermolysin | Sigma Aldrich | T-7902 | |
| OCT Compound | TissueTek | 4583 | |
| Trizol (aka. Denaturing solution – Acid guanidinium thiocyanate-phenol-chloroform extraction) | Invitrogen | 10296028 | |
| FastPrep FP120 | Thermo Scientific | ||
| Collagenase Type IV | CellSystems | LS004189 | 0.2 mg/ml and 57U/ml final conc. |
| Neutrale Protease (Dispase) | CellSystems | LS002104 | 0.2 mg/ml and 1.2U/ml final conc. |
| DNase I | Roche | 11 284 932 001 | 25 µg/ml final conc. |
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