Name: preparation and applications of organotypic thymic slice cultures
Uploaded: 2016-08-06T15:00:00.000+00:00
Duration: 10 min 10 s
Description: Watch this Scientific Journal Video about Preparation and Applications of Organotypic Thymic Slice Cultures at JoVE.com

We would like to thank Marilaine Fournier for her comments on the manuscript and Jos&#233;e Tessier for technical assistance. C57BL/6-Tg (OT-I)-RAG1&lt;tmMom&gt; #4175 were obtained through the NIAID Exchange Program, NIH. Support for this research is provided by a grant from the SickKids Foundation and CIHR-IHDCYN (NI15-002), an operating grant from the CIHR-III (MOP-142254), and start-up funds from the FRQS (&#201;tablissement de jeunes chercheurs) and H&#244;pital Maisonneuve-Rosemont Foundation to HJM. HJM is a junior 1 scholar of the FRQS, a CIHR New Investigator (MSH-141967), and a Cole Foundation Early Career Transition award recipient.

HOPITAL迈松内夫 - 罗斯芒特 - 适用于所有的动物研究协议是由动物护理委员会在该中心德RECHERCHE批准。 1.收获小鼠胸腺胸腺片的制备及单细胞悬浮液<ol><li>安乐死用CO 2鼠标颈椎脱位。 </li><li>在层流罩，针鼠标腹侧到夹层板。喷用70％乙醇鼠标。用纱布涂抹，以防止乙醇进入胸腔和破坏组织取出多余的酒精。 </li><li>解除在用一对镊子的胸骨的底部的皮肤，使通过皮肤切口。向上延伸的皮肤切口到每个前肢。 </li><li>使胸骨到膜片从肋骨分离的基础上额外削减。然后切向锁骨肋骨的两侧。翻转左肋过朝头部露出胸腔。的两个叶胸腺趴在心脏的顶部。 </li><li>使用镊子，显微解剖剪除去周围的胸腺裂片结缔组织和/或尖锐弯曲的剪刀。使用一对精尖弯钳从下方或通过剩余的结缔组织解除个别叶。 注意:不要直接掌握胸腺。 </li><li>放置在胸腺叶含PBS 15毫升的锥形管，直到需要预留冰上。 </li></ol> 2.琼脂糖嵌入和胸腺叶Vibratome切片<ol><li>制备4％的琼脂糖溶液为通过将2 G低低熔点琼脂糖在50ml无菌PBS中并在低设置微波直到琼脂糖完全溶解嵌入。覆盖在在55℃的水浴中铝箔和地点烧瓶直到准备使用。 </li><li>在组织培养罩，制备含有10％胎牛血清的完全RPMI-1640培养基（FBS），4毫摩尔L-谷氨酰胺，1×青霉素/链霉素和10μM的2-巯基乙醇。 </li><li>每孔加1.5毫升完全RPMI-1640培养基以6孔细胞培养板，并放置在每个一细胞培养插管井。在37℃下，直到需要拨该板在培养箱中。 </li><li>在冰桶准备与水和冰的泥泞冰水浴中。 </li><li>小心取出周围用细尖镊子各胸腺叶所有剩余的结缔组织。而胸腺瓣在PBS淹没在组织培养皿中，后转移到一个组织用PBS擦拭浸泡，或在解剖显微镜下做到这一点。 </li><li>允许琼脂糖冷却到低于40℃，当该烧瓶只是温暖的触感，以避免过热的组织。琼脂糖倒入冷却的4％到组织模具中的约1厘米的高度。 </li><li>用一对镊子的胸腺叶仔细转移到纸巾擦拭和轻轻摇动它以干燥它不破坏组织。确保组织完全干燥，否则将切片中滑出琼脂糖。 </li><li>小心将叶放入琼脂糖和其定位或者水平（以增加每个切片的表面积）或垂直于模具的底部（以增加片的数量）。放置在冰水模具5-10分钟，以使琼脂糖固化。 </li><li>在这段时间内，通过安装在缓冲盘，装配在试样盘，并插入vibratome刀片准备切断用vibratome。放置在试样盘上的片实验室胶带。消毒用70％的乙醇清洗，组装工作区。 </li><li>一旦琼脂糖凝固，翻转模具并轻轻按下，在其中心发布琼脂糖嵌入叶。 </li><li>使用锋利的刀片在底部修剪多余的琼脂糖围绕叶留下〜2毫米琼脂糖两侧各约0.5厘米</li><li>固定每个琼脂糖块与组织胶的样品盘上的一块胶布的下降。多个块可以粘在磁带上。 </li><li>对齐vibratome刀片无线TH琼脂糖块的顶部。用无菌PBS填充缓冲区托盘直到刀片和琼脂糖块（多个）完全浸没。 </li><li>部琼脂糖包埋的组织，得到切片的厚度为400-500微米。的vibratome设置0.225毫米/秒的速度，100赫兹的频率，和5°角。 </li><li>使用弯曲铲收集胸腺切片放入含有无菌PBS组织培养板，因为他们被切断。丢弃第一个和最后切片。 </li><li>检查在放大4倍光显微镜下的切片。选择与完整胸腺组织和周围琼脂糖（ 图1A）的片。 </li><li>转移片，用枪头轻轻弯曲铲胸腺片滑入细胞培养插管步骤2.3准备的板块。每个刀片可容纳〜3片。确保片不互相接触或细胞培养插入墙壁。 </li><li>直到需要保持在37℃的板。 </li></ol><p类="jove_title"&gt; 3。胸腺细胞的制备叠加<ol><li>隔离胸腺裂片如第1中所述手动解离波瓣使使用填充用5ml无菌PBS含2％FBS的灭菌15毫升组织研磨机单细胞悬浮液。转移细胞到15毫升锥形管中。 </li><li>离心将细胞在545×g离心在4℃下5分钟。弃去上清液，在室温下悬浮细胞在1ml 1×ACK裂解缓冲液（0.15M的氯化铵 ，10毫KHCO 3，0.1mM的钠2 EDTA）中的3分钟以溶解红细胞。 </li><li>所述管填充到15ml含2％FBS的PBS中。离心将细胞在545×g离心在4℃下5分钟。 </li><li>悬浮细胞在10毫升含2％FBS的PBS中，并通过255微米的筛网过滤器传递的细胞。 </li><li>算使用血球细胞。离心在545×g离心5分钟将细胞在4℃下，并在一个精矿重悬在PBS中含有2％FBS的细胞沉淀每毫升1×10 7个细胞的通货膨胀。 </li><li>标记将细胞与如果不使用胸腺小鼠表达同类系的标记或基因编码荧光报道从片内源性的细胞区分，根据制造商的协议蜂窝染料如羧基琥珀酰亚胺酯（CFSE）。 </li><li>标记的细胞的最后一次洗涤后，重悬在完全RPMI-1640培养基将细胞沉淀以每15微升1-3×10 6个细胞。 </li></ol> 4.胸腺细胞叠加到胸腺片<ol><li>用移液管抽吸周围的刀片胸腺切片的任何液体。 注:请注意，以避免损坏琼脂糖。如果该琼脂糖已损坏，重叠胸腺细胞不会附着到片表面由于表面张力的损失。 </li><li>如果没有枪头，覆盖触摸片15微升第3节准备到每个胸腺切片胸腺细胞。 </li><li>孵育解放军德在37℃下2小时，以使细胞迁移进入胸腺切片。 </li><li> 2小时后，冲洗胸腺切片3次，用1毫升的PBS以除去尚未迁移进入组织过量重叠的胸腺细胞。 </li><li>根据实验在37℃下的板直到胸腺切片准备收获，典型地1-72小时。 </li></ol> 5.胸腺切片流式细胞仪的解离<ol><li>加入的PBS 150微升含有2％FBS的准备为每个​​切片一个离心管中。 </li><li>加入1毫升含2％FBS的与胸腺切片组织培养插入物的PBS并用弯曲刮铲轻轻搅动以从细胞培养插入物的表面分离的片。 </li><li>从使用弯曲抹刀插入到微量离心管转移的切片。 </li><li>手动扰乱使用离心管样品杵切片。添加的PBS 150微升含有2％FBS的至最终体积300微升。 </li><li>过滤通过40微米的过滤器中的解离组织到一个新的离心管中。将过滤的细胞准备被染色流式细胞仪分析40。 </li><li>将它们传递上的流式细胞仪，以确保所有的琼脂糖被除去，不仪堵塞流之前进行过滤的细胞第二次。 </li><li>获取关于一个流动细胞仪和如先前已描述40分析数据。 </li></ol>

The authors declare that they have no competing financial interests.

在这里，我们描述了胸腺切片并通过流式细胞术重叠预选择-MHC I类限制性TCR转基因的胸腺细胞的高效阳性和阴性选择的代表性结果的制备的协议。此系统已用于具有类似的成功支持从预选DP胸腺细胞32 MHC II型限制的CD4 + T细胞的阳性选择，并且，在激动剂抗原，阴性选择和胸腺T 章发展11,12的存在， 36,38,39,43,44。该协议可以被修改为研究抑制剂，定义的肽的存在或不存在胸腺选择，以及遗传修饰的胸腺细胞或间质细胞群。胸腺片也适合于比胸腺子集的其他细胞群体的重叠。例如，最近表明覆盖在胸腺切片该树突细胞有效地迁移进入组织并能支持负的SelectIOn和调节性T发展39,43。有研究迄今使用的预选DP或总胸腺细胞以研究阳性和阴性选择。虽然T细胞发育可以继续在胸腺片至少4天，如果有意发起与较早祖细胞群的实验，但应考虑到，该系统的一个限制是，胸腺切片的质量开始后下降1-2天在培养由于细胞死亡和缺乏包封膜。 本文描述的协议也可用于从具有细微修改人胸腺组织生成切片。在琼脂糖嵌入前，人胸腺组织应当切成碎片，成年小鼠胸腺瓣的大致大小。值得注意的是，人胸腺样品富含结缔组织，以及，相对于鼠胸腺，它更难以制备良好品质片。根据我们的经验，而不是胎儿新生儿人类胸腺组织是切片准备更有利。它已经表明，人胸腺细胞亚群正确地定位于人类和小鼠胸腺片37。覆盖人​​胸腺细胞亚群的胸腺选择，然而，尚未报道在胸腺切片模式。阳性选择从多克隆群体的效率低，而且由于在胸腺切片重叠胸腺细胞的比例低（〜0.5-2％），这可能是难以评估阳性选择细胞的这样的小的数字。它可能是在重叠在人胸腺片来提高正选择效率现有人类T细胞祖细胞引入一个人的TCR转基因是可行的。这也并不排除在外源肽或其它细胞群和TCR信号的抑制剂，等等的存在或不存在内源性T细胞群的变化进行监测的可能性。 该协议也可以是广告艾普特的胸腺细胞迁移，TCR信号和细胞相互作用12,32-38的可视化。直到最近， 就地胸腺细胞行为大部分研究都仅限于皮质髓质位于市中心，在胸腺，刚刚超过检测的双光子显微镜的极限。与此相反，胸腺切片，所述切片具有完整的胸腺皮质和髓质区提供的独特优势和片表面允许通过双光子成像髓质的直接访问。此外，具有标记的钙指示剂染料如吲哚细胞重叠胸腺切片可用于监视使用细胞溶质钙水平通过双光子显微镜11,32,36与阳性和阴性选择相关联的TCR信号的一个指标的TCR 信号38,39,44。要为显微镜制备样品，切片可以直接在协议步骤4.5后使用。在这种情况下，应小心选择合适的荧光标记物スitable成像。 胸腺切片器官培养是研究小鼠和人类T细胞发育的各个方面在原地一个很好的工具。然而，该技术的成功应用，需要特别注意在协议的关键步骤。为了最大限度地提高用于高通量实验获得胸腺片的数目，所使用的小鼠的年龄，必须考虑到作为整个小鼠的寿命胸腺的大小变化。我们通常使用生成〜每只小鼠胸腺20片4-8周龄小鼠。然而，胎儿，新生儿或老年小鼠thymi理论上可以用于产生根据用户的实验需要切片的数量有限。为了获得良好的品质片，这是最重要的，除去周围胸腺叶所有结缔组织包埋琼脂糖之前。剩余的结缔组织不受vibratome刀片切割时有效地切断，并能损害胸腺叶和产生的片上。结缔组织的体积应胸腺收获期间被移除（步骤1.5），并允许瓣从胸腔除去未经组织本身的显著操纵。隔离叶后，小心地除去残留的结缔组织如步骤概述2.5。此外，保持围绕包埋组织琼脂糖的完整性是必要的片表面上有效地覆盖胸腺细胞。因此，它是用于在实验中使用选择的胸腺切片时，检查两胸腺片和周围琼脂糖至关重要。该vibratome通常位于组织培养罩之外，增加了污染的潜在危险。在实验之间彻底清洁vibratome并用70％的乙醇切片之前喷射的工作区域和vibratome可以限制污染的可能性。同样重要的是使用无菌PBS的步骤2.13和2.15中所述p提到rotocol。最后，它也是至关重要来区分迁移到从内源性的胸腺细胞中的组织覆盖胸腺细胞。这可以通过如在步骤3.7中提到标记的重叠细胞用荧光染料来实现。可替代地，从表达同类标记或基因编码荧光报道的小鼠胸腺细胞可以使用45,46。在一般情况下，然而，胸腺切片容易操纵并可以适应用户的特定实验需要，支持广泛的适用性已仅开始进行研究。

T细胞分化，通过在此期间，他们遇到几个检查点，以确保一个功能，自我耐受T细胞库1-3的产生胸腺一系列发育中间体。阳性选择促进与T细胞受体（TCR），能够识别的，具有低到中度亲和力，肽由皮质胸腺上皮细胞（CTEC）2,3-主要组织相容性复合体分子（MHC）呈现胸腺细胞的存活。阴性选择和调节性T（T REG）细胞发育有助于通过消除或转移强烈由MHC 2,4提出自身肽反应胸腺细胞的建立自身耐受。未成熟CD4 + CD8 +双阳性（DP）胸腺细胞表达了通过选择过程分化成成熟T细胞亚群的TCR，其中大部分是MHC I类限制性CD8 +细胞毒性或MHC II类限制性CD4 +辅助单阳性（SP）T细胞中，离开胸腺在次级淋巴器官1-3执行效应器功能之前。 添加到T细胞发育的复杂性是动态迁移并在整个基质细胞网络5-9显影胸腺细胞的细胞接触。这些基质细胞在胸腺细胞发展中发挥不同的作用，并在那里阳性和阴性选择发生10胸腺皮质和髓质区之间被差异分布。虽然正选择主要发生在皮质，有越来越多的证据认为DP胸腺细胞迁移到髓质和继续需要的TCR信号它们分化成成熟T细胞暗示髓质可提供必要的正选择和谱系完成附加的信号之前分化11,12。进一步，尽管表达专门髓质胸腺上皮细胞（MTEC）和本组织限制性抗原促进自身反应性胸腺13,14的缺失的情况下，阴性选择的相当大的比例发生在响应皮层到遍在表达自身肽树突呈现细胞15,16。因此，T细胞发育的准确的模型必须提供高度组织胸腺微环境，具有完整皮层和髓质区，便于胸腺细胞和基质细胞之间的相互作用，并支持胸腺细胞迁移，因为这些细胞经历阳性和阴性选择。 为了补充体外胸腺细胞分析作为研究阳性和阴性选择，一些在体外，原位的手段，并在 T细胞发育的体内模型已经开发17-22。它已经非常难以概括在体外的积极选择，但表达Notch配体干细胞群或基质细胞T细胞前体共培养，特别是OP9-DL1 / 4的细胞，具有支持T系的承诺和有限的正向选择的能力使其成为一个非常宝贵的体外模型研究T细胞发育23-25。本系统的限制，但是，包括这些细胞缺乏胸腺基质细胞上发现的唯一的肽加工机械和三维胸腺微环境的事实。 虽然在技术上更为繁琐，就地和胸腺选择的体内模型可以克服一些与体外系统的障碍。 Reaggregate胸腺器官培养（RTOC）包含定义的胸腺细胞和胸腺基质细胞18,26,27的混合物。这些胸腺上皮细胞reaggregates保持MHC I类和II表达并且可以支持DEVELOPME常规T细胞亚群的NT，但仍然缺乏定义的皮质和髓质结构。胎儿胸腺器官培养（FTOC）是T细胞发育的一个流行的模型，可以通过lymphodepleted胸腺叶或通过胸腺细胞注射悬滴文化的胸腺细胞被接种到lymphoreplete胸腺叶和支持CD4 +和CD8 + T的高效发展在细胞培养18,28-31时间。在胎儿胸腺瓣培养开始有mTECs的缺乏，但根据条件定义皮质和髓质结构可以随时间发展。一个重要的考虑是，该模型可优先支持胎儿对成人T细胞的发展。最后，在成年小鼠胸腺中定义的前体胸腺内注射在技术上具有挑战性，但显然提供了一个环境，以支持 T细胞发育体内 。这些原位 和体内模型是优秀的快工具Ø研究T细胞发育，其使用应在一个实验按实验基础上加以考虑。 胸腺片，然而，最近已成为一个通用的，互补的模型，研究胸腺选择就地与可能性，以适应独特的，复杂的，并且通常更高的吞吐量实验。胸腺片保持皮质和髓质区的完整性并提供基质细胞的框架，开发以及高效阳性和阴性选择11,32-39期间支持胸腺细胞迁移。添加顶上胸腺切片胸腺细胞亚群迁移到组织到相应的微环境利基34,37。的重叠胸腺细胞可以从胸腺切片内源性细胞经由同类标记或荧光标记来区分，并可以在培养物中维持数天。胸腺器官切片文化，可以用来研究的各个方面T细胞发展，包括胸腺选择，胸腺细胞行为（迁移和细胞相互作用），和胸腺细胞定位，等等。给生成每只小鼠〜20胸腺切片的能力，实验的可扩展性通常比在胸腺选择的原位模型其他更大。虽然胸腺切片的制备需要专门的设备，如vibratome，并且由于通过细胞死亡随着时间的推移细胞的损失和缺乏包封膜被限制在培养胸腺片的使用寿命，胸腺片提供了一个极好的模型对于一个成熟的胸腺微环境胸腺细胞同步群体的胸腺选择的分析。在这里，我们描述了胸腺片（包括收获的胸腺，胸腺裂片的琼脂糖嵌入和嵌入的组织的vibratome切片），隔离和胸腺细胞的重叠，并解离为流式细胞仪分析胸腺切片的制备方法。

<table><tbody><tr><td>Vibratome</td><td>Leica Biosystems</td><td>VT1000S&amp;nbsp;</td><td></td></tr><tr><td>NuSieve GTG Agarose</td><td>Lonza</td><td>50080</td><td>Low melting temperature agarose</td></tr><tr><td>Embedding Mold (Truncated - T12)</td><td>Polyciences</td><td>18986</td><td>22 mm x 22 mm square, truncated to 12 mm x 12 mm</td></tr><tr><td>Double Edge Prep Blades</td><td>Personna</td><td>74-0002</td><td></td></tr><tr><td>Tissue Adhesive</td><td>3M&amp;nbsp;</td><td>1469SB</td><td></td></tr><tr><td>0.4 &amp;micro;m Cell Culture Inserts&amp;nbsp;</td><td>BD Falcon</td><td>353090</td><td>Of several brands tested, these maintained the cells atop the slices the best</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>ThermoFisher</td><td>21600-010</td><td></td></tr><tr><td>RPMI-1640 with L-glutamine</td><td>Wisent</td><td>350-000-CL</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Wisent</td><td>080-110</td><td>Heat inactivated</td></tr><tr><td>L-Glutamine, 200 mM</td><td>Wisent</td><td>609-065-EL</td><td></td></tr><tr><td>Penicillin/Streptomycin, 100x</td><td>Wisent</td><td>450-201-EL</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Alfa Aesar</td><td>A15890</td><td></td></tr><tr><td>15 ml Tenbroeck Tissue Grinders</td><td>Wheaton</td><td>357426</td><td></td></tr><tr><td>Nylon Mesh Filter</td><td>Component Supply</td><td>U-CMN-255</td><td></td></tr><tr><td>Microcentrifuge Tube Sample Pestle</td><td>Bel-Art</td><td>F19922-0000</td><td></td></tr><tr><td>40 &amp;micro;m Nylon Cell Strainer</td><td>BD Falcon</td><td>352340</td><td></td></tr><tr><td>Forceps Inox Tip</td><td>Dumont&amp;nbsp;</td><td>RS-5047</td><td>Fine tip curved forceps, size .17 x .10 mm</td></tr><tr><td>Micro Forceps</td><td>Dumont&amp;nbsp;</td><td>RS-5090&amp;nbsp;</td><td></td></tr></tbody></table>

preparation and applications of organotypic thymic slice cultures

在产生了功能性的，自我耐受T细胞库的产生一种独特的和高度有组织的胸腺微胸腺选择前进， 在体外模型来研究T系承诺和发展已经提供了有价值的见解这一过程。然而，这些系统缺乏必需的T细胞发育的完整三维胸腺环境，因此， 在体内胸腺选择的不完全近似。一些涉及到建模T细胞发育的挑战可以通过使用原位模型提供了一个完整的胸腺微环境完全支持显影性T细胞的胸腺选择来克服。胸腺器官切片培养补充现有的原位技术。胸腺切片保存胸腺皮质和髓质区的完整性，并提供了一​​个平台，研究一个限定的发育阶段的或内源T C的重叠胸腺细胞发育一个成熟的胸腺微环境中厄尔。鉴于生成每只小鼠〜20片的能力，胸腺切片进行高通量实验的可扩展性方面呈现出独特的优势。此外，在生成胸腺切片和电位从不同遗传背景叠加不同胸腺子集或其它的细胞群相对容易提高该方法的多功能性。这里，我们描述的胸腺片，隔离和胸腺细胞的叠加，以及流式细胞仪分析胸腺片分离制备协议。这个系统还可以适于研究非常规T细胞的发育以及可视胸腺细胞迁移，胸腺间质细胞相互作用，并通过双光子显微镜与胸腺选择相关联的TCR信号。

T细胞发育的不同方面，如阳性和阴性选择胸腺切片支持分析。对于成功的实验中，胸腺切片的质量是至关重要的。 因此，应研究胸腺切片以确保胸腺组织的完整性和周围胸腺切片琼脂糖是完好（ 图1A）。当琼脂糖损坏导致在迁移进入组织胸腺细胞的数目显著减少表面张力就会受到损害。因此，具有组织损伤或刻痕的适应症胸腺片/眼泪在琼脂糖应该被丢弃（ 图1B）。 TCR转基因（TG）小鼠通常用于研究胸腺选择由于每胸腺细胞的表面上限定的，功能性的TCR，其会增大聚C的阳性选择的频率的表达lonal人口。为了捕捉正选择的每个阶段，预先选择的TCR TG胸腺细胞可以顶上胸腺片重叠，和成熟的发展，CD4 +或CD8 + SP T细胞，随后通过用流式细胞仪40的时间。从MHC I类限制的OT-I的TCR TG RAG1分离胸腺细胞- / - β2M - / -小鼠是在之前的胸腺选择在DP阶段被捕由于β2M的必要性关联与和稳定MHC I类重链41 42。当覆盖在β2M - / -胸腺切片，预选的OT-I的TCR TG胸腺细胞保持在DP的阶段，不产生显著的CD8 + SP T细胞（ 图2A，左）。与此相反，在含有内源性选择配体的WT胸腺片重叠时，该模型的支持正选择的能力是由CD8 + T细胞的发展在72小时证实（ 图2A，右）。曲CD8 + T细胞的发展作为读出阳性选择的antification示于图2B。 胸腺切片也可以用来研究负选择。阴性选择的很大一部分发生在应对无处不在抗原16。到阴性选择建模到无处不抗原，从OT-I的TCR TG RAG1总胸腺细胞- / -和野生型（WT）小鼠用CFSE标记和CTV，分别接在1:1混合比和重叠在WT片（ 图3A）。洗去过量的胸腺细胞后，胸腺切片放置在完全RPMI-1640媒体有或无1海里同源的，在OT-I TCR，SIINFEKL（OVA肽）激动剂肽。我表达细胞的MHC的所有类将呈递抗原，造型普遍表达抗原在胸腺中呈现。后在37℃下，OT-I的TCR TG CEL的比例温育24小时LS（％活CFSE +）以WT对照细胞（％活CTV +）通过流式细胞术（ 图3B）相比较。在OT-I TCR TG细胞的相对比例的降低代表针对OVA肽OTI TCR TG细胞的缺失。 要注意的是，由于在胸腺切片的大小异质性以及细胞进入胸腺片，应包括适当的分析和内部控制正常化为这些变量是重要的。这里，与OT-I的TCR TG细胞混合并覆盖顶上切片WT细胞用作作为这一人群不应显著影响的OVA肽的存在或不存在内部对照。基于比例而不是绝对数OTI TCR TG细胞负选择的程度进行定量。首先，WT OT-I TCR TG（％生活CFSE +）之间的比例在上存在各WT胸腺切片（％生活CTV +）细胞<html>或不存在的OVA肽的计算。这些比率中的每一个然后通过的OT-I的TCR TG（％活的CFSE +）之间的WT（％活CTV +）细胞上的WT片在不存在的OVA肽的比率的平均值除以。平均来说，如在图3C中所示的OT-I的TCR TG细胞的80％是在响应于无处不抗原耗尽。 <img alt="图1" src="/files/ftp_upload/54355/54355fig1.jpg" /> 图1: 从一个垂直的嵌入式胸腺叶制备胸腺切片的代表性图像 。 （ 一 ）质量好片与嵌入式组织和周围的琼脂糖完好无损。 （ 二 ）质量差的切片与因切片在包埋组织的撕裂损伤。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54355/54355fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> ove_content"FO:保together.within页="1"&gt; <img alt="图2" src="/files/ftp_upload/54355/54355fig2.jpg" /> 图 2: 在胸腺切片阳性选择的流式细胞术分析的OT-I的TCR TG RAG1 - / - β2M - / -胸腺细胞用CFSE标记和覆盖在非选择β2M - / -或选择WT切片。在37℃下72小时培养后，CD8 + T细胞的发展是通过流式细胞术分析。 （A）活CFSE从β2M+TCRβ 高胸腺细胞表面的CD4和CD8表达的有代表性的数据- / -和WT片。 （ 二 ）对WT片现场CFSE +TCRβ 高 CD8 + T细胞发育的量化相比，非选择控件。每个点代表一个独立的胸腺切片和线表示平均值。 （*** P &lt;0.001，配对t检验）ove.com/files/ftp_upload/54355/54355fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54355/54355fig3.jpg" /> 图3: 在胸腺切片阴性选择的流式细胞术分析 ，A 1:总CFSE标记的OT-I的TCR TG RAG1 1的比例- / -和CTV标记WT胸腺细胞中的存在或不存在被覆盖在WT片OVA肽SIINFEKL。后在37℃下温育24小时，重叠的细胞的相对比例，通过流式细胞术进行分析。 （A）中的实验装置表示的CTV标记野生型细胞（蓝色）覆盖混合1的示意:1与CFSE标记的OT-I的TCR TG细胞（绿色）到在存在或不存在的WT胸腺切片OVA的肽。 （B）的有代表性的数据，描绘活的CFSE + OT-I的比例TCR TG细胞和CTV + WT细胞从孵育有或没有的OVA肽切片。 （C）的数据被切片之间标准化，并示出带电的CFSE + OT-I的TCR TG细胞活CTV + WT细胞的相对比例。误差线表示标准偏差。 N = 3每个条件。 （* P &lt;0.05，非配对t检验）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54355/54355fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Preparation and Applications of Organotypic Thymic Slice Cultures at JoVE.com

Preparation and Applications of Organotypic Thymic Slice Cultures

我们描述的是，在用流式细胞仪组合，可以被用来建模显影T细胞的阳性和阴性选择胸腺切片的制备方法。胸腺片也可以适用于胸腺细胞迁移，本地化的原位分析，并通过免疫荧光和双光子显微镜信令。

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 ...

preparation-and-applications-of-organotypic-thymic-slice-cultures

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

8.0K 次观看.  Hôpital Maisonneuve-Rosemont. 我们描述的是，在用流式细胞仪组合，可以被用来建模显影T细胞的阳性和阴性选择胸腺切片的制备方法。胸腺片也可以适用于胸腺细胞迁移，本地化的原位分析，并通过免疫荧光和双光子显微镜信令。 

视频: Preparation and Applications of Organotypic Thymic Slice Cultures

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

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

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

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 ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

神经科学

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 ...

癌症研究

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

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 ...

生物学

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. Tissue&#160;slices immediately cut from freshly resected tumors preserve spatial aspects of intratumor heterogeneity (ITH), thus making them useful surrogates of in vivo biology. Careful optimization of tissue slice preparation and cultivation conditions is fundamental for the predictive diagnostic potential of tumor slice explants. In this regard, murine models are valuable, as these provide a consistent flow of tumor material to perform replicate and reproducible experiments. This protocol describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. Our previous work showed that the use of rotating incubation units improves the viability of tissue compared to other culture methods, particularly floating slices and stagnant filter supports. Here, we further show that slice thickness influences the viability of cultured slices, suggesting that optimization of slice thickness should be done for different tissue types. Pronounced ITH in relevant oncogenic functions, such as signaling activities, stromal cell infiltration or expression of differentiation markers, necessitates evaluation of adjacent tissue slices for the expression of markers altered by drug treatment or cultivation itself. In summary, this protocol describes the generation of murine lung tumor slices and their culture on a rotating incubation unit and demonstrates how slices should be systematically analyzed for the expression of heterogeneous tissue markers, as a prerequisite prior to drug response studies.

We provide a method for the generation, cultivation and systematic analysis of organotypic slices derived from murine lung tumors. We also describe how to optimize for slice thickness, and how to select drug concentrations to treat tumor slices.

肿瘤切片提取物的建立与分析是诊断检测的前提

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the ...

免疫与感染

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

脊髓 Cocultures 丘脑-皮质轴突分支和突触形成的可视化研究

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

髓鞘有机小脑片培养的制备及免疫染色

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...