这项工作是由美国国立卫生研究院的一部分授予R21 AI113000（WSM）和R01 AI123392（YF）的支持。

在实验中使用的所有动物饲养在阿勒格尼歌手研究院由阿勒格尼健康网/阿勒格尼歌手研究院的机构动物护理和使用委员会审查和批准议定书下的动物设施。 1.消化胶原酶与胸腺<ol><li>嘉实胸腺。 <ol><li>安乐死在二氧化碳3-5周龄C57BL6 / J小鼠（CO 2）的腔室收获胸腺。详细地说，放置小鼠在CO 2的诱导下腔室3-5分钟，并通过验证的心脏或呼吸停止确认死亡。 </li><li>铺设在它的后面的胎体和用70％乙醇润湿体。做一个小的水平切口用一把锋利，平直解剖它的腹部通过皮肤剪刀。拉两端（头部和腿），以便它在里朝外翻转皮肤。 </li><li>做一个横向切断与解剖剪刀刚下最低的肋骨，并通过隔膜横着切。持用钳子剑突切肋两侧继续向上的中间。拉起肋/胸骨使得胸腺是清晰可见。 注意:胸腺直接放置在心脏的顶部，由2叶和是一个白色半透明色。 </li><li>用弯钳，轻轻舀拉胸腺瓣关闭结缔组织和洗涤液转移（见材料清单）。 </li></ol></li><li>通过混合9毫升的RPMI-1640（罗斯韦尔园区纪念研究所介质-1640），0.025毫克/毫升的纯化的胶原酶，10毫米的HEPES [4-（2-羟乙基）-1-哌嗪乙磺酸]，和0.2毫克/毫升制备消化溶液DNA酶I在50毫升离心管中。保持在37℃水浴中消化溶液直到使用。 注:制备的消化溶液的量是足以为最多五个成年小鼠thymi，其可集中在一个5毫升聚苯乙烯圆头孵育底部的管。 </li><li>转移胸腺含5-6毫升RPMI-1640 60毫米组织培养皿。 </li><li>解剖与28克的胰岛素注射器针头成小块（约1 立方毫米见方）胸腺让淋巴细胞流出。 </li><li>冲洗胸腺件用RPMI-1640是在培养皿​​用玻璃巴斯德吸管。倾斜的菜，让胸腺片段沉淀在底部。慢慢吸并用玻璃吸管丢弃的RPMI-1640上清液。重复此冲洗步骤用5-6毫升的RPMI-1640与玻璃吸管4-5次，直到溶液是较少不透明的。 注:玻璃移液器在这一步推荐的，因为胸腺片段倾向于坚持塑料，导致胸腺组织中过多的损失。有关该过程的其余部分，用塑料吸管。 </li><li>最后冲洗后，如在步骤1.5中所述弃上清。添加3毫升消化溶液和胸腺片和溶液转移到5毫升的圆底polystyrene管。 </li><li>放置在试管旋转器的管中，在12 rpm的旋转速度，在37℃孵育6分钟。 </li><li>温育后，让胸腺片段通过重力沉降到管底部。收集用巴斯德吸管将上清液并转移到50毫升离心管中，用10ml的洗涤溶液。离心机在300×g离心5分钟，弃去上清，重悬在10ml洗涤液中的细胞。置于冰上。 </li><li>在5毫升的圆底聚苯乙烯试管的胸腺片段重复步骤1.6-1.8两次。池上清液在同样的50毫升管。 注意:离心是没有必要的，第二和第三次洗涤。 </li><li>最终消化后，吸管上下用2ml塑料血清吸管在管和传送任何剩余片段分解到50ml离心管中。 </li><li>离心机50毫升管在300×g离心5分钟，吸出上清液，重悬在10ml洗涤buffe的r和过滤到100微米的细胞过滤。置于冰上。 </li></ol> 2.肾小管上皮的富集连续密度梯度<ol><li>通过混合2.4毫升密度梯度介质（60％w / v的水碘克沙醇）和9.6毫升洗涤液制备21％的密度梯度溶液中。 </li><li>离心在50 ml收集管中的解离的胸腺细胞从步骤1.11，在300×g离心在2.5ml洗涤缓冲液5分钟，重悬。 </li><li> 20毫升RPMI-1640培养基添加到细胞悬浮液。 </li><li>填10ml的血清吸管加入12ml 21％的密度梯度溶液与电子移液器。放置血清吸管的尖端在50ml含离心管细胞的底部，并允许密度梯度溶液排放到通过重力管的底部。 <ol><li>轻轻地从吸管排出密度梯度溶液来完成产生不同密度的两层。 </li></ol></li><li>离心机600 XG FOř在RT在吊桶式转子20分钟。设置在最低水平的减速度。 </li><li>顶层和接口转移到一个新50ml管中。 注意:大多数的TECs的被保留在接口。淋巴细胞会沉淀到离心后在管的底部。如果这些细胞将被用于其他目的，冲洗用20ml洗涤溶液的沉淀三次。悬浮细胞在10ml洗涤缓冲液中。 </li><li>高达40毫升400 XG添加洗涤液向管在步骤2.6和离心6分钟，在4℃。除去上清液，吸用吸管。 </li><li>如步骤2.7中所述重复洗涤和抽吸2次以上。 </li><li>重悬在2ml的洗涤液和计数与血球细胞。 </li></ol> 3.隔离的TEC用FACS <ol><li>调整从步骤2.9细胞以1×10 6个细胞/ 100微升用洗涤溶液和转让的浓度将细胞以5毫升的圆底管（聚丙烯或聚苯乙烯，所推荐的流分拣机的说明）。 </li><li>添加2微升抗Fc受体IgG的每1×10 6个细胞，并在4℃下孵育10分钟。 </li><li>加抗CD45（1:150稀释，每个样品10微升）和抗-EpCAM（1:100稀释，每个样品10微升）的抗体，并在4℃下孵育超过20分钟。 </li><li>洗用2ml洗涤液并离心将细胞在400×g离心6分钟，4℃。吸用吸管将上清液。 </li><li>重悬在1ml 1×磷酸盐缓冲盐水（PBS）的细胞。 </li><li>通过100微米滤网过滤。 </li><li>应用该细胞的分选仪。选择淋巴细胞和TEC人口不包括侧向散射光（SSC）/前向散射光（FSC）面板（死细胞），然后为CD45 -的EpCAM +选定人口分拣13上栅极的最小单元。 </li></ol> 4.摹TEC / EAK骨料eneration 注:执行试剂的制备和涉及细胞层流罩下混合步骤。 <ol><li>通过在无菌1.5毫升微量离心管中混合4微升抗His-标签的IgG，8微升抗EpCAM抗体和2.6微升pA的/ G的制备pA的/ G适配器配合物，并在室温孵育5分钟。 </li><li>调整从步骤3.7的排序的TECs与洗涤液，和吸管100μl至低蒸发，96孔圆底组织培养板的一个孔5×10 4细胞/ ml。添加14.6微升pA的/ G适配器配合到细胞中。放置板在摇摆平台上在4℃下20分钟。 </li><li>用200微升洗涤液洗一次。离心板在400×g离心6分钟。丢弃用微量上清。 </li><li>用200微升10％蔗糖溶液洗一次。离心在400 xg离心6分钟。丢弃用微量上清。 </li><li>悬浮细胞在30微升10％S每孔terile蔗糖溶液。 </li><li>添加EAKIIH6 10微升（7.5毫克/毫升），并在室温下孵育5分钟。 </li><li>添加EAK16-II的20微升（10毫克/毫升）到TEC混合物。 </li><li>添加100微升完全培养基到体系内，引发胶凝。放置在平台摇动该板在室温15-20分钟。 </li><li>放置在孵化该板在37℃和5％的CO 2。 2-4小时后，再添100微升完全培养基。 </li><li>更换培养基隔日直到使用。 <ol><li>抽吸100μl的从表面介质的不使用微量并且轻轻放置枪头井的壁添加100μl的预热介质的干扰凝胶。 </li></ol></li></ol> 5.表征TEC / EAK骨料<ol><li>要检查体内 TEC / EAK聚集的功能，收集O / N培养和移植后TEC / EAK水凝胶的无胸腺裸鼠的肾囊下鼠标，以前使用描述的过程11,14。 </li><li>通过收获50-100微升的血液样品进入含有EDTA血液收集管从裸鼠4-6周后移植和染色用抗CD4的鸡尾酒，-CD8，-CD45监测T细胞在外围的发展和-CD 3抗体12。 </li><li>分析T淋巴细胞的百分比在与流式细胞仪的外周血白细胞（FCM），使用单细胞分析软件12。 </li></ol>

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Authors declare no conflict of interest.

同时的TECs是胸腺基质的主要人口和播放的胸腺的结构和功能重要作用，它们仅占大约0.1％-0.5％的总胸腺细胞构成的。它们也可用作细胞死亡的高百分数以下胶原酶消化偶尔观察到的， 即 ，治疗以解离来自细胞外基质（ECM）的TECs脆弱的细胞。其稀有性（〜20余万小鼠胸腺）和脆弱使其成为具有挑战性的任务来隔离的TEC。高度纯化的胶原酶了TEC隔离最近利用已显著增加活细胞的以下酶消化<su...

胸腺是负责病原体 - 反应性，自我耐受T细胞的不同群体的产生即获得性免疫系统的功能所必需的主要淋巴器官。它是一个动态的器官，其中显影胸腺细胞，从骨髓如淋巴细胞祖细胞移居，通过胸腺基质的海绵状三维（3-D）矩阵迁移，经受谱系说明书和分化，最终移民如成熟T细胞。此井编程过程的成功在很大程度上取决于迁移胸腺细胞和住宅胸腺上皮细胞（TECs的）中，胸腺基质的主要群体是建立和维护的胸腺微环境的完整性必不可少之间的串扰。 根据他们的解剖位置和独特的功能，的TECs可以分为两个子集:所述的TECs在皮层（cTECs）是responsible选择自身MHC（主要组织相容性复合物）限制性T细胞（阳性选择），并且在髓质是用于消除自身反应性T细胞（阴性选择）1,2-必需的的TECs（mTECs）。许多因素（ 如老化，感染，辐射，药物治疗）可能会导致不可逆的损伤胸腺上皮细胞，导致受损适应性免疫。尽管多次努力，恢复胸腺功能一直具有挑战性，由于重现胸腺微环境的难度。值得注意的是，胸腺三维（3-D）的配置是的TECs的生存和功能是至关重要的，而在2维环境中培养的TECs迅速降低基因thymopoiesis 3,4-临界的表达。 EAK16-II（AEAEKAKAEAEAKAK）及其C末端histidinylated类似物EAKIIH6（AEAEKAKAEAEAKAKHHHHHH）是低分子量，两亲性寡肽可溶于去离子水:R，但是当暴露在离子强度超过20毫摩尔NaCl（在人体液正常盐浓度为154毫摩尔）更高发生凝胶化，形成β-纤维。此环境响应特性使得他们多才多艺的积木，形成三维结构。上EAKII-H6 His标签提供一个对接机构，通过该抗His的IgG / Fc结合的重组蛋白A / G（αH6:PA / G）的配合物可以作为一个适配器锚蛋白药物和其它生物分子（ 例如 ，在水凝胶复合5-8抗体）。 我们以前表明，锚定在水凝胶的荧光标记IgG分子可以在注射部位长达13天9被保留。此外，当αH6:用抗CD4的IgG锚pA的/ G适配器加到EAK16-II / EAKIIH6（EAK）水凝胶，CD4 + T细胞是专门捕获10。使用类似的技术，我们最近证明的TECs的c的3-D聚合乌尔德在EAK水凝胶促进携带特定的TEC-抗-EpCAM抗体复合适配器（αH6:PA / G:αEpCAM）。当裸鼠的肾囊下移植，嵌在EAK水凝胶将TEC集群可以有效地支持体内11,12官能性T细胞的发展。 在这里，我们示出了我们的方法来快速并有效地净化用荧光激活细胞分选（FACS）的TECs和生成与EAK水凝胶系统的3-D TEC聚集体。

<table><tbody><tr><td>&lt;strong&gt;TEC Isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70% Ethanol</td><td>Decon Laboratories</td><td>2701(1 Gallon)</td><td>Ethanol 200 Proof, deionized water</td></tr><tr><td>Dissecting scissors, straight</td><td>Fine Science Tools, Inc.</td><td>91460-11</td><td></td></tr><tr><td>Graefe forceps, straight</td><td>Fine Science Tools, Inc.</td><td>11053-10</td><td></td></tr><tr><td>Graefe forceps, curved</td><td>Fine Science Tools, Inc.</td><td>11052-10</td><td></td></tr><tr><td>Washing solution</td><td></td><td></td><td>1x PBS, 0.1% BSA, 2&amp;nbsp;mM EDTA</td></tr><tr><td>1x PBS (Phosphate buffered saline)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>BSA (Bovine serum albumin)</td><td>Sigma</td><td>A1470-100G</td><td></td></tr><tr><td>EDTA (ethylenediaminetetraacetic acid)</td><td>Invitrogen</td><td>15575-038</td><td></td></tr><tr><td>Digestion solution</td><td></td><td></td><td>9 ml RPMI-1640, 0.025&amp;nbsp;mg/ml Liberase TM Research grade, 10&amp;nbsp;mM HEPES, 0.2&amp;nbsp;mg/ml DNaseI</td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>11879-020</td><td></td></tr><tr><td>Liberase TM Research Grade</td><td>Roche</td><td>05 401 127 001</td><td>referred as &quot;purified collagenase&quot;</td></tr><tr><td>1&amp;nbsp;M HEPES</td><td>Lonza</td><td>17-737E</td><td></td></tr><tr><td>Dnase I</td><td>Roche</td><td>10 104 159 001</td><td></td></tr><tr><td>50&amp;nbsp;ml Centrifuge tube</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>60&amp;nbsp;mm tissue culture dish</td><td>Falcon</td><td>353002</td><td></td></tr><tr><td>1/2&amp;nbsp;cc U-100 Insulin syringe 28&amp;nbsp;1/2&amp;nbsp;G</td><td>Becton Dickinson</td><td>329461</td><td></td></tr><tr><td>5&amp;nbsp;ml Polystyrene round-bottom tube</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>5&amp;nbsp;ml glass pipet</td><td>Fisher Healthcare</td><td>13-678-27E</td><td>Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets.</td></tr><tr><td>MACSmix tube rotator</td><td>Miltenyi</td><td>130-090-753</td><td></td></tr><tr><td>100&amp;nbsp;&amp;micro;m Cell strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>Density gradient medium: OptiPrep</td><td>Axis-Shield</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cell Sorting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5&amp;nbsp;ml Polypropylene round-bottom tube</td><td>Falcon</td><td>352063</td><td></td></tr><tr><td>Anti-mouse CD16/CD32 (Fc Block)</td><td>BD Biosciences</td><td>553142</td><td>Use as undiluted, 2&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>Anti-mouse CD45-PercpCy5.5</td><td>eBioscience</td><td>45-0451-80</td><td>Use at 1:150, 10&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>Anti-mouse CD326 (EpCAM)-PE</td><td>eBioscience</td><td>12-5791-82</td><td>Use at 1:100, 10&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>BD Influx</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Single cell analysis software</td><td>FlowJo</td><td></td><td></td></tr><tr><td>&lt;strong&gt;EAK Gel Assembly&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-His-Tag</td><td>AnaSpec</td><td>29673</td><td>&quot;anti-His-Tag IgG&quot;</td></tr><tr><td>Purified anti-mouse CD326 (EpCAM)</td><td>BioLegend</td><td>118202</td><td>&quot;anti-EpCAM IgG&quot;</td></tr><tr><td>Recombinant protein A/G</td><td>Pierce Biotechnology</td><td></td><td></td></tr><tr><td>1.5&amp;nbsp;ml Safe-Lock Tubes, Biopur, Sterile</td><td>Fisher Healthcare</td><td>05-402-24B</td><td>referred as &quot;1.5&amp;nbsp;ml microcentrifuge tube&quot;</td></tr><tr><td>96-well, Tissue culture plate, Round-bottom with low evaporation lid</td><td>BD Falcon</td><td>353917</td><td></td></tr><tr><td>Rocking platform: Nutator Mixer no.1105</td><td>BD Clay Adams</td><td></td><td></td></tr><tr><td>10% sucrose</td><td>Sigma</td><td>S0389</td><td>Prepare with sterile distilled water</td></tr><tr><td>EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH&lt;sub&gt;2&lt;/sub&gt;)</td><td>American Peptide Company&amp;nbsp;</td><td></td><td>custom synthesized, 10&amp;nbsp;mg/ml</td></tr><tr><td>EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH&lt;sub&gt;2&lt;/sub&gt;)</td><td>American Peptide Company&amp;nbsp;</td><td></td><td>custom synthesized, 7.5&amp;nbsp;mg/ml</td></tr><tr><td>Complete medium</td><td></td><td></td><td>RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5&amp;nbsp;mM HEPES, 50&amp;nbsp;&amp;micro;M 2-Mercaptoethanol</td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>11879-020</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Atlanta Biologicals</td><td>S11150</td><td>Heat inactivated before use</td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>L-glutamine 200&amp;nbsp;mM (100x)</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>NEAA (non-essential amino acid) 100x</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>1&amp;nbsp;M HEPES</td><td>BioWhittaker</td><td>17-737E</td><td></td></tr><tr><td>2-Mercaptoethanol (100x)</td><td>Millipore</td><td>ES-007-E</td><td></td></tr><tr><td>Platform shaker: The Belly Dancer</td><td>Stovall Life Sciences Inc.</td><td>model: USBDbo</td><td></td></tr></tbody></table>

promoting 3-d aggregation of facs purified thymic epithelial cells with eak 16-ii/eakiih6 self-assembling hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

为了检查使用密度梯度分离协议以丰富的CD45 - 间质细胞的有效性，同时从界面和沉淀的淋巴细胞粒料收集的细胞用抗CD45和抗EpCAM抗体染色。既防荆豆凝集素1（UEA1）和抗MHC II类抗体也包括在染色鸡尾酒进一步标识CTEC和MTEC子集。正如图1所示，我们能够接近常规实现到的TECs的15-20倍富集。 要检查的TECs在EAK水凝胶的?...

Watch this Scientific Journal Video about Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel at JoVE.com

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

促进FACS纯化的胸腺上皮细胞的3-D聚合，EAK 16-II / EAKIIH6自组装凝胶

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

promoting-3-d-aggregation-facs-purified-thymic-epithelial-cells-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.5K 次观看.  Allegheny Health Network. This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

视频: Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

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 ...

科研

发育生物学

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

生物工程

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

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

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

组织工程细胞在三维凝胶封装

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

用于可重复模式形成的混合凝胶立方体器件中的初始三维单元群集控制

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

<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 ...

促进FACS纯化的胸腺上皮细胞的3-D聚合，EAK 16-II / EAKIIH6自组装凝胶

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Reaggregate胸腺文化

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用于可重复模式形成的混合凝胶立方体器件中的初始三维单元群集控制

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