この作品は、国立衛生研究所によって部分的にサポートされていたR21 AI113000（WSM）とR01 AI123392（YF）を付与します。

実験で使用した全ての動物は、アレゲニー健康ネットワーク/アレゲニーシンガー研究所の施設内動物管理使用委員会によって審査および承認されたプロトコルの下でアレゲニー・シンガー研究所の動物施設に収容しました。 1.コラゲナーゼで胸腺を消化<ol><li>収穫胸腺。 <ol><li>胸腺を収穫するために、二酸化炭素（CO 2）チャンバー内で3-5週齢のC57BL6 / Jマウスを安楽死させます。具体的には、3〜5分間、CO 2の誘導の下でチャンバー内にマウスを置き、心臓や呼吸停止を検証することによって、死亡を確認。 </li><li>その裏に死体を置き、70％エタノールで体を濡らします。皮膚を通してその腹部に鋭い、ストレート解剖ハサミで小さな水平切開を行います。それは裏返しされるように両端（頭部と脚）の皮膚を引きます。 </li><li>すぐ下に解剖ハサミで水平カットを作ります最低リブとは、横振動板をカット。ピンセットで剣状突起を持ち、両側にまで進んリブの真ん中を切りました。胸腺がはっきり見えるように肋骨/胸骨を引き上げます。 注：胸腺、心臓の上に直接位置する2葉からなり、白色半透明色のものです。 </li><li>湾曲した鉗子を使用して、静かに（材料のリストを参照してください）​​洗浄液中の結合組織および転送のオフ胸腺ローブをスクープし、引き出します。 </li></ol></li><li> 9mLのRPMI-1640（ロズウェルパーク記念研究所培地-1640）、0.025 mg / mlで精製されたコラゲナーゼ、10mMのHEPES [4-（2-ヒドロキシエチル）-1-ピペラジンエタンスルホン酸]、および0.2 mg / mlで混合することにより、消化液を調製50ml遠心管中のDNase I。使用するまで37℃の水浴中で消化溶液を保管してください。 注：準備消化溶液の量は、1 5ミリリットルポリスチレン丸で一緒にインキュベートすることができる最大5つの成体マウスの胸腺、のために十分です下部チューブ。 </li><li> 5〜6ミリリットルRPMI-1640を含む60ミリメートルの組織培養皿に胸腺を転送します。 </li><li>リンパ球が流出できるように小片（約1mm 3の正方形）に28 Gインスリン注射針で胸腺を解剖。 </li><li>ガラスパスツールピペットでペトリ皿で​​あるRPMI-1640と胸腺の作品をすすぎます。皿を傾け、胸腺断片が底に沈殿してみましょう。ゆっくり吸引し、ガラスピペットを用いて、RPMI-1640上清を捨てます。解決策はあまり不透明になるまで、ガラスピペットを用いて4-5回5〜6ミリリットルRPMI-1640でこのすすぎ工程を繰り返します。 注：胸腺断片は、胸腺組織の過剰な損失をもたらす、プラスチックに固執する傾向があるので、ガラスピペットは、この段階で推奨されています。残りの手順については、プラスチック製のピペットを使用しています。 </li><li>ステップ1.5で説明したように最後のすすぎの後、上清を捨てます。 3ミリリットルの消化溶液を追加し、5ミリリットル丸底Pに胸腺片とソリューションを転送olystyreneチューブ。 </li><li>チューブ回転にチューブを置き、12回転の回転速度で6分間37℃でインキュベートします。 </li><li>インキュベーション後、胸腺断片は、重力によって、チューブの底に沈殿してみましょう。パスツールピペットを用いて上清を回収し、洗浄溶液10mlと50mlの遠心管に移します。 5分間、300×gで遠心分離し、上清を捨て、10mlの洗浄溶液で細胞を懸濁します。氷の上に保管してください。 </li><li>繰り返しは、5ミリリットル丸底ポリスチレンチューブに胸腺断片に二回1.6から1.8を繰り返します。同じ50ミリリットルチューブに上清をプール。 注：遠心分離は、第2および第3の洗浄のための必要はありません。 </li><li> 50mlの遠心管にチューブ、転送中の任意の残りのフラグメントを打破するために、最終的な消化の後、アップピペットと2ミリリットルのプラスチック血清学的ピペットを用いてダウン。 </li><li> 5分間、300×gで遠心分離50ミリリットルチューブ、上清を吸引、洗浄ブフェの10ミリリットルで再懸濁100μmのセルストレーナーを通してrおよびフィルタ。氷の上に保管してください。 </li></ol>不連続密度勾配でのTECの2濃縮<ol><li> 2.4ミリリットルの密度勾配培地（水中のイオジキサノールのw / vの60％）と9.6ミリリットル洗浄溶液を混合することにより、21％密度勾配溶液を調製します。 </li><li> 2.5ミリリットル洗浄緩衝液中で5分間再懸濁し、300×gでステップ1.11から50ミリリットルコレクションチューブに解離した胸腺細胞を遠心します。 </li><li>細胞懸濁液に20ミリリットルRPMI-1640培地を追加します。 </li><li>電子ピペッターで10ミリリットル血清学的ピペットで21％密度勾配液の12ミリリットルを記入してください。細胞を含有する50mLの遠心チューブの底に血清学的ピペットの先端を配置し、重​​力を介してチューブの底に排出するための密度勾配溶液を可能にします。 <ol><li>穏やかに異なる密度の二層を生成終了するピペットからの密度勾配溶液を排出。 </li></ol></li><li> 600×gでFOで遠心分離スイングバケットローター中で、室温で20分rは。最も遅いレベルでの減速度を設定します。 </li><li>新しい50ミリリットルチューブにトップ層との界面を転送します。 注：のTECのほとんどは界面に保持されます。リンパ球は、遠心分離後、チューブの底に沈殿します。これらの細胞は、他の目的に使用する場合、3回20mLの洗浄溶液でペレットを洗浄。洗浄緩衝液10ml中に細胞を再懸濁。 </li><li> 4℃で6分間、400×gでステップ2.6および遠心機でチューブに40ミリリットルに洗浄液を追加します。ピペットで上清を、吸引を削除します。 </li><li>ステップ2.7で説明したように洗浄し、吸引2回繰り返します。 </li><li>洗浄液の2ミリリットル中に再懸濁し、血球計数器で細胞をカウント。 </li></ol> FACS 3.分離のTEC <ol><li>洗浄液や転送に1×10 6細胞/ 100μlの濃度でステップ2.9から細胞を調整します（フローソーターの指示により推奨されるように、ポリプロピレン、ポリスチレン、）5ミリリットル丸底チューブに細胞。 </li><li> 1×10 6個の細胞当たり2μlの抗Fc受容体のIgGを添加し、4℃で10分間インキュベートします。 </li><li> （：150希釈、サンプルあたり10μlの1）および抗EpCAM（1：100希釈、サンプルあたり10μl）を抗体および以上4℃で20分間インキュベート抗CD45を追加します。 </li><li> 4℃で6分間、400×gで2ミリリットルの洗浄液と遠心で細胞を洗浄します。ピペットで上清を吸引除去します。 </li><li> 1ml中の細胞1×リン酸緩衝生理食塩水（PBS）に再懸濁します。 </li><li> 100μmのストレーナーを通して濾過。 </li><li>仕分け機器に細胞を適用します。その後、側方散乱光（SSC）/前方散乱光（FSC）パネル（死細胞）、13をソートするためCD45-のEpCAM +のために選択された集団にゲート上の最小のセルを除くリンパ球とTEC人口を選択します。 </li></ol> 4. GTEC / EAK骨材のeneration 注：試薬調製と層状フードの下で混合セルを伴う手順を実行します。 <ol><li>滅菌1.5ミリリットルマイクロチューブ中の抗HisタグIgGの4μlの、抗EpCAM IgGの8μlのとPA / Gの2.6μLを混合することにより、PA / Gアダプター複合体を準備し、そして室温で5分間インキュベートします。 </li><li>洗浄溶液、およびピペット低蒸発、96ウェル丸底組織培養プレートの1ウェルに100μlの5×10 4細胞/ mlにステップ3.7からソートのTECを調整します。細胞に14.6μlのPA / Gアダプター複合体を追加します。 4℃で20分間ロッキングプラットフォーム上でプレートを置きます。 </li><li> 200μlの洗浄液で1回洗浄。 6分間400×gでプレートを遠心。マイクロピペットで上清を捨てます。 </li><li> 200μlの10％スクロース溶液で1回洗浄します。 6分間400×gで遠心分離します。マイクロピペットで上清を捨てます。 </li><li> 30μlの10％s内の細胞を再懸濁しウェルあたりショ糖ソリューションterile。 </li><li> EAKIIH610μlの（7.5ミリグラム/ ml）を加え、室温で5分間インキュベートします。 </li><li> TEC混合物にEAK16-II（10mg / ml）の20μlのを追加します。 </li><li>ゲル化を開始するために、システムへの完全培地100μlを加えます。室温で15〜20分間プラットフォームシェーカー上でプレートを置きます。 </li><li> 37℃のインキュベーターでプレートを置き、5％CO 2。 2-4時間後、完全培地の別の100μlのを追加します。 </li><li>使用時まで一日おきに培地交換。 <ol><li>吸引除去し、表面からの培地100μlのマイクロピペットを用いてゲルを乱すことなく、ゆっくりとでは、ウェルの壁にピペットチップを配置することによって、予め温めた培地100μlを追加します。 </li></ol></li></ol> TEC / EAK骨材の5キャラクタリゼーション<ol><li> in vivoでの TEC / EAK凝集体の機能を調べるために、無胸腺ヌードの腎臓被膜下O / N培養と移植後のTEC / EAKヒドロゲルを収集以前に記載された手順11,14を用いて、マウス、。 </li><li> 4-6週間は、抗CD4、-CD8のカクテルで-CD45を移植し、汚れを投稿ヌードマウスからEDTA含有採血管に50〜100μlの血液サンプルを採取することにより末梢におけるT細胞の発生を監視します、および-CD3 12は抗体。 </li><li>単一細胞解析ソフトウェア12を使用して、フローサイトメトリー（FCM）で末梢血白血球におけるTリンパ球の割合を分析します。 </li></ol>

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</ol>

Authors declare no conflict of interest.

TECは、胸腺間質の優勢な集団であると胸腺の構造と機能のために重要な役割を果たしているが、それらは、総胸腺細胞充実の約0.1から0.5パーセントを表します。彼らはまた、細胞死の高いパーセンテージは時折すなわち 、治療は、細胞外マトリックス（ECM）からのTECを解離するために、コラゲナーゼ消化後に観察されている脆弱な細胞です。その希少性（マウス胸腺あたり200,000）お?...

胸腺は、獲得免疫系の機能に必須である病原体反応、自己寛容T細胞の多様な集団の生成を担う主要なリンパ器官です。それは、胸腺細胞の開発、リンパ球前駆細胞として骨髄から移住動的な器官である、胸腺間質のスポンジ状の三次元（3-D）マトリックスを通って移動し、系統仕様と分化を起こし、最終的にのように移住します成熟T細胞。このよくプログラムされたプロセスの成功は、移行胸腺細胞や住宅胸腺上皮細胞（TECを）、胸腺微小環境の整合性を確立し、維持するために必須である胸腺間質の優勢な集団間のクロストークに大きく依存します。 その解剖学的位置とユニークな機能に基づいて、TECのは、2つのサブセットに分けることができます。皮質でのTEC responある（cTECs）1,2- T細胞（正の選択）を制限し、自己MHC（主要組織適合遺伝子複合体）を選択するためのsible、および自己反応性T細胞を除去するために不可欠である髄質（mTECs）中のTEC（負の選択）。多くの要因（ 例えば 、老化、感染症、放射線照射、薬物治療）は妥協適応免疫が得られ、胸腺上皮に不可逆的な損傷を引き起こす可能性があります。多くの試みにもかかわらず、胸腺機能を回復することは、胸腺微小環境を再現する難しさに挑戦してきました。 2次元環境で培養のTECが急激胸腺形成3,4のための重要な遺伝子の発現を減少させる一方、特に、胸腺三次元（3-D）の構成は、のTECの生存および機能に重要です。 EAK16-II（AEAEKAKAEAEAKAK）およびそのC末端histidinylatedアナログEAKIIH6（AEAEKAKAEAEAKAKHHHHHH）は低分子量であり、脱イオンWATEに可溶性である両親媒性オリゴペプチドRが、20mMのNaClを（ヒト体液中の正常な塩濃度は、154 mMで）より高いイオン強度に曝されたとき、β原線維を形成するためにゲル化を受けます。この環境応答性は、3D構造を形成するように多目的なビルディングブロックを作ります。複合タンパク質薬物および他の生体分子を固定するためのアダプタとして機能することができる（：EAKII-H6上のHisタグは、ドッキング機構を、これにより抗HisのIgG / Fc結合組換えタンパク質A / G（PA / GはαH6）提供します例えばヒドロゲル複合5-8に、抗体）。 我々は以前に、ヒドロゲルに固定蛍光標識されたIgG分子は、最大13日間9のための注入部位に保持することができることを実証しました。 αH6時更に、抗CD4 IgGを用いて固定PA / GアダプタがEAK16-II / EAKIIH6（EAK）ヒドロゲルに添加し、CD4 + T細胞を特異的に10を捕捉しました。同様の技術を使用して、我々は最近のTEC Cの3-Dの凝集を実証しています（：PA / G：αEpCAMαH6）TEC-特定の抗EpCAM抗体を運ぶアダプター複合体とEAKハイドロゲルに昇格するウルド。ヌードマウスの腎臓カプセル下に移植した場合、EAKハイドロゲルに埋め込 ​​まれたTECクラスタが効果的に生体内11,12に機能的なT細胞の開発を支援することができます。 ここでは、迅速かつ効果的に蛍光活性化細胞選別（FACS）でのTECを浄化し、EAKハイドロゲルシステムと3次元TEC凝集体を生成するために、我々の方法を示します。

<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.

CD45-間質細胞を濃縮するために密度勾配分離プロトコルを使用しての有効性を調べるために、インタフェースと沈殿リンパ球ペレットの両方から採取した細胞を、抗CD45及び抗EpCAM抗体で染色しました。抗ULEX Europaeusアグルチニン1（UEA1）および抗MHCクラスII抗体の両方がさらにCTECとMTECサブセットを識別するために、染色カクテルに含まれていました。 図1?...

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.

EAK 16-II / EAKIIH6自己組織化ヒドロゲルとFACS精製した胸腺上皮細胞の3-D集約を推進

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

Research

JoVE Journal

Immunology and Infection

6.5K ビュー.  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.

ビデオ: 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.

髄質胸腺上皮細胞の増殖、分化および無差別遺伝子発現を支える3D器官共培養モデル

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

発生生物学

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.

胸腺文化を再集合させる

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.

組織工学のための3Dハイドロゲルの細胞のカプセル化

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

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

Xeno 無料条件下におけるさまざまな剛性とポリビニル アルコール Co イタコン酸ゲルのひと多能性幹細胞文化

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

生物工学

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.

カプセル化と 3 D の細胞の免疫染色のエラスチン様タンパク質ゲルの生産

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

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

腎臓皮質細胞外マトリックス由来ハイドロゲルを製造

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

<meta charset="utf8"></head><body>iPS細胞由来オルガノイドによる胸腺微小環境の再現

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>著者スポットライト:ヒト胸腺オルガノイドによる胸腺上皮細胞とT細胞研究の進歩

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.

三次元フィブリンハイドロゲルにおけるヒト胸腺オルガノイドの形成

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

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

ハイドロゲルアレイにより、3D腫瘍モデルにおけるマトリックス成分および治療薬の効果をスクリーニングするためのスループットの向上が可能

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

EAK 16-II / EAKIIH6自己組織化ヒドロゲルとFACS精製した胸腺上皮細胞の3-D集約を推進

要約

さらに動画を探す

髄質胸腺上皮細胞の増殖、分化および無差別遺伝子発現を支える3D器官共培養モデル

胸腺文化を再集合させる

組織工学のための3Dハイドロゲルの細胞のカプセル化

Xeno 無料条件下におけるさまざまな剛性とポリビニルアルコール Co イタコン酸ゲルのひと多能性幹細胞文化

カプセル化と 3 D の細胞の免疫染色のエラスチン様タンパク質ゲルの生産

腎臓皮質細胞外マトリックス由来ハイドロゲルを製造

三次元フィブリンハイドロゲルにおけるヒト胸腺オルガノイドの形成

三次元フィブリンハイドロゲルにおけるヒト胸腺オルガノイドの形成

三次元フィブリンハイドロゲルにおけるヒト胸腺オルガノイドの形成

ハイドロゲルアレイにより、3D腫瘍モデルにおけるマトリックス成分および治療薬の効果をスクリーニングするためのスループットの向上が可能

EAK 16-II / EAKIIH6自己組織化ヒドロゲルとFACS精製した胸腺上皮細胞の3-D集約を推進

要約

さらに動画を探す

髄質胸腺上皮細胞の増殖、分化および無差別遺伝子発現を支える3D器官共培養モデル

胸腺文化を再集合させる

組織工学のための3Dハイドロゲルの細胞のカプセル化

Xeno 無料条件下におけるさまざまな剛性とポリビニル アルコール Co イタコン酸ゲルのひと多能性幹細胞文化

カプセル化と 3 D の細胞の免疫染色のエラスチン様タンパク質ゲルの生産

腎臓皮質細胞外マトリックス由来ハイドロゲルを製造

三次元フィブリンハイドロゲルにおけるヒト胸腺オルガノイドの形成

三次元フィブリンハイドロゲルにおけるヒト胸腺オルガノイドの形成

三次元フィブリンハイドロゲルにおけるヒト胸腺オルガノイドの形成

ハイドロゲルアレイにより、3D腫瘍モデルにおけるマトリックス成分および治療薬の効果をスクリーニングするためのスループットの向上が可能

Xeno 無料条件下におけるさまざまな剛性とポリビニルアルコール Co イタコン酸ゲルのひと多能性幹細胞文化