Name: 3d organotypic co-culture model supporting medullary thymic epithelial cell proliferation, differentiation and promiscuous gene expression
Uploaded: 2015-07-30T13:00:00.000+00:00
Duration: 6 min 47 s
Description: Watch this Scientific Journal Video about 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression at JoVE.com

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

本研究では、Regierungspräsidiumカールスルーエの倫理委員会によって承認されています。すべての動物は、ドイツ癌研究センター（DKFZ）で特定の病原体を含まない条件下で飼育しました。生後1~7日に至るまで、すべての培養実験の仔マウスのために使用しました。 胸腺からmTECsの1の単離注：以前に1滅菌条件下で説明したように、次のように次の消化工程はいくつかの変更を加えて実施しました。 <ol><li>仔マウスを刎ねると胸腺を削除します。 RPMI（5％FCSを含む）1640培地を含むペトリプレートに氷上で胸腺を置きます。 </li><li> 〜5〜30ミリリットルRPMI培地で丸底チューブに細かい、小さな作品や場所に胸腺をカットし、穏やかに室温で10分間、磁石を用いて撹拌します。 </li><li>その後、主に胸腺細胞を含む上清をデカントし、collagenaの1ラウンドで残りの組織を順次ダイジェスト37℃で25分ごとに、コラゲナーゼ/ディスパーゼ（0.2 mg / mlの1.2 U / mlの最終濃度）、続いて37℃でそれぞれ（0.2 mg / mlとし、最終concentartion 57 U / ml）で15分間SEタイプIV、胸腺まで磁気撹拌しながら水浴中でCは完全に消化されます。三つの胸腺への2つあたり1ミリリットルの酵素を使用してください。 </li><li>パスツールピペットで一度、7-10分の組織を攪拌します。コラゲナーゼ/ディスパーゼの画分をプールし、70ミクロンガーゼでろ過します。 </li><li>磁気細胞ソーティング（MACS）によりmTECsを豊か。以前11に記載され、 図1に示すように、磁気セルソーティングによってmTECsの精製を行います。 注：（1で使用し、G8.8：100希釈）および抗EpCAMバイオ：抗CD80-PE（100希釈16-10A1、1で使用します）mTECsの磁気選別のために、我々は、以下の抗体を使用した12。 MACSを使用して未成熟および成熟mTECsは次のように定義された： -のEpCAM + CD80 -及びCD45 - CD45 CD80 + respectivはエリー。 </li><li>以下に説明するようにMACS精製後（未熟mTECsの純度= 83.1±6.3％と成熟mTECs = 79.23±3.42パーセント）は、（2.3節）器官培養物にmTECsをシード。 </li><li>以下の抗体を用いて、CD45 MACS枯渇後（100ミクロンノズルを使用して）FACSによるあるいは、ソートmTECs：抗CD45-PerCPを（30-F11、1で使用：100希釈）、抗Ly51-FITC（6C3、1で使用：100希釈）、抗EpCAM-Alexa647（G8.8、1で使用：500希釈）および抗CD80-PE（16-10A1、1で使用：100希釈）。 （4日目）：ヨウ化プロピジウム（5,000 1）を使用して、死細胞を除外します。それぞれのEpCAM + CD80 + - CD45 - - Ly51 -のEpCAM + CD80 -およびPI - CD45 - Ly51 PI：FACSを使用して、未成熟および成熟mTECsは、次のように定義されました。 </li></ol> 2. 3D器官共培養（OTCs） 注：keratinocytの器官培養のための3D-真皮構築物先に説明したように9,13 ESを調製しました。すべてのステップで、細胞を37℃、5％CO 2でインキュベートしました。次のようにmTECsを使用してOTCsはわずかな変更を加えて調製しました。 <ol><li>ヒト線維芽細胞の調製 注：以前に9説明したように、ヒト皮膚線維芽細胞は、脱epidermised真皮の外植片培養物から得られました。 <ol><li>簡潔には、ヒトの皮膚（〜5cmの長さ）のストリップを切断し、サーモリシン、4℃でO / N（10mMのヘペス（pH7.4）で生理食塩水中の0.5mg / ml）で処理します。 </li><li>その後、ピンセットを用いて真皮から表皮を分離します。 </li><li>細かく10センチメートルペトリ皿に小片、所定の位置に真皮をカットし、無菌気流下で1〜2時間乾燥させます。 DMEM、20％FBSを含むと定期的に外植片を補います。 </li><li>外に成長している線維芽細胞を分割すると、コンフルエント植片は、さらに詐欺のためにプレートに付着し続けることを確認して、0.1％トリプシンを使用して（通常は約3週間）伸長のsecutiveラウンド。 </li><li> 14それらを、10％FBSを含むDMEMで最大3回、同じ外植片からの線維芽細胞を展開し、クライオ保ちます。各実験シリーズの線維芽細胞の同じバッチを使用してください。 注：線維芽細胞は、真皮同等物のセットアップのために照射されませんでした。 </li></ol></li><li>足場の調製<ol><li>正確に12ウェルフィルターインサートに適合するように鋭利な11ミリメートルの直径の金属パンチャーを使用して、よく画定円に0.4〜0.6ミリメートルの厚さのビスコース、不織繊維材料（Excelシートを支える製品の詳細を）カット。その後、足場として、12ウェルフィルターインサート（ポリエステル毛管細孔膜、3μmの孔サイズ）に配置します。滅菌12ウェルプレートに、完全なフィルタ設定を置きます。 </li><li>フィブリノーゲンおよびトロンビ​​ンの組み合わせからなる手術のためにフィブリン糊キットを使用してフィブリンゲルを準備します。フィブリノーゲンならびに8 mg / mlで、10のキットのトロンビン成分を事前に希釈それぞれ単位/ ml、。以下のように12ウェルプレートの1ウェル進行。 <ol><li> Ca 2+およびMg 2+、pH7.0のせずに、100μlのリン酸緩衝生理食塩水（PBS）を用いて100μlのフィブリノーゲン（8ミリグラム/ ml）を希釈します。 27万線維芽細胞を含む100μlのFCSで100μlのトロンビン（10単位/ ml）を希釈します。 </li><li>を2mg / mlのフィブリノーゲンおよび2.5単位/ mlのトロンビンの最終濃度で、その結果、（1混合物1）200μlのフィブリノゲンを追加している、足場上に細胞ミックス線維芽細胞を含むトロンビンの200μLを分注します。よく混ぜ、穏やかにピペッティング（1日目）によって足場の全域に​​わたって均等に分配します。 注：37℃で30分後、線維芽細胞を囲む血餅が完全に足場の内部空間を充填し、滑らかな上面を形成し、形成されているであろう。 </li></ol></li></ol></li><li> mTECsとの共培養<ol><li>前培養のために、器官培養を沈めますメディアの変化と、10％FBS、50μg/ mlのL-アスコルビン酸および1 ngの/ mlのTGF-β1を含むDMEM 4-5日間一日おきに。 </li><li> MTEC播種の日に、RFAD媒体によってメディアを交換します（1：1 DMEM + DMEM / F12）を、10％FBSを、10 -10 M、コレラ毒素、0.4 mg / mlのヒドロコルチゾン、50μg/ mlのL-アスコルビン酸、したがって、線維芽細胞によって分泌されるセリンプロテアーゼによって早熟線維素溶解を防止するRANKリガンド（を0.1μg/ ml）および500単位/ mlのアプロチニン、。 </li><li> 7-8時間後、セットアップ共培養を、線維芽細胞の足場の上にウェル当たり100μlの容量で25万mTECs（完全mTECs、またはCD80 LOおよびCD80 こんにちはサブセットのいずれか）を播種することにより。ノイバウアー室（4日目）を用いて細胞数を計測します。 注：胸腺3D器官培養物を空気持ち上げられる皮膚OTCsとは異なり、メディア内に浸漬されているすべての回で。 </li><li> 24時間のインキュベーションの後、今containi（ステップ2.3.2）上記のような培地で培養（総培地交換）を供給アプロチニン、250単位/ ml（5日目）の減少量をngの。 </li><li> mTECsの増殖活性を評価するために、培養の終了前4時間OTCsにEDU（6.7μM/ mlの、 すなわち、10μM/ウェル）を追加します。各培養標本のか流れによって2つのセクションでいずれかのカウントK14 + EDU +細胞による、mTECsの増殖指数を決定ケラチン14の共染色と組み合わせたエドゥイメージングキットに記載されているように店頭凍結切片の染色を行いますフローサイトメトリー（EDU 1.7節ではなくのLy51-FITCが1でCDR1-PB 15を使用し、フローサイトメトリー：100希釈し、2.3.6.4）。 </li><li>共培養の4-7日後、RNA分離、凍結切片またはFACS分析（日8-11）のためOTCs、処理を終了します。 <ol><li>ウェルインサートのフィルターから足場/真皮同等物を分離し、ピンセットを用いて培養を終了します。 </li><li>クライオセクショニングについては、OCT化合物で全体OTCを埋め込むとLiで凍結クライオセクショニング前に頭の良い窒素蒸気。使用するまで-20℃でクライオスタットとストアを使用して、5-7ミクロンの厚さのOTCのセクションを準備します。凍結切片OTCsの免疫組織化学（AF64 1で使用：1,000希釈）抗ケラチン14を使用してください：抗体、および抗ビメンチン（100希釈GP53、1で使用します）。それぞれの二次抗体を用いた間接免疫組織化学染色を行います。 </li><li> RNA単離のために、溶液2 mlのRNaseフリーのチューブのスクリューキャップで（フェノール及びチオシアン酸グアニジンを含む）を変性1ミリリットルを追加します。メスで小片に全体OTCをカットし、変性溶液を含むチューブに追加します。機械的には、（サンプルは、この時点の後に-80℃で保存することができる）2分間氷上の間に配置し、6.0の速度で30秒間回FASTPREP器具でOTCsを細断処理しました。 -80℃で凍結した場合は、氷上で解凍。 4℃で10分間、11,500-13,000 rpmでチューブを遠心。 RTおよびFで5分間インキュベートし、新しいチューブに上清を移し製造業者によって記載されるように酸性チオシアン酸グアニジニウム - フェノール - クロロホルム抽出を用いollow RNA単離プロトコール。 </li><li> FACS分析のために、足場を取り外し、フィブリン/線維芽細胞/ MTECゲルから膜を分離します。メスで細かく切断し、ゲルを〜20分間、または完全に磁気撹拌しながら水浴中で37℃にてコラゲナーゼ/ディスパーゼ2mlで消化されるまで、FACSチューブにそれを消化します。一度5分パスツールピペットを有する酵素液を攪拌します。完全消化した後、70μmのフィルターを通して細胞懸濁液をフィルタリングします。 1.7に記載のように抗体を用いて、単一細胞懸濁液を染色し、フローサイトメトリーによって分析します。 </li></ol></li></ol></li></ol>

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RTOCsと並んで、3D OTCsはTEC分化およびPGEのメンテナンス/誘導他（I）と比較した（ 表1）「単純化された3Dの文化」を使用しての面ではるかに優れていたことで-線維芽細胞を、単独で足場なし。二次元システムを用いて-のTEC 10と線維芽細胞/フィーダー細胞を共培養し、TECのクローンの開発が、PGEが失われた式（III）3T3-J2細胞、（IV）マトリゲルまたは（V）ECM成分（未発表?...

残りの2％を一括胸腺間質（ すなわち、上皮細胞、樹状細胞、マクロファージ、B細胞、線維芽細胞、内皮細胞）を構成する種々の細胞で構成されていながら、開発胸腺細胞は、胸腺の約98％を占めています。外側皮質上皮細胞（cTECs）は、骨髄、多能プレT細胞におけるT細胞系譜の誘導と自己MHCのポジティブ選択からプロT細胞の移民を調達未熟胸腺細胞を制限しました。内側の髄質胸腺上皮細胞（mTECs）はいずれかの誘導陰性選択またはT調節細胞系譜へのそれらの偏差による自己ペプチド/ MHC複合体のための高親和性TCRを有するもの胸腺細胞の寛容の誘導に関与しています。中央寛容誘導のコンテキストでは、mTECsは、彼らがこのように周辺自己ミラーリング組織限定自己抗原（TRAS）の広いスペクトルを表現するという点でユニークです。この現象は、無差別遺伝子発現（PGE）と呼ばれています1,2。 様々な短期の2D培養システムは、必ず最初の2日3-6内PGEおよびMHCクラスII、FoxN1とアイレのような重要な調節分子の喪失をもたらしたように、この魅力的な細胞型の最新の研究は、ex vivoで単離された細胞に依存しています。これは、特定の成分および胸腺の無傷の3次元網目構造の特徴は、2次元モデルで欠落した、しかし不明でした。再凝集胸腺器官培養（RTOC）は無傷の胸腺の微小環境7に、他方では、これまでのところ唯一の3D一方で、T細胞の発達の研究を可能にするシステム、および間質細胞生物学でした。しかし、RTOCsは一定の制限がある、 すなわち、それらは既に、細胞の複合混合物を含む、胎児間質細胞の入力を必要とし、5〜10日の最大の培養期間に耐えます。 体外培養系における還元主義の欠如は、の研究を妨げていますT細胞の発達と胸腺器官形成のいくつかの側面はなく、少なくとも分子PGEの調節およびmTECsの発生生物学との関係。 皮膚および胸腺の上皮細胞の構造化された組織の近接関連性のために、我々は、in vitroでケラチノサイトの分化をエミュレートするため、真皮等価物を作成するために独自に開発された3D器官培養（OTC）のシステムを選択しました。 OTCシステムは、ケラチノサイトが8,9を播種し、その上にフィブリンゲル中に閉じ込められている真皮線維芽細胞を重層不活性足場マトリックスで構成されています。ここでは、精製されたmTECsでケラチノサイトを置き換えます。このモデルの基本的な機能を維持しながら、私たちは、特定のパラメータを最適化します。 採用店頭モデルmTECsでは、増殖分化を受け、MTECのアイデンティティとPGEを維持し、このように密接にインビボ mTECs開発の模倣<sup&gt; 10。このテクニカルノートでは、胸腺OTCsの段階的なセットアップを可能にする詳細なプロトコルを提供します。

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

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

イントラ胸腺T細胞の発達は、様々な間質細胞、 すなわち、非T細胞から構成される複雑な三次元網目構造を必要とします。胸腺細胞は、末梢に先立ってエクスポートに、順次義務のチェックポイントを通過しながら、高度に調整された時間的、空間的順序でT細胞受容体レパートリーの生成と選択に続いて、すなわち、T細胞系列のコミットメントを、この足場を横断します。この足場を形成する2つの主要な居住細胞型（cTECs）皮質及び髄質胸腺上皮細胞（mTECs）です。 mTECsの重要な特徴は、多数の組織限定抗原のいわゆる無差別式です。これらの組織制限抗原は、それぞれ、自己寛容を生じ、mTECsまたは胸腺樹状細胞によって直接的または間接的に胸腺細胞の未成熟するために提示されます。 cTECsとmTECsの発生経路と機能をエミュレートし、in vitroでの適切なモデルは、現在、LACですキング。適切な実験モデルの欠如は、例えば、まだ不十分な細胞および分子レベルで理解されて無差別遺伝子発現の分析を妨げました。我々は、単離されたmTECs ex vivoで培養するために3Dの器官共培養モデルを適応しました。このモデルは、もともとインビトロでの皮膚同等物を生成するような方法で、ケラチノサイトを培養するために考案されました。 、CD80 こんにちは 、アイレ陽性mTECs、RANKLへの（ii）の反応性、およびFoxN1の（iii）の持続的な式に（I）の増殖およびCD80 LO、アイレ陰性の分化：3DモデルはMTEC生物学の重要な機能的特徴を保存しますアイレとCD80 こんにちは mTECsで組織限定遺伝子。

私たちは、もともとケラチノサイト9 のインビトロ長期培養のために開発された3D器官の共培養モデル（3D OTC）を採用しました。 MACS富化mTECsは（MACS濃縮スキーム図1参照）フィブリンゲルおよび封入された線維芽細胞からなる足場に播種しました。線維芽細胞は、 インビトロで mTECsをサポートする必須の細胞外マトリックス（ECM）を提供します。 MTECsは（OTC?...

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

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

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

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 ...

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

Research

JoVE Journal

Developmental Biology

10.6K ビュー.  German Cancer Research Center (DKFZ). Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression. 

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

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

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

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

マウス誘導多能性幹細胞由来腫瘍抗原特異的胸腺移民を生成する三次元胸腺培養システム

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

がん研究

Reaggregate Thymus Cultures

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

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

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

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処理した胸腺器官培養の準備

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

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

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

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

細胞外マトリックス内に埋め込まれたエンジニアリング三次元上皮組織

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

生物工学

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

微小トリプシン法によるモザイク乳腺オルガノイドの生成

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

発生生物学

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

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

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

細胞外マトリックスベースおよび細胞外マトリックスフリーのマウス精巣オルガノイドの生成

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

Preparation and Applications of Organotypic Thymic Slice Cultures

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

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

調製及び器官胸腺スライス培養の応用

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

免疫・感染学

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

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

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

上皮癌細胞のスキャフォールドフリー3D培養におけるヘテロ多細胞間質細胞による浸潤の促進

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

<meta charset="utf8"></head><body>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 ...

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

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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