We would like to acknowledge Connecticut Children's Medical Center Strategic Research Funding for supporting this work.

インフォームドコンセントは、小児患者の両親/保護者から入手し、治験審査委員会（IRB＃13から094）に従ってた後食道生検が得られました。 1.殺菌楽器とゼラチン溶液<ol><li>オートクレーブ鉗子、かみそりの刃及び汚染を防止するために、組織を処理する前に鋏。 </li><li> 0.1％ゼラチン溶液200mLを作るために、ゼラチンを0.2gと蒸留水200mLを組み合わせます。使用前にオートクレーブし、冷却。 </li></ol> 2.コーティング組織培養プレート<ol><li>前の細胞単離に約2時間、組織培養皿、（100 mm）をカバーし、細胞培養インキュベーター中、37℃でインキュベートするのに十分な0.1％のゼラチンを加えます。 注：これらのプレートはまた、事前に作製し、使用する前に、インキュベーター内で維持することができます。 </li></ol> 3.解離のための酵素溶液を作ります<ol><li>前細胞単離するのに約30分、秤量バイアル上ミリグラム濃度あたりの単位に基づいてバランスにディスパーゼの10単位。 37℃の水浴中で10 mLのダルベッコ改変イーグル培地（DMEM）のと場所で15 mLコニカルチューブ内の粉末を配置します。 </li></ol> 4.メイキング細胞培養培地<ol><li> F12の35ミリリットル（ハム培地）、DMEMの11.5 mLを、L-グルタミンの0.5 mLを、100×ペニシリン/ストレプトマイシンの0.5 mLを、2.5mLの：無菌コニカルチューブに以下の成分を混ぜて、条件付き再プログラミング媒体の50mLのを作るために、ウシ胎児血清（FBS）の、ヒトインスリンの250μgのヒト上皮成長因子（EGF）の500 ngの。 </li><li> 3T3培地の500ミリリットルを作るために、FBSの50ミリリットル、100×ペニシリン/ストレプトマイシン及びDMEM 500mLのとL-グルタミンの5 mLを5 mLのを混ぜます。 </li><li>ケラチノサイト無血清培地の500ミリリットルを作成するには、EGF及び（メディアで提供）は、ウシ下垂体抽出物（BPE）のサプリメントを解凍。基本培地の500ミリリットルボトルにサプリメントを追加し、10の5 mLを加え0xのペニシリン/ストレプトマイシン</li></ol>フィーダーソースとして5.培養し、照射3T3細胞<ol><li> DMEM、10％FBS、1×ペニシリン/ストレプトマイシンおよび2mM L-グルタミンを含む、3T3培地を準備します。 </li><li> 文化少なくとも3日間患者サンプルの到着前組織で処理されたT-75フラスコ中の3T3細胞。 <ol><li>前患者サンプルの到着、フラスコ当たり0.25％トリプシンEDTA 5mLのを使用してトリプシン処理3T3細胞へと5分間、またはほとんどの細胞が浮遊するまで37℃でインキュベートするだけ</li></ol></li><li>反応を停止さ3T3培地の5ミリリットルを追加します。細胞懸濁液を吸引し、15 mLコニカルチューブに移します。 300×gで5分間スピン。 </li><li>培地を吸引し、細胞計数のためにトリパンブルーの定義された量で細胞を再懸濁します。死細胞を排除するためにトリパンブルーの10μLで細胞の10μLを兼ね備えています。細胞計数のための血球計数器の負荷、この溶液10μLを。 注：このプロトについてcolが、照射された3T3細胞のための播種密度は100ミリメートルプレート60万の細胞に等しい、1cm 2当たり10,900細胞です。 </li><li> 50 mLコニカルチューブに3T3培地25mLに患者サンプルを再懸濁当たりの100mmプレートにプレートし、3000ラドで照射するのに必要な細胞の数をアリコート。 </li><li>照射後、100ミリメートルのプレートあたり5 mLで条件付き再プログラミング培地で300×gで再懸濁で5分間細胞をスピンダウン。 </li><li>患者の細胞のメッキは、プロトコルの後半に行われるまで脇にチューブを設定し（ステップ7.6を参照してください）。 </li></ol> 6.小児食道生検の保存や輸送、入手方法<ol><li>コニカルチューブ15 mLに100μg/ mLのprimocinとの完全なケラチノサイト無血清培地5 mLを加え、氷上で医療施設にそれを持って来ます。 注：媒体のこれらの管は、最大2ヶ月間4℃で保存することができます。 </li><li> TIで約8食道生検を取得内視鏡検査の私と、以下のように区切ります。 <ol><li> 100μg/ mLのprimocinと濡れた氷の上の場所との完全なケラチノサイト無血清培地を含む15 mLコニカルに6生検を置きます。 </li><li>濡れた氷の上に緩衝ホルマリンと場所の5mLに1生検を置きます。 </li><li>最適切削温度化合物とcryomold小さな生検で1生検を置きます。 <ol><li>直ちに液体窒素中に配置されたイソペンタンのビーカーに、この金型をドロップします。いったん凍結し、標本袋にcryomoldを置き、発泡スチロール容器内のドライアイス上で保存します。 </li></ol></li></ol></li><li>密封可能な袋の内側とウェット氷を含む輸送用ボックスに培地またはホルマリンで生検を含むチューブを置きます。密封し、バイオハザードラベルと箱にラベルを付け、実験室に運びます。 注：ドライアイスを含むコンテナおよび冷凍生検はまた、密封し、バイオハザード標識で標識されています。 </li></ol> 7.処理ダウンストリーム文化と分析のために、患者の組織<ol><li>実験室に到着すると、下流の切片またはRNA抽出のためのCの冷凍庫-80にドライアイス上で凍結した生検を転送します。ホルマリンにサンプルを置きます。 4℃の冷蔵庫に保管し、一晩固定することができます。 </li><li> 300×gで、残りの生検をスピンダウンし、培地を吸引除去します。円錐管、シールのディスパーゼ溶液10mlを加え、それを15分間、水浴中で37℃でインキュベートすることを可能にします。 </li><li>インキュベーション後、300×gで生検をスピンダウン。 0.05％トリプシンEDTA 5mLの培地を再懸濁生検を吸引します。 </li><li> 100ミリメートル滅菌プレートに生検を含むトリプシンEDTA 5mLのを転送し、可能な限り細かいとして2滅菌カミソリの刃を使用して生検をミンチ。 </li><li>新しい15 mLコニカルにみじん切りの生検を含むトリプシンEDTA 5mLのを転送します。 0.05％トリプシンEDTAの追加5 mLでプレートを2回すすぎ、円錐に追加チューブ。 37℃の水浴中で10分間チューブをインキュベートします。 </li><li>インキュベーション後、5分間300×gで（1.4 RPM）で生検をスピンダウンし、培地を吸引除去します。円錐管だけでなく、放射線照射フィーダー細胞を含む条件付き再プログラミング培地5 mLに条件付き再プログラミング媒体の5 mLを加え（ステップ5.7を参照してください）。 </li><li>組織培養皿からゼラチンを吸引した後、細胞懸濁液とプレートの10 mLにROCK阻害剤（1μL/ mLで）を追加します。 37℃でインキュベートします。 </li></ol> 8.細胞を培養すると拡大<ol><li>文化の約5日後、刻んだ生検断片を含む初期培地を吸引し、5mLの滅菌PBSでシャーレに2-3回すすいでください。 10μMでのROCK阻害剤を含むプレートに新しいメディアを追加します。 </li><li> 70％の密集度に到達すると、細胞を拡大。プレートに0.05％トリプシンEDTA 5mLのに加え、37℃のインキュベーターに目を削除するには、no以上2.5分間インキュベートEフィーダー細胞。そのインキュベーション後、トリプシンEDTAを吸引し、PBS 5mLでプレートをすすぎます。 </li><li> PBSを吸引し、プレートに0.25％トリプシンEDTA 5mLのを追加し、5〜10分間、または細胞がプレートからルーズになるまでインキュベートします。反応を停止し、50 mLコニカルチューブに流体のすべてを譲渡し、5分間、300×gでスピンダウンするプレートに培地の等量を追加します。 </li><li>条件付き再プログラミング媒体の定義された量で細胞を再懸濁し、トリパンブルーの10μLとサスペンションの10μLを混合します。総細胞数を決定するために、血球計数器でカウントします。 </li><li> 、細胞を拡大120万と一緒に150ミリメートルプレートに100万人の食道細胞を追加するには15ミリリットルの全容量で3T3細胞を照射しました。 10μMで新鮮なROCK阻害剤を追加し、ゼラチンコートした150ミリメートル皿に細胞懸濁液を追加します。 </li></ol> 9.フリーズおよびストアヒト食道上皮細胞<ol><li>凍結培地にするために、10％を追加条件付き再プログラミング媒体にDMSO。トリプシン処理し、計数した後、新しい50 mLコニカルチューブに凍結し、5分間、300×gでスピンダウンする細胞を分取。 </li><li>培地を吸引し、クリオバイアル当たり2ミリリットルで培地を凍結追加します。細胞懸濁液が均質になったら、前標識されたcyrovials 10 6細胞のアリコート。長期液体窒素貯蔵にバイアルを移動する前に、少なくとも24時間、-80℃で凍結容器内に配置します。 </li></ol>

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The authors declare they have no competing financial interests.

患者の生検から食道上皮細胞を分離し、拡大するために最も重要な手順は次のとおりです。1）適切最小限の細胞死と生検組織を解離させます。 2）ROCK阻害剤は、すべての培地交換で細胞培養培地に添加されていることを確認します。 3）推奨よりも多くのフィーダー細胞を使用しないでください。 4）クリーンな無菌の文化を維持します。 5）コンフルエンスに到達する直前に継代細胞。 <...

食道組織工学と好酸球性食道炎（EoEのは）過去10年間で多くの研究室での研究の焦点となっています。このような食道閉鎖症などの先天性の欠陥は、1を食べることができないことにつながる食道の不完全な開発につながる約1 4000で出生、に見られます。 EoEのの発生率と罹患率は1993年に病気のエンティティの識別EoEのの発生率は、人年あたり0.7から10 / 100,000に変化し、有病率は0.2〜43 /10万2の範囲であって以来、増加しています。長いギャップ食道閉鎖症を治療する新しい魅力的外科的アプローチは、患者自身の細胞を用いた移植のための組織構築物を生成することにあります。合成足場に関連してこれらの細胞は、免疫抑制を必要としない自己の構築物を生成します。いくつかのグループには、すでに私たちを調査し始めています7 -食道組織工学3だけでなく、ネイティブの食道上皮細胞の使用のための幹細胞様細胞のeは粘膜4を再入力します。小児患者の食道内に存在している疾患は、多くの場合、診断または介入なしで勉強するのは難しいです。また、利用する動物モデルまたはin vitroで、正確な疾患の病因または患者特異的な差異8を包含しないEoEのような小児疾患の細胞株モデルを不死化。したがって、抗原を誘発する特定の疾患を同定する基礎となるメカニズムを評価し、薬物治療を調べるために、インビトロで患者の疾患の過程を研究する能力は、新規であると患者の治療を助けることができる情報を臨床医に提供します。 TISSUにおける使用のために提案されてきた多くの自己または患者特異的細胞型がありました電子工学およびヒト疾患の病因を研究。しかしながら、これらの細胞型のいくつかは、大きな足場をシードまたはインビトロ試験で高いスループットを実行するために特定の表現型の十分な細胞を生成する能力が限られています。多能性又は多分化能性幹細胞の使用は、多くの研究の議論の主題となっているが、これらの細胞を使用するための制限および欠点がよく9に記載されています。ヒト胚性幹細胞の使用は非常に議論し、多くの倫理的な問題を提示されています。最も重要なことは、これらの細胞は、それらが前の生きたホスト10にそれらを提供すること、それらの多能性状態と区別されていない場合、腫瘍に類似している奇形腫を形成します。また、胚性幹細胞の使用は、患者特異的ではないであろうと、同種異系応答および免疫抑制10の必要性を誘発する可能性があります。人工多能性幹細胞（iPS細胞）は、そのでき多能性細胞であります患者自身の細胞から誘導することができます。このような皮膚細胞などの体細胞は、統合および非統合の様々な技術を用いて、多能性状態に誘導することができます。次いで、これらの細胞を組織工学または疾患の研究のための患者特異的細胞源として働きます。これらの細胞内への不必要な遺伝物質の統合は、多くが記載されているとの配列が完全に除去性IPSCは、彼らが11を誘導された細胞型に向けてエピジェネティックな「記憶」を節約するために表示されている場合でも問題です。移植11の前に分化していない場合、これらの細胞は、 生体内で奇形腫を形成します。多くの分化プロトコルは、しかし、分化の終了時に得られた細胞型が均一とOではないことに注意することは非常に重要であり、上皮系統12、13、14に着目し研究されていますNLY目的の細胞型の割合を有します。このことは、低い収率および所望の細胞型を精製する必要が生じます。 iPS細胞は、潜在的な患者特有の細胞源であるが、組織工学又は疾患の調査のどちらかのために目的の細胞型を得るためのプロセスは非常に非効率的です。 肺15、胸16、小腸17、結腸18、膀胱19および食道20：ヒト上皮細胞が正常に含む人体の両方の罹患および非罹患種々の組織から単離されています。ヒト初代細胞の表現型は、21、22を維持する通路の有限数があることを留意することが重要です。残念ながら、このことは、疾患の研究のために又は操作足場に播種するために必要なセルの数移植のために達成されない場合があります。したがって、新たな技術が依然として上皮表現型を維持しながら、患者の細胞を拡大するために必要とされます。フィーダー細胞及びROCK阻害剤を用いた、正常および癌性上皮細胞の再プログラミング条件は、Liu らにより2012年に記載されました。 2 3。この技術は、照射されたフィーダー細胞を、ROCK阻害剤および条件付き再プログラミング媒体を使用して、前立腺癌および乳癌の生検から得られた癌性上皮細胞を拡大するために利用されました。目標は、このような薬物スクリーニングなどのin vitroアッセイのための十分な細胞を生成することでした。この技術は、幹細胞または高度に増殖性である前駆体のような状態へのこれらの細胞の「再プログラミング」によって無期限に上皮細胞を拡大することが可能です。これらの細胞は、非腫瘍形成性であり、奇形23、24を形成する能力を有さないことが実証されています。また、無染色体異常または遺伝子操作は、この技術23,24を用いて培養中のこれらの細胞を継代した後に存在しました。最も重要なことは、これらの細胞は、目的のネイティブな細胞型に分化することができます。したがって、この技術は不死化を必要とせずに、疾患の調査や組織工学のための患者特有の上皮細胞の大規模な貯水池を提供しています。 疾患過程を研究するために、特定の器官の上皮組織を入手することは、患者のリスクを常に頻繁に制限されないことが可能です。食道疾患や欠陥を患っている患者では、内視鏡生検の取得は解離し、条件付きでその患者の食道の粘膜に特異的である不定細胞源を提供するために再プログラムすることができ上皮組織を得るための低侵襲性アプローチです。これは、その後、in vitro試験を可能にします上皮細胞の潜在的な治療のための疾患過程と画面を評価しました。非常にこのアプローチから利益を得ることができる1つの疾患過程は、食道8のアレルギー性疾患として記載された好酸球性食道炎です。アレルギー試験ならびに治療的アプローチは、患者自身の上皮細胞を用いてin vitroで評価することができ、このデータは、個別の治療計画を開発するために、治療する医師に渡すことができます。小児患者からの内視鏡生検を得ることに関連して、条件付き再プログラミングの技術は、任意の患者から無期限に正常食道上皮細胞を拡張する機能を提供しています。この細胞源は、したがって、欠陥、病気や外傷のための患者固有外科的選択肢を提供するために、天然または合成の足場と一緒に組んですることができました。不定細胞数を持つことは、完全に再播種持って食道のコンストラクトを設計に役立つだろう残りの細胞タイプの再生を促進するのを助けるために、食道の上皮細胞でルーメン。

<table><tbody><tr><td>Primocin</td><td>InVivogen</td><td>ant-pm2</td><td></td></tr><tr><td>Isopentane</td><td>Sigma Aldrich</td><td>277258-1L</td><td></td></tr><tr><td>Gelatin From Porcine Skin</td><td>Sigma Aldrich</td><td>G1890-100G</td><td></td></tr><tr><td>DMEM</td><td>Thermofisher Scientific</td><td>11965092</td><td></td></tr><tr><td>Cryomold</td><td>TissueTek</td><td>4565</td><td></td></tr><tr><td>Cryomatrix OCT</td><td>Thermofisher Scientific</td><td>6769006</td><td></td></tr><tr><td>15 mL Conical Tubes</td><td>Denville Scientific</td><td>C1017-p</td><td></td></tr><tr><td>Complete Keratinocyte Serum Free Medium</td><td>Thermofisher Scientific</td><td>10724011</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Thermofisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Glutamax</td><td>Thermofisher Scientific</td><td>35050061</td><td></td></tr><tr><td>Insulin Solution</td><td>Sigma Aldrich</td><td>I9278-5ml</td><td></td></tr><tr><td>Human Epidermal Growth Factor (EGF)</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>ROCK Inhibitor (Y-27632)</td><td>Fisher Scientific</td><td>125410</td><td></td></tr><tr><td>F-12 Medium&amp;nbsp;</td><td>Thermofisher Scientific</td><td>11765054</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Denville Scientific</td><td>FB5001</td><td></td></tr><tr><td>Dispase</td><td>Thermofisher Scientific</td><td>17105041</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25300062</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25200072</td><td></td></tr><tr><td>100 mm Dishes</td><td>Denville Scientific</td><td>T1110-20</td><td></td></tr><tr><td>150 mm Dishes</td><td>Denville Scientific</td><td>T1115</td><td></td></tr><tr><td>50 mL Conicals</td><td>Denville Scientific</td><td>&amp;nbsp; C1062-9&amp;nbsp;</td><td></td></tr><tr><td>Phosphate Buffered Saline Tablets</td><td>Fisher Scientific</td><td>BP2944-100</td><td></td></tr><tr><td>5 mL Pipettes</td><td>Fisher Scientific</td><td>1367811D</td><td></td></tr><tr><td>10 mL Pipettes</td><td>Fisher Scientific</td><td>1367811E</td><td></td></tr><tr><td>25 mL Pipettes</td><td>Fisher Scientific</td><td>1367811</td><td></td></tr><tr><td>9&quot; Pasteur Pipettes</td><td>Fisher Scientific</td><td>13-678-20D</td><td></td></tr><tr><td>NIH 3T3 Cells</td><td>ATCC</td><td>CRL1658</td><td></td></tr></tbody></table>

conditional reprogramming of pediatric human esophageal epithelial cells for use in tissue engineering and disease investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

患者の生検から食道上皮細胞を単離における重要なステップの概要を図1に要約されています。上皮細胞のコロニーは約4-5日で形成され、線維芽細胞フィーダー細胞（ 図2A）に囲まれます。これらのコロニーが拡大するにつれ、彼らはより大きなコロニー（ 図2B）を形成するために他のコロニーと合併します。培養物が70％コンフ?...

Watch this Scientific Journal Video about Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation at JoVE.com

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

条件付き再プログラミングを利用した人の小児食道上皮細胞の拡大は、欠陥やけがを治療し、治療的スクリーニングアッセイのためのリザーバとして機能する自家移植のために食道の構築物を設計するために利用することができる細胞の患者特有の人口を持つ研究者を提供します。

ティッシュ・エンジニアリングと疾病調査に使用するための小児のヒト食道上皮細胞の条件付き再プログラミング

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

conditional-reprogramming-pediatric-human-esophageal-epithelial-cells

Research

JoVE Journal

Developmental Biology

6.9K ビュー.  UConn Health. 条件付き再プログラミングを利用した人の小児食道上皮細胞の拡大は、欠陥やけがを治療し、治療的スクリーニングアッセイのためのリザーバとして機能する自家移植のために食道の構築物を設計するために利用することができる細胞の患者特有の人口を持つ研究者を提供します。 

ビデオ: Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

子供からプライマリ鼻上皮細胞を培養し、人工多能性幹細胞に再プログラム

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

発生生物学

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

生産、特性評価および3D組織工学ヒト食道粘膜モデルの使用可能性

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

生物工学

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

心臓細胞療法のメカニズムを研究するために定義されたヒューマン・エンジニア心臓組織の構築

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

全組織生検からヒト上皮EnteroidsとColonoidsの確立

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

医学

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人ヒト線維芽細胞の導出と拡張神経前駆細胞へのそれらの直接変換

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

神経科学

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

ラットにおける円周食道再建のための組織工学型移植片

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

バイオエンジニアリング気道組織を生成するためのイメージング誘導バイオリアクター

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

オープントップ臓器チップ上でのヒト上皮組織の細胞構造と機能の再構成

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

人間の食道オルガノイドおよび気液界面の培養のプロトコール

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

著者スポットライト:好酸球性食道炎の病態生理学の調査

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.