国立心肺血液研究所（賞＃R00HL093358にLDB） - この作品は、国立衛生研究所によってサポートされていました。

<ol>
	<li>Stegemann, J. P., Hong, H., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical,+biochemical,+and+extracellular+matrix+effects+on+vascular+smooth+muscle+cell+phenotype.">Mechanical, biochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype.</a> Journal of applied physiology. 98, 2321-2327 (2005).</li><li>Guarnieri, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalently+immobilized+RGD+gradient+on+PEG+hydrogel+scaffold+influences+cell+migration+parameters.">Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters.</a> Acta. 6, 2532-2539 (2010).</li><li>Krebs, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+poly(lactic-co-glycolic)+acid+scaffolds+with+in+situ+pore+formation+for+tissue+engineering.">Injectable poly(lactic-co-glycolic) acid scaffolds with in situ pore formation for tissue engineering.</a> Acta. 5, 2847-2859 (2009).</li><li>Zimmermann, W. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+heart+tissue+grafts+improve+systolic+and+diastolic+function+in+infarcted+rat+hearts.">Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts.</a> Nature. 12, 452-458 (2006).</li><li>Chung, C., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Three-Dimensional+Hyaluronic+Acid+Stem+Cell+Chondrogenesis.">Influence of Three-Dimensional Hyaluronic Acid Stem Cell Chondrogenesis.</a> Tissue engineering. 15, (2009).</li><li>Black, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-induced+alignment+augments+twitch+force+in+fibrin+gel-based+engineered+myocardium+via+gap+junction+modification.">Cell-induced alignment augments twitch force in fibrin gel-based engineered myocardium via gap junction modification.</a> Tissue engineering. 15, 3099-3108 (2009).</li><li>Syedain, Z. H., Weinberg, J. S., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+distension+of+fibrin-based+tissue+constructs:+evidence+of+adaptation+during+growth+of+engineered+connective+tissue.">Cyclic distension of fibrin-based tissue constructs: evidence of adaptation during growth of engineered connective tissue.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 6537-6542 (2008).</li><li>Falvo, M. R., Gorkun, O. V., Lord, S. 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D., Oegema, T. R., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fibrin-based+arterial+media+equivalent.">A fibrin-based arterial media equivalent.</a> Journal of biomedical materials research. 66 (Part A. , 550-561 (2003).</li><li>Barocas, V. H., Tranquillo, R. 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利害の衝突は宣言されません。

心筋システムのin vitroモデルにおける一貫性と実行可能な三次元におけるフィブリンゲルの結果の新生児ラット心筋細胞のカプセル化。細胞が捕捉されたとき、彼らは代謝活性と、コンパクト化改造およびネイティブの心臓組織12との一貫性のある細胞外マトリックスを再作成することが可能であるため、フィブリンが望ましい生体材料です。我々は心筋細胞はこの環境で自分自身を整列することを可能にするので、それらの機能は、等方性の組織6に比べてより大きな収縮力で、その結果心筋のより特徴的です。潜在的な治療のアプリケーションでは、それは実行可能性と機能性の両方を促進する物質の中で細胞をカプセル化する必要があります。プロトコルは、3次元の微小環境における心筋細胞の挙動を制御するために、フィブリンネットワークを作成するための効率的かつ正確な手段を示すここで紹介。 いくつかの潜在的な問題は、これらのコンストラクトの作成と培養中に発生する可能性があります。つの潜在的な問題は大幅に構造の機能に影響を与える前に、カプセル化に細胞の生存率の維持、である。努力が分離し、フィブリンゲル内の細胞のカプセル化の間の時間を減らすことによって、分離後の細胞死を制限するためになされるべきである。構文は、厳密なスケジュールで一日おきにメディアを提供する必要があります。さらに、使用されるソリューションのすべてに均一性を確保することが重要です。フィブリノーゲン、トロンビンと細胞の混合物は、異機種環境を生成する場合、ECMを改造する細胞の能力は、機械的に夫婦と契約が潜在的に遮断されていない。エンジニアリング組織の継続性の乱れを防ぐために、構造の注入時の気泡の形成を防止することも重要です。この問題を緩和する一つの方法は、シリンジに構造を作り、ゆっくりと注入する必要以上のゲルを描くです。最後に、一度フィブリンマトリックスが設定されていると24時間培養液中にされている、それはフィブリンゲルの細胞ベースの圧縮を促進するため、リング型の側面から構造を切り離すことが不可欠です。金型の中央に構造を維持すること、ガスと栄養素の交換が容易になります。リングの金型の側面への付着はまた、所望の細胞整列が中断される場合があります。 その意識断頭は国立衛生研究所の両方からのガイドラインの下で承認された方法とアメリカ獣医学協会であるこのプロトコルでは安楽死の方法として使用されている点が重要です。しかし、一部の金融機関は、新生児ラットの断頭続いて麻酔を使用することをお勧めします。それは、摘出した心臓組織/細胞のための低酸素条件下での時間の最小量を保証するため、我々は意識的な斬首を選択している。低酸素の比較的少量では虚血と有意にこのプロトコルの結果に影響を与える可能性のある潜在的に心筋細胞死を、筋細胞につながることができます。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>ナトリウム塩化</td><td>シグマ</td><td> S7653 </td><td> PBS </td></tr><tr><td>カリウム塩化</td><td>シグマ</td><td> P9333 </td><td> PBS </td></tr><tr><td>ナトリウム二塩基性リン酸</td><td>シグマ</td><td> S7907 </td><td> PBS </td></tr><tr><td>一塩基性リン酸カリウム</td><td>シグマ</td><td> P5655 </td><td> PBS </td></tr><tr><td>グルコース</td><td>シグマ</td><td> G5400 </td><td>分離</td></tr><tr><td>止血剤</td><td>ファイン科学ツール</td><td> 91308〜12 </td><td>分離</td></tr><tr><td>細かいピンセット</td><td>ファイン科学ツール</td><td> 11251〜20 </td><td>分離</td></tr><tr><td>大きなはさみ</td><td>ファイン科学ツール</td><td> 91401〜14 </td><td>分離</td></tr><tr><td>マイクロはさみ</td><td>ファイン科学ツール</td><td> 91501〜09 </td><td>分離</td></tr><tr><td>メスハンドル</td><td>ファイン科学ツール</td><td> 10008〜13 </td><td>分離</td></tr><tr><td>メス刃</td><td>フィッシャーサイエンティフィック</td><td> 08から918 - 5A </td><td>分離</td></tr><tr><td>吸収ベンチunderpad </td><td> VWR </td><td> 56617-014 </td><td>分離</td></tr><tr><td>滅菌ドレープ</td><td>フィッシャーサイエンティフィック</td><td> GM42526 </td><td>分離</td></tr><tr><td>オートクレーブバッグ</td><td>フィッシャー</td><td> 01-812-54 </td><td>分離</td></tr><tr><td>ガーゼのパッド</td><td>フィッシャーサイエンティフィック</td><td> 13-761-52 </td><td>分離</td></tr><tr><td> betadine </td><td>パーデュー大学の製品</td><td> 67618-150-01 </td><td>分離</td></tr><tr><td>滅菌手袋</td><td>フィッシャーサイエンティフィック</td><td> 19から020 </td><td>分離</td></tr><tr><td>無菌トランスファーピペット</td><td>フィッシャーサイエンティフィック</td><td> 9962 </td><td>分離</td></tr><tr><td>コラゲナーゼ</td><td>ワージントン</td><td> CLS2 </td><td>分離</td></tr><tr><td>テフロンロッド1 / 4インチ直径</td><td>マクマスター - カー</td><td> 8546K11 </td><td>型の部</td></tr><tr><td>テフロンチューブ1 / 4インチID、1 / 2インチOD </td><td>マクマスター - カー</td><td> 8547K31 </td><td>型の部</td></tr><tr><td>シリコンO -リング1 / 4インチID、1 / 2インチOD </td><td>マクマスター - カー</td><td> 9396K204 </td><td>型の部</td></tr><tr><td>テフロンチューブ1 / 4インチID、5 / 16インチ外径</td><td>マクマスター - カー</td><td> 52355K14 </td><td>型の部</td></tr><tr><td>ケンドールmonojectシリンジ6cc </td><td>フィッシャーサイエンティフィック</td><td> 05-561-41 </td><td>型の部</td></tr><tr><td> BDシリンジ3CC </td><td>フィッシャーサイエンティフィック</td><td> 309585 </td><td>型の部</td></tr><tr><td>ウシフィブリノーゲン</td><td>シグマ</td><td> F8630 </td><td>構築する</td></tr><tr><td>ウシトロンビン</td><td>シグマ</td><td> T7513 </td><td>構築する</td></tr><tr><td> 1 M HEPES </td><td>シグマ</td><td> H0887 </td><td>構築する</td></tr><tr><td>塩化ナトリウム</td><td>シグマ</td><td> S7653 </td><td>構築する</td></tr><tr><td> DMEM </td><td>インビトロジェン</td><td> 10569 </td><td>構築する</td></tr><tr><td>プルロニックF - 127 </td><td>シグマ</td><td> P2443 </td><td>構築する</td></tr><tr><td>塩化カルシウム</td><td>シグマ</td><td> 383147 </td><td>構築する</td></tr><tr><td> 0.2ミクロンフィルター</td><td>フィッシャーサイエンティフィック</td><td> SCGVT05RE </td><td>構築する</td></tr><tr><td> 40ミクロ​​ンのセルストレーナー</td><td>フィッシャーサイエンティフィック</td><td> 22-363-547 </td><td>構築する</td></tr><tr><td> 0.45ミクロンのボトルトップフィルター</td><td>コー​​ニング</td><td> 430627 </td><td>構築する</td></tr><tr><td>ガラスプレフィルター</td><td>ミリポア</td><td> AP2007500 </td><td>構築する</td></tr><tr><td> 18G 1 1 / 2インチの長い針</td><td>フィッシャーサイエンティフィック</td><td> 14から826 - 5D </td><td>構築する</td></tr><tr><td> 21G 1インチの針</td><td>フィッシャーサイエンティフィック</td><td> 14 - 826C </td><td>構築する</td></tr><tr><td> jarファイルを構築する</td><td>フィッシャーサイエンティフィック</td><td> 2116 </td><td>構築する</td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td>インビトロジェン</td><td> 15140 </td><td>メディアの</td></tr><tr><td>馬血清</td><td>シグマ</td><td> H1138 </td><td>メディアの</td></tr><tr><td>ウシ胎仔血清</td><td>インビトロジェン</td><td> 16000 </td><td>メディアの</td></tr><tr><td>アミノカプロン酸</td><td>アクロスオーガニック</td><td> 103305000 </td><td>メディアの</td></tr><tr><td>アスコルビン酸</td><td>シグマ</td><td> A5960 </td><td>メディアの</td></tr><tr><td>インスリン</td><td>シグマ</td><td> I9278 </td><td>メディアの</td></tr><tr><td>パラホルムアルデヒド、16％ </td><td>電子顕微鏡学</td><td> 15710 </td><td>組織学</td></tr><tr><td>最適な切削温度（OCT） </td><td>テッドペラ</td><td> 2705​​0 </td><td>組織学</td></tr><tr><td> 2 - メチルプタン</td><td>フィッシャー</td><td> 03551〜4 </td><td>組織学</td></tr><tr><td>マウスMYH1/2/4/6一次抗体</td><td>サンタクルスバイオテクノロジー</td><td> SC - 32732 </td><td>組織学</td></tr><tr><td>ウサギコネキシン43の一次抗体</td><td>セルシグナリングテクノロジー</td><td> 3512 </td><td>組織学</td></tr><tr><td> Dylight 549 -結合ロバ抗マウス二次抗体</td><td>ジャクソンイムノ研究所</td><td> 715-505-151 </td><td>組織学</td></tr><tr><td> Dylight 488標識Donke抗ウサギ二次抗体</td><td>ジャクソンイムノ研究所</td><td> 711-485-152 </td><td>組織学</td></tr><tr><td> LIVE / DEADアッセイ</td><td>インビトロジェン</td><td> L - 3224 </td><td>分析</td></tr></tbody></table>

encapsulation of cardiomyocytes in a fibrin hydrogel for cardiac tissue engineering

三次元ハイドロゲルの環境で培養する細胞は組織工学のための構造を開発するだけでなく、in vitroで種々の培養条件下で細胞応答を研究するための重要なテクニックです。三次元環境では、より密接に細胞が原因で、すべての次元1の機械的および化学的刺激のアプリケーションに、in vivoで観察するものを模倣している。三次元のヒドロゲルは、どちらかのようなPEG - DA 2およびPLGA 3またはそのようなコラーゲン4、ヒアルロン酸5またはフィブリン6,7のような天然のタンパク質の数などの合成高分子から行うことができます。フィブリン、天然由来の血液凝固タンパク質、から作成されたハイドロゲルは、身体の自然な創傷治癒のプロセス8の一部であるメッシュを形成するために重合することができます。フィブリンは、組織工学のために理想的な一時的な足場作り、細胞分解性と潜在的に自家9です。 ここでは、3つの日齢の仔ラットと組織工学のためのフィブリンハイドロゲルの構造でカプセル化するための細胞の調製から詳細に新生児心筋細胞の単離を説明します。新生児の心筋細胞は、心臓組織の形成と工学4のin vitro試験で使用される一般的な細胞源です。フィブリンゲルは、酵素トロンビンと混合フィブリノゲンによって作成されます。トロンビンは、他のモノマー10との相互作用の結合部位を明らかに、フィブリノーゲンからfibrinopeptides FPAとFPBを切断する。これらの相互作用は、単量体は、ヒドロゲルのメッシュを形成する繊維に自己集合させる。この酵素反応のタイミングがフィブリノゲンにトロンビンの比、またはトロンビンへのカルシウムの比率を変えることによって調整できるので、一つは射出成形金型は、異なる形状11,12の数で構築できます。さらに我々は文化は13ゲルを制約する方法により、結果として組織のアライメントを生成することができます。 培養静的条件下での二週間のための工学心臓組織の構成要素の後に、心臓の細胞が構造を改造し始めていると電気的ペーシングの条件6歳未満の収縮力を生成することができます。このプロトコルの一部として、我々はまた、機能的な心筋によって生成されたアクティブな力の分析、最終的な細胞の生存を決定するための電気刺激だけでなく、方法によって構築する（ライブデッド含む培養期間後に心筋の設計組織を分析するための方法を説明します。アッセイ）と収縮（ミオシン重鎖またはMHC）と心筋細胞間の細胞結合（コネキシン43またはCx43）のための重要な典型的なタンパク質の発現と形態を調べるために免疫組織染色。

Watch this Scientific Journal Video about Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering at JoVE.com

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

我々は、新生児の心筋細胞と組織工学のためのフィブリンハイドロゲルの構造でカプセル化するための細胞の調製の分離を説明します。我々は、電気刺激と細胞の生存および免疫組織染色により発生する活性力を含む培養期間後に心筋培養組織を分析するための方法を説明します。

心臓組織工学のためのフィブリンゲルにおける心筋細胞のカプセル化

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

encapsulation-cardiomyocytes-fibrin-hydrogel-for-cardiac-tissue

Nanyang Technological University

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Bioengineering

24.1K ビュー.  Tufts University. 我々は、新生児の心筋細胞と組織工学のためのフィブリンハイドロゲルの構造でカプセル化するための細胞の調製の分離を説明します。我々は、電気刺激と細胞の生存および免疫組織染色により発生する活性力を含む培養期間後に心筋培養組織を分析するための方法を説明します。

ビデオ: Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

タンパク質分解的に分解性3次元システムにおける卵胞のカプセル化と文化のための方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

生物工学

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

ASCの分化を制御するための二層ゲルをエンジニアリング

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

組織工学のためのエレクトロ繊維のポスト処理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

マウスモデルにおける腸のティッシュエンジニアリング

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

心臓組織工学のためのキャピラリーフォースリソグラフィ

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

人間の3次元の組織工学<em&gt; in vitroで</em&gt;腫瘍テストシステム

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

中に組み込み血管新生による組織工学<em&gt;インビボ</em&gt;ティッシュ・エンジニアリング商工会議所

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

内耳組織工学用マウス蝸牛を Decellularizing するためのプロトコル

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

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 ...