快くこのプロジェクトで使用される試料を供給するためオルブリッチ庭園のジョン ・ ワースに感謝したいと思います。この作品は、国民の中心、肺および血の協会で一部サポートされて (G.R.G.、R01HL115282)全米科学財団 (DGE1144804 J.R.G、G.R.G.)、ウィスコンシン大学外科教室と同窓会基金 (H.D.L.)。この作品は、環境保護庁 (星グラント号 83573701)、国立衛生研究所 (R01HL093282 01A1 と UH3TR000506)、全米科学財団 (IGERT DGE1144804) 部分で支えられましたまた。

1. 洗剤ベースのアプローチを使用して植物組織の decellularization
<ol><li>
新鮮なまたは冷凍F. オオバアサガラ葉のサンプルを使用します。-20 ° C のフリーザー (1 年) 将来の使用のための店で未使用の新鮮な試料を凍結します。 注: は、ほぼあらゆる目的の植物の茎や葉の組織を使用します。拡張ストレージ時間は、組織への損傷を引き起こす可能性が。<ol>
<li>サイズとサンプルの意図されていた使用に基づいて処理されるサンプルの形状が決まります (短冊状にカット サンプルすなわち機械的テスト アプリケーションに適しています、一方 8 mm ディスク サンプル ウェル培養アプリケーションに有用であります。).室温 (20-25 ° C) 脱イオン H2o ・水没中パンチ シャープ、きれいな生検で 8 mm ディスクに葉をカットします。 注: このプロトコルは、全体の葉や茎で使用できます。ただし、小さいサンプルが高速 decellularize します。</li>
		<li>室温 (20-25 ° C) 脱イオン H2O の洗浄および/またはそれらを解凍する低速設定に振るプレート セットで 5-10 分のためのサンプルをインキュベートします。十分な脱イオン H2O を使用すると、すべてのサンプルは徹底的に接液します。</li>
	</ol>
</li>
	<li>
適当な容器 (ガラスまたはプラスチックの皿が理想的です) のサンプル 10% (w/v) ドデシル硫酸ナトリウム (SDS) 脱イオン H2o ・場所でのソリューションを準備し、SDS サンプルを完全にカバーするソリューションを追加します。室温 (20-25 ° C) で 5 日間のサンプルへの損傷を防ぐための低速度に振るプレート セットのサンプルをインキュベートします。 注: は満杯になるコンテナー decellularization プロセスを遅らせるし、SDS によって不均一な治療につながることができます。この手順では、サンプルは、茶色の色調を獲得すべき。<ol>
<li>5 日後に置き換えます SDS ソリューション脱イオン H2o ・加温残留 SDS ソリューションを徹底的に洗い流す振るプレートに追加の 10 〜 15 分のためのサンプル。</li>
	</ol>
</li>
	<li>1% (v/v) の非イオン性界面活性剤 10% (v/v) の漂白剤溶液を準備します。500 mL の溶液を作る、非イオン性界面活性剤の 5 mL の漂白剤 50 mL を混ぜて、脱イオン H2o ・ サブマージ 445 mL を加える作りたてのソリューションのサンプルします。 注: 非イオン性界面活性剤/漂白剤の解決は従って長い貯蔵寿命がない、準備の 48 時間以内に使用する必要があります。</li>
	<li>
サンプルが完全にクリアされるまで、非イオン性界面活性剤/漂白剤ソリューションすべての 24 h を置き換える (視覚的比較のため図 1 aを参照してください)。余分な非イオン性界面活性剤/漂白剤溶液を洗い流す純水を 2 分間振るプレート上にサンプルをインキュベートします。振動板を試料の損傷を防ぐために低設定に設定します。 注: 使用サンプルをクリアするために必要な時間は、種類や植物の種類によって異なります。サンプルの完全な変色は、完全 decellularization の示す (徹底したクリアリングを確保するため DNA の定量化を行う)。<ol>
<li>年までそれらを格納するサンプルを凍乾 (液体窒素を用いて凍結フラッシュをお勧め、-80 ° C のサンプルを凍結も可能です)、室温 (20-25 ° C) の低湿度で保管します。</li>
		<li>トリス-HCl (10 mM、pH 8.5) のサンプルを再構成十分なコートのサンプルを使用します。優しく使用 (すなわち使用 150-300 μ L/標準 24 well プレート リンスイン) する前にマイクロ ピペットを使用して血清自由な媒体のサンプル 2-3 回をすすいでください。 注: トリス塩酸バッファーと無血清メディア (すなわちDMEM がメディアを置き換えることができます任意の基本的な細胞培養) 脱イオン H2O だけで治療を受けた人よりも扱われる SDS サンプルの細胞生存率への影響を下げることに効果的です。</li>
	</ol>
</li>
</ol>2. 洗剤無料 Decellularization アプローチを使用して試料の調製
注: この手順の最初の手順は、手順 1.1 1.1.2 (上記参照) と一致します。
<ol><li>5% (v/v) の漂白剤 (NaClO) と 3% (w/v) (NaHCO3) 重炭酸ナトリウム溶液を準備します。F. オオバアサガラの葉のサンプルにホット プレート攪拌しながら 8 mm ディスクにカットの 60-70 ° C に発煙のフード ソリューションを温めます。 注: 重炭酸ナトリウムは、炭酸ナトリウム (Na2CO3)、または水酸化ナトリウム (NaOH) と代替可能します。所望の温度は大きく (部屋温度 90 ° C) から変化し、使用するサンプルの特性に応じて調整する必要があります。</li>
	<li>ソリューションには、所望の温度範囲が達すると、サンプルが水没、それらが損傷しないように攪拌速度を減らします。サンプルが目に見えてクリア後 (視覚的比較のため図 1 bを参照)、お風呂から慎重に取り外します。脱イオン H2O に余分な漂白剤溶液を削除する 1-2 分で一度サンプルをインキュベートします。 メモ: サンプルをオフに必要な時間はかなり異なることができます。たとえば、全体の葉の茎は、高温のお風呂は完全にオフの時間を取ることができるように、カット パセリが厚い、大きなサンプル中の 10-15 分でクリアできます。</li>
	<li>サンプルを凍結乾燥し、室温 (20-25 ° C) の低湿度で保管します。</li>
</ol>

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<li>Ma, J., Holden, K., Zhu, J., Pan, H., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Application+of+Three-Dimensional+Collagen-Scaffolds+Seeded+with+Myoblasts+to+Repair+Skeletal+Muscle+Defects%5BTitle%5D)+AND+%22Journal+of+Biomedicine+and+Biotechnology%22%5BJournal%5D)">The Application of Three-Dimensional Collagen-Scaffolds Seeded with Myoblasts to Repair Skeletal Muscle Defects.</a> Journal of Biomedicine and Biotechnology. 2011, 1-9 (2011).</li>
<li>Tyree, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+hydraulics%3A+the+ascent+of+water%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Plant hydraulics: the ascent of water.</a> Nature. 424 (6943), 923(2003).</li>
<li>Raven, P., Evert, R., Eichhorn, S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRaven%2C+P+%22Biology+of+Plants%22">Biology of Plants</a>. , 7th edition, (2005).</li>
<li>Turrell, F. <a target="_blank" href="https://www.jstor.org/stable/2435976">The area of the internal exposed surface of dicotyledon leaves.</a> American Journal of Botany. 23 (4), 255-264 (1936).</li>
</ol>

著者が明らかに何もありません。

ここで、植物組織を decellularize に 2 つの方法を説明します。ここでは、提示される結果25先行研究の結果と相まって出すプロトコルが可能性があることをお勧めの広いスペクトルに適用される植物種と茎と葉の両方で実行できます。これらの手順は単純な特殊な機器は必要ないので植物 decellularization は、ほとんどの実験室で行うことが。それは decellularization 後、足場必要が...

組織工学生体組織を作成する 1980 年代の代理および可能性のあるアドレス重要な臓器不足1。1 つの戦略は、足場を刺激し、行方不明の組織や臓器を再生する体をガイドに使用しています。高度な 3次元印刷を作り出した独特な物理的性質の足場などにアプローチを製造、達成可能な物理的な生物的特性の多様な範囲の足場を製造する能力がチャレンジ2,3。 また、機能血管ネットワークの不足は、これらの技術が限定されていました 3 次元組織を再生します。足場として脱動物と人間組織の使用が回避この問題4,5,6,7で支援します。ただし、高コスト、バッチ間の可変性および限られた可用性の広まった使用を制限可能性があります脱動物骨格の8。患者に潜在的な病気の感染やいくつかの哺乳類ティッシュの脱9に対する免疫学的反応の懸念もあります。
セルロース、植物および細菌のソースから派生は、再生医療で幅広い用途のためのバイオマテリアルを生成する広く使用されています。いくつかの例があります: 骨10、11、軟骨12,13,14と創傷治癒の15。足場を構成するセルロースの耐久性と耐性を哺乳類細胞によって分解されている付加的な利点があります。これは、哺乳類細胞はセルロース分子を分解に必要な酵素を生成しないためにです。比較では、足場はコラーゲンなどの細胞外マトリックスから高分子を使用して生産、16容易に分解され、長期的なアプリケーションには適していない場合があります。コラーゲンの足場は、化学架橋により安定化することができます。ただし、足場17の生体適合性に影響を与えるゲルビーズの固有の毒性のためのトレードオフがあります。逆に、セルロースには、哺乳類細胞18,19,20による酵素分解に不浸透ではないため長時間の注入のサイトで存在を維持する可能性があります。これは加水分解前処理の分解の率およびセルラーゼ21足場の共同配信をチューニングすることによって変更できます。植物由来セルロースの脱足場体内の生体適合性は、マウス22に行われた研究で実証されています。
進化の何百万年の何百も、植物はその構造と流体輸送と保存の効率を高めるための構成を洗練しています。工場血管は、小型船の場合、マレーの法23によると哺乳類の血管のように分岐することで油圧抵抗を最小限に抑えます。Decellularization 後、植物の相互接続された毛穴と血管の複雑なネットワークは維持されます。、すぐに利用できる異なる植物種の膨大な数を考慮した植物由来足場現在組織工学24,25の足場に影響を与える設計上の制限を克服するために可能性があります。例えば、Modulevskyらは脱アップル組織は皮下22マウスの背中に移植されたときに血管新生や細胞の移行が発生したことを示した。同様に、Gershlakらは、血管内皮細胞を脱葉24の血管内で成長するかもしれないことを示した。別の実験では、Gershlakらは心筋細胞が葉の表面上に成長させることができる、24を契約することができたことを示すことができるも。
植物には、複雑な組織を細胞からマクロ スケールで培った高度な製造技術とも達成するために困難であるにも含まれます。植物組織の複雑な階層設計、それらを彼らの成分26の合計よりも強く。植物は堅く、堅い茎等に至る種々 の機械的特性の茄多を持っている、27の葉よりフレキシブルで柔軟なものにします。葉は、サイズの面で種によって異なります、形状、破壊強度、血管新生、性、親水性の程度が異なるを運ぶことができます。全体的にみて、これらの植物のプロパティは、脱植物がティッシュ工学足場などのユニークで高機能な医療機器として使用できることを提案します。
このプロトコルは植物組織を decellularize に 2 つの方法に焦点を当てて、ようの葉し、茎、再生組織工学における足場として使用するため。最初の方法は DNA および哺乳類 decellularize し、組織6,22,25 を植物に広く使われている技術から適応されている携帯電話の問題を削除する風呂のシリーズを使用して洗剤ベースの技術 ,28,29,30。2 番目のメソッドは洗剤無料、一般に葉31の軟部組織を削除するために使用「白骨」プロトコルから適応。前の仕事を示したことは血管の周囲の軟部組織の31からの分離を促進漂白剤と炭酸水素ナトリウムの溶液に葉をぐつぐつ煮えます。この手法は、17回と 18th世紀、アルベルトゥスマグヌス セバ32とエドワード ・ パリッシュ33の仕事などで行う実験に引用できます。これらの実験は、葉や果物などの植物の問題を残すことのまわりの中心は長時間水に浸漬時間 (週か月) と当然のことながら朽ち果てるに柔らかい組織を許可します。ここで「白骨化」アプローチは、細胞残渣を削除する、かなり軟部組織構造を崩すことを避けるために低温培養時間が長くなるなど、穏やかな条件を使用する適応されます。ここに詳細な実験、3 つの植物のタイプを使用しました:イチジク オオバアサガラ、パキラアクアティカとフクギの種。DNA の定量化、機械テスト、および両手法による細胞の代謝活性に与える影響の結果を説明します。

<table><tbody><tr><td>Sodium dodecyl sulfate</td><td>Sigma Life Science</td><td>75746-1KG</td><td></td></tr><tr><td>Triton X-100</td><td>MP Biomedicals, LLC</td><td>807426</td><td>Non-ionic surfactant referenced in paper. Very viscous reagent, can help to cut end of pipette tip when drawing it up.</td></tr><tr><td>Concentrated bleach (8.25% sodium hypochlorite)</td><td>Clorox</td><td>Item #: 31009</td><td>Standard concentrated bleach.</td></tr><tr><td>Sodium bicarbonate</td><td>Acros Organics</td><td>217120010</td><td>Can be substituted with sodium hydroxide or sodium carbonate.</td></tr><tr><td>8 mm Biopunch</td><td>HealthLink</td><td>15111-80</td><td>Cuts samples that fit well in 24 well plate</td></tr><tr><td>Belly Dancer-Shake table</td><td>Stovall Life Sciences</td><td>BDRAA115S</td><td>Use low speeds to not damage tissues. Can use any model/brand of shake table.</td></tr><tr><td>Isotemp hot/stir plate</td><td>Fisher Scientific</td><td></td><td>Can use any style/brand of hot/stir plate.</td></tr><tr><td>Beaker</td><td>Any</td><td></td><td>Can use any size beaker as long as it will fit your samples and not overcrowd them.</td></tr><tr><td>Tris Hydrochloride</td><td>Fisher Scientific</td><td>BP153-500</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>MT50003PC</td><td></td></tr><tr><td>Quant-iT Picogreen dsDNA assay</td><td>Life Technologies</td><td>P11496</td><td>Can use any dsDNA quantification mehtod on hand.</td></tr></tbody></table>

two methods for decellularization of plant tissues for tissue engineering applications

組織置換のための足場として使用されて自己、合成、動物由来の移植率の低い、貧しい生体適合性、コストなどの制限があります。植物組織に適して表面積が大きく、優れた水の輸送と保存などの足場として相互接続された気孔率、既存の血管ネットワーク、および機械の広い範囲を使用して、それら有利な特性を持っています。プロパティ。組織工学用植物 decellularization の 2 つの成功した方法は以下のとおりです。最初の方法は、洗剤お風呂使用の哺乳類ティッシュをクリアする以前に確立された方法に類似している携帯電話の問題を削除するに基づいています。2 番目の葉の血管系を分離し、葉と茎をクリアする塩浴と温水の漂白剤の使用を含むプロトコルから適応洗剤無料メソッドです。両方のメソッドは、同等の機械的性質と低細胞代謝の影響より良い自分の用途に合ったプロトコルを選択するユーザーをできるように足場を得られます。

両方の方法は、細胞培養と組織エンジニア リング アプリケーションにより適していた足場を得られました。図 1は、洗剤手法や洗剤を使用しない方法のために切断された試料 (直径 8 mm) をそのまま葉を使用して decellularization プロセスの一般的なワークフローを示します。フィカス オオバアサガラ組織両方の方法を次の成功した decellulariz...

Watch this Scientific Journal Video about Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications at JoVE.com

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

組織工学用植物組織の Decellularization の 2 つの方法

ここで私たちは、現在し、コントラストの 2 つのプロトコルを使用して植物組織を decellularize: 洗剤ベースのアプローチと洗剤無料アプローチ。両方の方法は、組織工学用の足場として利用することができます使用、植物組織の細胞外のマトリックスを残します。

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

two-methods-for-decellularization-plant-tissues-for-tissue

Research

JoVE Journal

Bioengineering

14.5K ビュー.  University of Wisconsin-Madison. ここで私たちは、現在し、コントラストの 2 つのプロトコルを使用して植物組織を decellularize: 洗剤ベースのアプローチと洗剤無料アプローチ。両方の方法は、組織工学用の足場として利用することができます使用、植物組織の細胞外のマトリックスを残します。

ビデオ: Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

全肝臓の細胞化とRecellularization

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

生物工学

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

逆行性冠灌流によってブタ心臓の脱細胞化するための手順

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

ナチュラル無細胞腸基質の産生のための脱細胞化の方法論

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

生体外研究における 3 D のようなプラットフォームとして胎児と成人の心筋組織を外植体の対等な Decellularization

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. 
One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. 
When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

細胞培養のためのマウス胎児骨格筋からの3D脱細胞化マトリックスの調製

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various ...

発生生物学

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

Decellularization とひと伏在静脈の Recellularization 方法

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

逆方向に加圧ポーチ内全体の人間の心の decellularization

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Decellularization for the Preparation of Highly Preserved Human Acellular Skin Matrix for Regenerative Medicine

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of surrounding cells due to its content of diverse bioactive molecules. Due to these characteristics, the ECM has been recently considered a promising candidate for the creation of biological scaffolds to boost tissue regeneration. Emerging studies have demonstrated that decellularized human tissues could resemble the native ECM in their structural and biochemical profiles, preserving the three-dimensional (3D) architecture and the content of fundamental biological molecules. Hence, decellularized ECM can be employed to promote tissue remodeling, repair, and functional reconstruction of many organs. Selecting the appropriate decellularization procedure is crucial to obtain acellular tissues that retain the characteristics of the ideal microenvironment for cells. 
The protocol described here provides a detailed step-by-step description of the decellularization method to obtain a reproducible and effective cell-free biological ECM. Skin fragments from patients undergoing plastic surgery were scaled down and decellularized using a combination of sodium dodecylsulfate (SDS), Triton X-100, and antibiotics. To promote the regular and homogeneous transport of the solution through the samples, they were enclosed in embedding cassettes to ensure protection from mechanical insults. After the decellularization procedure, the snow-white color of skin fragments indicated complete and successful decellularization. Additionally, decellularized samples showed an intact and well-preserved architecture. The results suggest that the proposed decellularization method was effective, fast, and reproducible and protected samples from architectural damages.

<meta charset="utf8"></head><body>再生医療のための高保存ヒト無細胞皮膚マトリックスの調製のための脱細胞化

Decellularized human skin is suitable for tissue regeneration. A major issue of decellularization is the preservation of the native architecture, along with the appropriate content of structural proteins, glycosaminoglycans (GAGs), and growth factors. The method proposed allows fast and effective decellularization, producing decellularized skin with well-preserved native features.

再生医療のための高保存ヒト無細胞皮膚マトリックスの調製のための脱細胞化

Extracellular matrix (ECM) provides biophysical and biochemical stimuli to support self-renewal, proliferation, survival, and differentiation of ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

カスタマイズされた灌流バイオリアクターにおけるブタ血管化フラップの調達と灌流-脱細胞化

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...

組織工学用植物組織の Decellularization の 2 つの方法

要約

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全肝臓の細胞化とRecellularization

逆行性冠灌流によってブタ心臓の脱細胞化するための手順

ナチュラル無細胞腸基質の産生のための脱細胞化の方法論

生体外研究における 3 D のようなプラットフォームとして胎児と成人の心筋組織を外植体の対等な Decellularization

細胞培養のためのマウス胎児骨格筋からの3D脱細胞化マトリックスの調製

Decellularization とひと伏在静脈の Recellularization 方法

逆方向に加圧ポーチ内全体の人間の心の decellularization

再生医療のための高保存ヒト無細胞皮膚マトリックスの調製のための脱細胞化

再生医療のための高保存ヒト無細胞皮膚マトリックスの調製のための脱細胞化

カスタマイズされた灌流バイオリアクターにおけるブタ血管化フラップの調達と灌流-脱細胞化