著者はブローガンゲスト、ミシェル·ウィーバー、そしてクリステンリッペルトを承認​​したいと思います。この研究のための資金は、NIHグラントR03EB009237ならびにNIHの訓練助成生体イメージングとバイオの国立研究所からT32EB001026-06とT32HL076124-05によって提供されました。

1。組織の準備と実験セットアップ<ol><li>食肉処理場や研究施設からすぐに安楽死の後に収穫豚の臓器と過剰血を洗い流す。心房と大動脈をそのまま維持し、余分な脂肪や組織の中心部をトリミングします。離れて大動脈から肺動脈を分離する脂肪トリミング。組織内の任意のカットがある場合は、適切に廃棄してください。 </li><li>冷凍庫の紙で個別にそれぞれの心をラップし、完全凍結を確実にするために、少なくとも24時間、-80℃の冷凍庫内のすべての組織を保管してください。 </li><li>ときに使用する（通常3ヶ月未満）、一晩4℃で4リットルのビーカーに沈めタイプ1の雪解け水1そのまま冷凍ブタ心の準備ができて</li><li>心が完全に解凍された後、心が乾いたなで心を量ると、体重を記録する。市場重量豚の心臓部は、約375〜450グラムの重量を量るべきである。 </li><li>とげのある減速機の¼ &quot;最後までサイズで18マスターフレックスチューブを接続します。有刺鉄線減速機を挿入し、大動脈内側チューブ。ちょうど腕頭動脈の下、大動脈周囲に2ホースクランプまたはセキュアジップタイを置きます。減速機とチューブは大動脈弁ので、冠状動脈が灌流することができます（図1）以上を保つ必要があります。 </li><li>タイプIの水でチューブを埋めるために30または60 mlの注射器を使用しています。そのおおよその中点でマスターフレックスローラーポンプのカートリッジ内にチューブを挿入します。流入水2.5リットルを充填した4リットルのビーカーの底にチューブの端とチューブを固定沈める。 </li><li>水、気泡を除去するためにプライムポンプを入れたビーカーに心を置きます。気泡がチューブが挿入されている大動脈から来て認められた場合には、大動脈には、追加の関係で再配置または確保する必要があるかもしれません。気密シールは脱細胞化処理（図2）の間に十分な圧力を維持することが重要です。 </li><li> 0.2パーセントTrypsin/0.05％EDTA/0.05％のNaN 3 Lを含む4リットルのビーカーを置きプレートをかき混ぜ、脱細胞化処理の準備で37℃に温めて上の3ソリューションを提供します。 </li></ol> 2。組織リンス<ol><li>正しいチューブサイズを選択していることを確認、400ミリリットル/分の流量にポンプを設定します。 15から25分間、私は水を入力して心臓をフラッシュします。ポンプが開始されると、心臓が心室から血液を膨潤し、発散させるべきである。新鮮なソリューションは、すべての5〜10分間に置換、あるいは心臓から削除される血液の量に基づいて、必要に応じなければならない。血液は心臓から流出していない場合は、必要に応じてチューブとクランプを調整します。 </li><li>ポンプを停止し、緩衝生理食塩水（PBS）2Xリン酸で満たされた別のビーカーに心を移す。チューブを溶液中に浸漬された後、ポンプを起動し、700 ml / minに流量を増加させる。心臓は、ソリューションごとに5分を変え、15分間溶液中に残るはずです。各ソリューションの変更は、組織とチューブを移動させながら、ポンプを一時的に停止させておく必要があり新しいビーカーに。 </li><li> 10分間、私は水を入力し、750 ml / minに流量を増加するために、心臓を転送します。 </li></ol> 3。脱細胞化とソリューション灌流<ol><li> 0.2パーセントTrypsin/0.05％EDTA/0.05％で、37のNaN 3℃の入ったビーカーに心を移す1200ミリリットル/分にポンプ速度を増加させ、ポンプを起動します。ビーカー中の溶液を循環させるためにビーカーの底に置か攪拌棒を使用しています。心臓は3時間の合計を37℃で0.2パーセントTrypsin/0.05％EDTA/0.05％のNaN 3溶液中に残るはずです。 1時間後、1,500 ml / minにポンプ速度を増加させる。追加の時間後、1,800 ml / minにポンプ速度を増加させる。組織は徐々に条件を組織に増加灌流速度を施し、血管の破裂を防止しています。心臓が膨張し、ほぼプロトコルのこのステップの間にサイズが2倍になります。組織は目を通して心房から頂点へと進行する、その自然な色を失うことになるメールプロトコル（図3）。 </li><li>各ソリューション灌流後、すすぎ2ステップは、細胞破片、化学残留物、援助、細胞溶解を除去するために行われている。すすぎが10分続くタイプI水ですすぎ、10分で構成されてそれぞれが室温で2X PBS溶液ですすいでください。各洗浄は、ソリューションをすすぎ追加、水没心臓を入れたビーカー内に灌流液を循環させ、元のビーカーから溶液の除去で構成されています。 1900ミリリットル/分で0.2パーセントTrypsin/0.05％EDTA/0.05％のNaN 3溶液、水灌流したあと、これを1950ミリリットル/分で2X PBSを灌流する。 </li><li>室温で3％トリトンX-100/0.05％EDTA/0.05％のNaN 3の溶液に心を移してください。 1時間2000ミリリットル/分であり、灌流液にポンプの速度を増加させる。ビーカーから溶液を除去し、新鮮な溶液と交換し、2100 ml / minにポンプ速度を増加させ、さらに1時間半の新鮮な溶液を灌流、合計時間を持ち込む3パーセントトリトンX-100/0.05％EDTA/0.05％NaN 3を 2.5時間に。 </li><li> 10分ごとに2180ミリリットル/分で2150ミリリットル/分と2XのPBSでタイプI水にティッシュを洗い流す。 </li><li>室温で4％デオキシコール酸ナトリウム液に心を移す。 2200ミリリットル/分および3時間灌流液にポンプの速度を増加させる。 </li><li>各ソリューションの5から10分後にソリューションを変更すると、15分ごとに2200ミリリットル/分でタイプIの水でと2X PBS中で組織を洗浄します。説明灌流手順が二回リンス工程を実施し、4℃で一晩添付のチューブと一緒に心を格納することにより、複数の日にわたって分割して、タイプIの水中に沈んでいる可能性があります。 </li><li>次の日は、5分、5分、1,500 ml /分で1×PBSですすぎ、続いて750ミリリットル/分でタイプIの水で洗い流し行う。プロトコルは、その後、適切な解決策で説明流量で継続することができる。 </li></ol> 4。消毒と最終処理<ol><li> 0.1％peracに心を移すETIC酸/ 2200ミリリットル/分で1.5時間、4％のエタノール溶液と灌流液。 </li><li>組織のための最終的なリンスは、すべての2200ミリリットル/分の速度で実行されます。タイプIの水に2分5回洗浄し、15分のための1×PBSで組織を灌流する。リンスのこのシリーズは、溶液灌流手続きを完了するために、もう一度繰り返されます。 </li><li>ポンプの電源をオフにして、心を排出するために、溶液から心臓を削除します。大動脈からの関係を断つ、すべてのチューブを取り外し、1時間排水するために、空のビーカーに心を置く。余分な液体は、定期的に排水する必要があります。完全に心を排出する吸収パッド（図4）に心を置きます。 </li><li>水の大部分が除去された後、心臓の細胞外マトリックス（C-ECM）の重量を記録する。心臓を脱細胞化処理中に、その初期重量の約20〜25％を失うことが予想される。 </li><li>右心室と左心室、ならびにDNA quantificatiための心室中隔を解剖上および組織学的処理の順序で組織の完全な脱細胞（図5）を確認します。 </li><li>凍結乾燥前に少なくとも2時間、-80℃で、C-ECMを凍結。 </li></ol>

<ol>
	<li>Ott, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+orthotopic+transplantation+of+a+bioartificial+lung.">Regeneration and orthotopic transplantation of a bioartificial lung.</a> Nat. Med. 16, 927-933 (2010).</li><li>Ott, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat. Med. , (2008).</li><li>Petersen, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+lungs+for+in+vivo+implantation.">Tissue-engineered lungs for in vivo implantation.</a> Science. 329, 538-541 (2010).</li><li>Uygun, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat. Med. , (2010).</li><li>Wainwright, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cardiac+extracellular+matrix+from+an+intact+porcine+heart.">Preparation of cardiac extracellular matrix from an intact porcine heart.</a> Tissue Eng. Part C Methods. 16, 525-532 (2010).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32, 3233-3243 (2011).</li><li>Gilbert, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+tissue+and+organ+decellularization.">Strategies for tissue and organ decellularization.</a> Journal of cellular biochemistry. , (2012).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27, 3675-3683 (2006).</li><li>Akhyari, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quest+for+an+optimized+protocol+for+whole-heart+decellularization:+a+comparison+of+three+popular+and+a+novel+decellularization+technique+and+their+diverse+effects+on+crucial+extracellular+matrix+qualities.">The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel decellularization technique and their diverse effects on crucial extracellular matrix qualities.</a> Tissue Eng. Part C Methods. 17, 915-926 (2011).</li><li>Weymann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+perfusion+decellularization+porcine+heart+model--generation+of+3-dimensional+myocardial+neoscaffolds.">Development and evaluation of a perfusion decellularization porcine heart model--generation of 3-dimensional myocardial neoscaffolds.</a> Circulation journal : official journal of the Japanese Circulation Society. 75, 852-860 (2011).</li><li>Cortiella, J., et al. <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+acellular+natural+lung+matrix+on+murine+embryonic+stem+cell+differentiation+and+tissue+formation.">Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation.</a> Tissue Eng. Part A. 16, 2565-2580 (1089).</li><li>Remlinger, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrated+xenogeneic+decellularized+tracheal+matrix+as+a+scaffold+for+tracheal+reconstruction.">Hydrated xenogeneic decellularized tracheal matrix as a scaffold for tracheal reconstruction.</a> Biomaterials. 31, 3520-3526 (2010).</li><li>Sellaro, T. L., Ravindra, A. K., Stolz, D. B., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+hepatic+sinusoidal+endothelial+cell+phenotype+in+vitro+using+organ-specific+extracellular+matrix+scaffolds.">Maintenance of hepatic sinusoidal endothelial cell phenotype in vitro using organ-specific extracellular matrix scaffolds.</a> Tissue Eng. 13, 2301-2310 (2007).</li><li>Wainwright, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Right+ventricular+outflow+tract+repair+with+a+cardiac+biologic+scaffold.">Right ventricular outflow tract repair with a cardiac biologic scaffold.</a> Cells, tissues, organs. 195, 159-170 (2012).</li></ol>

博士ギルバートは、研究が行われている間にACell、Inc。で科学諮問委員会にあったし、最近の研究開発担当副社長となりました。 ACell社は、膀胱行列を販売しており、本研究では商業的な利害関係はありません。

現在の研究では、ブタの心臓の一貫性のある効率的な脱細胞化するための方法論を説明した。プロトコルは、以前に発表した報告書1への変更であり、再現性の高い結果が得られた流量と圧力の上昇に長い露出が含まれていました。結果として脱細胞化組織は、組織2が正常細胞化のために発行基準のすべてを満たした。頻繁なソリューションの変更は組織に細胞物質の再導入?...

<table border="1"><tbody><tr><td> 試薬/材料の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>トリプシン</td><td>ギブコ</td><td> 15090 </td><td></td></tr><tr><td> EDTAを</td><td>フィッシャー</td><td> BP120-500 </td><td></td></tr><tr><td> NaN 3を </td><td>シグマ</td><td> S2002-500G </td><td></td></tr><tr><td>トリトンX-100 </td><td>シグマ</td><td> X100-1L </td><td></td></tr><tr><td> 10X PBS </td><td>フィッシャー</td><td> BP399-20 </td><td></td></tr><tr><td>デオキシコール酸ナトリウム</td><td>シグマ</td><td> D6750-500G </td><td></td></tr><tr><td>過酢酸</td><td>ファルツとバウアー</td><td> P05020 </td><td> 35％CAS番号79-21-0 </td></tr><tr><td>エタノール</td><td> Pharmco </td><td> 111000200 </td><td></tD&gt; </tr><tr><td>マスターフレックスポンプドライブ</td><td>コー​​ルパーマー</td><td> SI-07524から50 </td><td></td></tr><tr><td>マスターフレックスチューブ</td><td>コー​​ルパーマー</td><td> 96400から18 </td><td>サイズ18 </td></tr><tr><td>有刺鉄線リデューサー</td><td>コー​​ルパーマー</td><td> EW-30612から20 </td><td></td></tr><tr><td> 4Lビーカー</td><td>フィッシャー·サイエンティフィック</td><td> 02-540T </td><td></td></tr></tbody></table>

procedure for decellularization of porcine heart by retrograde coronary perfusion

主に足場のネイティブアーキテクチャを維持しながら、灌流ベースの全臓器の脱細胞化は最近、サイト固有の細胞外マトリックスの足場を作成するための手段として、組織工学の分野で関心を集めています。現在までに、このアプローチは、心臓、肺、肝臓1月5日を含めて、臓器の様々なシステムで利用されている。簡単にアクセスできる血管網なしで組織のための前の脱細胞化法は、界面活性剤、酸、または周囲の細胞外環境6月8日から細胞および核成分を除去するための手段として、酵素処理のソリューションに対する組織の長期の暴露に依存してきた。しかし、拡散を介して組織に浸透するソリューションの能力に応じてこれらの方法ヒンジ式の有効性。 decellularizatiのとは対照的に、自然の血管系による臓器の血流を効果的に拡散距離を減少させ、促進輸送組織のうち、組織および細胞成分にエージェント上。ここで、我々は完全に冠状逆行灌流を通して無傷ブタの心臓をdecellularizeする方法について述べる。プロトコルは、無傷の心臓の3次元構造を完全に脱細胞化された心臓の細胞外マトリックス（C-ECM）の足場を得た。我々の方法は、酵素、界面活性剤、および細胞の溶解および除去を助けるため高張低張とリンスと相まって酸のシリーズを使用していました。プロトコルは、細胞物質の除去を助けるためにTriton X-100およびデオキシコール酸ナトリウム溶液に続くマトリックスから細胞を分離するためにトリプシン溶液を使用していました。説明されたプロトコルはまた、長時間の2リットル/ min以上の灌流速度を使用します。細胞破片の汚染なし、組織への薬剤の輸送許可ソリューションの変更と相まって、高流量、および組織のリンス効果を確実にした。記載された方法はNATIからすべての核物質を除去様々なアプリケーションに使用することができ、サイト固有の心臓ECMの足場を作成VEブタ心臓組織、。

全体ブタの心臓に脱細胞化の効果は自然に大きさの違い、圧力、容器の配置によって変化する。従って、細胞外マトリックス由来の足場の正確な組成は、心臓から心臓に同じにはなりません。説明されたプロトコルの完了は、細胞材料の損失を示す、白色または半透明表示される心臓を得ることができます。しかし、それは広く組織はさらにいくつかの定量的なパラメータ8の組み合?...

Watch this Scientific Journal Video about Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion at JoVE.com

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

迅速かつ完全に逆行灌流を通して無傷ブタ心臓から細胞成分を除去するための方法が記載されている。この方法は、複数の臨床アプリケーションで使用するための可能性を秘めているサイト固有心筋細胞外マトリックス足場が得られます。

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 ...

procedure-for-decellularization-porcine-heart-retrograde-coronary

Research

JoVE Journal

Bioengineering

23.8K ビュー.  McGowan Institute for Regenerative Medicine. 迅速かつ完全に逆行灌流を通して無傷ブタ心臓から細胞成分を除去するための方法が記載されている。この方法は、複数の臨床アプリケーションで使用するための可能性を秘めているサイト固有心筋細胞外マトリックス足場が得られます。

ビデオ: Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

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 Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

振動圧力潅流装置内のラット肝臓の脱細胞化のための手順

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

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 ...

Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery,&#160;mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 &#176;C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

分離と全体ブタ膵臓の Decellularization

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

ナノ製剤中のエンドトキシン検出カブトガニ リムラス ライセート (ラル) 試金を使用して

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

ブタ心臓における容量性免疫プローブによる神経ペプチドダイナミクスの時間分解 インビボ 測定

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical ...

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 ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

血管化複合同種移植のための 生体外 灌流ベースのバイオリアクターを用いたラット後肢の調達と脱細胞化

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

搭載型ブタ大動脈弁の流体力学的特性の評価

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a method for mounting a porcine aortic valve in a pulse duplicator to evaluate its hydrodynamic properties. These properties can then be evaluated before and after the procedure under investigation is performed and/or the investigative medical device is applied. Securing the inflow segment presents some difficulty owing to the lack of circumferential myocardium in the left ventricular outflow tract. This method addresses that issue by securing the inflow segment using the anterior leaflet of the mitral valve and then suturing the left ventricular free wall around the inflow fixture. The outflow segment is secured simply by inserting the fixture into an incision in the superior aspect of the aortic arch. We found that specimens had significantly different hydrodynamic properties before and after tissue fixation. This finding induced us to use fresh specimens in our testing and should be considered when using this method. In our work, we used this method to test novel intracardiac patch materials for use in the valvular position by performing an aortic valve neocuspidization procedure (Ozaki procedure) on the mounted porcine aortic valves. These valves were tested before and after the procedure to assess the change in hydrodynamic properties in comparison to the native valve. Herein, we report a platform for hydrodynamic testing of experimental aortic valve procedures that enables comparison with the native valve and between different devices and techniques used for the procedure under investigation.

著者スポットライト:大動物実験に着手する前に心臓手術試験を進める 

We present a method for mounting a porcine aortic valve on a pulse duplicator to test its hydrodynamic properties. This method can be used to determine the change in hydrodynamics after the application of an experimental procedure or novel medical device prior to use in a large animal model.

実験的大動脈弁処置および新規医療機器の流体力学的試験のための ex vivo 豚モデル

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...

Methods Article

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