The authors would like to gratefully thank Steffen Lippert, Khalid Aliyev, Korinna J&#246;hrens and Katharina Struecker for their help during this project.

/ 0365レッグ号のO;動物は、実験医学（FEM、シャリテ、ベルリン、ドイツ）のための施設で維持し、全ての実験プロトコルが見直され、健康と地方事務のステートオフィス（LAGeSo、ベルリン、ドイツによって承認されました。 11）。 1.肝臓の収穫<ol><li>手術前の準備<ol><li>固定のためにコルク板を使用してください。プレートに医療ドレープを入れてください。 4針を使用して、術中の吸入麻酔のためのコルク板に吸入マスクを固定します。 </li></ol></li><li>ポータル静脈カニューレの前準備<ol><li>三方活栓に末梢静脈カテーテル（G 20）を接続します。静脈カテーテルは、約の長さで、斜めにカットされるべきです。 1.5センチメートル。 </li><li>デ空気リンゲル液を充填した10mLのシリンジを使用してシステム。ガス塞栓症が否定灌流脱細胞化に影響を与える可能性があるので、慎重に脱空気に非常に重要です。 </li></ol></li><li>談合動脈カニューレの<ol><li>蝶のカニューレを使用し、5mmの長さに針をカット。 5ミリメートルの針にし、瞬間接着剤で固定し、5cmの管（外径0.96ミリメートルID 0.58ミリメートル）を取り付け。大きなチューブにし、瞬間接着剤で固定し2センチ長い管（OD 0.61ミリメートルID 0.28ミリメートル）を挿入します。 </li><li>アバットメントとしてこの構造の上に別のチューブをスリップし、瞬間接着剤で固定します。デ空気リンゲル液を充填した5mLのシリンジを用い動脈カニューレ。 </li></ol></li><li>麻酔や鎮痛<ol><li> 1.5 L /分の流量で酸素3.5％イソフルランによる麻酔誘導チャンバー内のラットの麻酔を誘導します。 </li><li>動作中に安全な鎮痛を確保するために、腹腔内にメタミゾールた（100mg / kgの体重）及び低用量のケタミン/メデトミジン（10mg / kgの体重あたり0.1 mg）を注入します。 </li><li>外科麻酔法のその後のメンテナンスのための吸入マスクを介して1リットル/分の酸素中の1.5％イソフルランを供給する。 </li></ol></li><li>術前アレンジメント&lt;オール&gt; <li>電気せん断機で自由に腹部を剃ります。消毒用の70％エタノールで腹部に剃毛切開部位を湿ら。 </li><li> 、準備されたプレート上に仰臥位で動物を置き吸入マスクにヘッドを挿入し、医療テープとピンと手足を固定します。ガーゼ湿布で過剰のエタノールや毛皮を削除します。 </li></ol></li><li>麻酔評価<ol><li>呼吸数を見て、麻酔の深さを評価します。麻酔は（40〜60呼吸/分）十分な深思わ場合には、鎮痛は、両方の後肢の足の指の間の皮膚を挟ま手術用鉗子を使用して、つま先のピンチ反射をトリガーしようとすることで十分であるかどうかを確認してください。 </li></ol></li><li>腹部アプローチ<ol><li>膀胱上記尾腹壁に正中切開を行います。 V字状に両側肋骨のアーチには、この点からカット。振動板に切断しないように注意してください。胸の上にカット腹壁を引き出します。 </li&gt; <li> 、オーバーホルトクランプで剣状突起を修正頭側に引き、ゴムバンドで固定します。腸を排出するために湿った綿棒を使用してください。濡れたガーゼ包帯で腸を包みます。また、濡れた包帯を持つすべての腹部のマージンをカバーしています。 </li></ol></li><li>肝臓の準備<ol><li>鎌状靭帯を解剖。頭側振動板のドームの下で内側と左肝葉を保持するために、湿ったガーゼの包帯を使用してください。肝十二指腸間膜および上部大網肝葉を公開します。 </li><li>ローブが携帯するまで慎重に上部大網葉に付着した靱帯を分析するために、2つのマイクロピンセットと春のはさみを使用してください。代わりに、内側と左葉を保持する、ガーゼ湿布の下ウェット綿棒を使って葉を移動します。 </li><li>上部の大網肝葉の動員後、左に胃を移動します。クランプで胃を修正して、マイクロピンセットと春のはさみとマイナー大網を分析。 </li><li>下omentumの複数形を動員L肝葉、モバイルであり、その後、その空洞内にバックローブを移動するまで。十二指腸を保持するために、バックハウスクランプを使用してください。ストレッチや肝十二指腸間膜を露出させるために左に十二指腸を移動します。延伸肝十二指腸間膜内の総胆管を取り囲みます。 </li><li>脂肪および膵臓組織からの胆管を解剖。胆管分岐部から胆管1.5センチカット。周囲の脂肪組織から門脈を分析。胃十二指腸静脈を露出させます。 </li><li>シルク6-0で二回、胃十二指腸静脈を結紮し、2合字間の静脈を分割。すべての動脈枝が明らかにされるまで、周囲の組織から腹腔動脈と総肝動脈を解剖。 </li><li>周囲の組織から胃十二指腸動脈を解放。互いに離れタイで、絹6-0で二回胃十二指腸動脈を結びます。 2合字の間に胃十二指腸動脈を解剖。 </li><li>脾動脈を解放周囲の組織から。互いに離れタイで、絹6-0で二回脾動脈を結びます。 2合字の間に脾動脈を解剖。 </li><li>周囲の組織からの左胃動脈を遊離。互いに離れタイで、絹6-0で二回、左胃動脈を結びます。 2合字間動脈を解剖。 </li><li>右腎と右横肝葉の間の下大静脈を囲みます。後腹膜腔からの静脈を解剖。腹腔動脈に従うことによって大動脈を露出させます。 </li><li>後腹膜脂肪組織を解剖。下大静脈に1ミリリットルの生理食塩水で千IEヘパリンを注入します。カニューレを撤回し、綿パッドで切開を閉じます。ヘパリンの全身的な効果のために1〜2分待ってください。 </li><li>ピンセットで大動脈を囲みます。大動脈を解剖し、ラットを放血。また、腎臓から遠位下大静脈を解剖。 </li></ol></li><li>門脈カニューレ挿入（セクション1.2で調製したカニューレを使用してください） <ol><li>遠位門脈魚口切開を行います。鉗子で切開を開き、門脈にカニューレを挿入。肝臓脱色するまで20ミリリットルリンゲル液で肝臓をフラッシュし、合字（絹6-0）でカニューレを固定します。 </li></ol></li><li>動脈カニューレ挿入（セクション1.3で調製したカニューレを使用してください） <ol><li>腹腔動脈上記の大動脈を解剖。後腹膜腔からの大動脈セグメントを解放します。大動脈パッチを生成するために、縦方向に大動脈セグメントをカットします。静的ホルダーに自作のカニューレを挿入します。 </li><li>カニューレを介して大動脈パッチをスリップする2ピンセットを使用してください。カニューレ上のシルク6-0と肝動脈を結紮し、突合せ部でパッチを修正。 </li></ol></li><li>肝外植<ol><li> 、絞りを開いた円形の切開を行い、周囲の構造から肝臓を分析。 </li></ol></li><li>ストレージ<ol><li> STO使用するまで350ミリリットルリンゲル液で満たされた500mlの滅菌槽内の肝臓再。 </li></ol></li></ol> 2.洗剤溶液<ol><li> 10％トリトンX-100の原液<ol><li> 900ミリリットルの水ビデと千ミリリットルのボトルを埋めます。 、100mlのトリトンX-100（99.9％）を追加します。トリトンX-100を溶解するために撹拌機を使用してください。水ビデでボトルを埋めます。千ミリリットルの全容量に達するまで。 </li></ol></li><li> 1％トリトンX-100 <ol><li>千ミリリットルボトルにストック溶液100ml（10％）を加えます。 900ミリリットルの水ビデを追加します。二回ボトルを振ります。滅菌フィルターを介して、1％トリトンX-100液をフィルタリングします。 </li></ol></li><li> 10％SDS原液<ol><li> 900ミリリットルの水ビデと千ミリリットルのボトルを埋めます。および66.99グラムのSDS（99.9％、密度0.67）を追加します。 SDSを溶解するために撹拌機を使用してください。水ビデでボトルを埋めます。千ミリリットルの全容量に達するまで。 </li></ol></li><li> 1％SDS <ol><li>千ミリリットルボットにストック溶液100ml（10％）を追加TLE。 900ミリリットルの水ビデを追加します。二回ボトルを振ります。滅菌フィルターを介して、1％SDS溶液をフィルタリングします。 </li></ol></li></ol> 3.脱細胞化<ol><li>潅流装置のセットアップ<img alt="図1" src="/files/ftp_upload/53029/53029fig1.jpg" /></li></ol> 図1：発振の圧力条件の下で選択的動脈、フォーラット肝臓の門脈灌流のための灌流装置のスキーム （Struecker ら 14から変更されました。）。 <ol><li>三方活栓および充填レベルを調節し、500ミリリットル0.9％NaClで反応器に充填するハイデルベルグの拡張子にドレインをリンクします。 <img alt="図2" src="/files/ftp_upload/53029/53029fig2.jpg" /></li></ol> 図2：（4）門静脈アクセスを介して気泡トラップ（5）（2）または動脈を介して交流先端に灌流セットアップスキーム灌流チャンバーのリンク（1400センチメートル3）（1）セス（3）。気泡トラップは、（5）先端の閉塞（4）とベント（6）を装備する必要があります。ハイデル拡張（7）に気泡トラップ（5）を接続します。ポンプセグメント（8）にハイデルベルグの拡張子をリンクします。また、別のハイデルベルグ拡張（9）にポンプセグメント（8）を接続してください。界面活性剤溶液（10）のボトルに最後のハイデル拡張（9）を挿入します。 （または複数の灌流の場合には圧力分配器）灌流チャンバーに人工呼吸器（11）を接続します。原子炉流体を排出するために、廃棄ボトル（12）への流出を接続します。 <ol><li>ポンプ内にポンプセグメントを挿入します。 PBSでサイクルを埋めるためにポンプを起動し、気泡トラップの先端を閉じて、通気口を開きます。泡の底部がしっかりとPBS（2-3センチ）で覆われている場合には、通気孔を閉じて、気泡トラップの遠位端を開きます。 </li><li>肝臓のインストール<ol><li>関連リアクターアクセスに門脈カニューレの三方活栓をリンクします。 C言語動脈アクセスに動脈カニューレONNECT。全く空気がシステムに入らないことを保証するために注意してください。 </li></ol></li><li>振動の圧力条件の適用<ol><li> 15 BPMの周波数と26ミリバール（分4ミリバール、最大30ミリバール）の振幅と呼吸器を使用してください。圧力分布室に呼吸器を接続し、反応器に、チャンバを接続します。圧力アプリケーションを起動します。 </li></ol></li><li>脱細胞化プロトコル<ol><li> /分5ミリリットルの流量で灌流手順を実行します。 90分間、1％トリトンX-100と肝臓の灌流に脱細胞化を開始します。灌流チャンバーを一定の内側と灌流肝臓上記流体レベルを維持するために潅流装置の流出を介して廃棄物の流出を可能​​にします。 90分後、1％SDSに界面活性剤を変更します。 <img alt="図3" src="/files/ftp_upload/53029/53029fig3.jpg" /> <img alt="表1" src="/files/ftp_upload/53029/53029table1.jpg" /></li></ol></li></ol> &lt;P&gt; 図3：すべての実験グループの灌流プロトコル。

<ol>
	<li>Struecker, B., Raschzok, N., Sauer, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+support+strategies:+cutting-edge+technologies.">Liver support strategies: cutting-edge technologies.</a> Nat Rev Gastroenterol Hepatol. 11, 166-176 (2014).</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>Wang, X., 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=Decellularized+liver+scaffolds+effectively+support+the+proliferation+and+differentiation+of+mouse+fetal+hepatic+progenitors.">Decellularized liver scaffolds effectively support the proliferation and differentiation of mouse fetal hepatic progenitors.</a> J Biomed Mater Res A. , (2013).</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. 16, 814-820 (2010).</li><li>Shupe, T., Williams, M., Brown, A., Willenberg, B., Petersen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+decellularization+of+intact+rat+liver.">Method for the decellularization of intact rat liver.</a> Organogenesis. 6, 134-136 (2010).</li><li>Bao, 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=Construction+of+a+portal+implantable+functional+tissue-engineered+liver+using+perfusion-decellularized+matrix+and+hepatocytes+in+rats.">Construction of a portal implantable functional tissue-engineered liver using perfusion-decellularized matrix and hepatocytes in rats.</a> Cell transplantation. 20, 753-766 (2011).</li><li>Baptista, P. 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=The+use+of+whole+organ+decellularization+for+the+generation+of+a+vascularized+liver+organoid.">The use of whole organ decellularization for the generation of a vascularized liver organoid.</a> Hepatology. 53, 604-617 (2011).</li><li>Soto-Gutierrez, A., Wertheim, J. A., Ott, H. C., 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=Perspectives+on+whole-organ+assembly:+moving+toward+transplantation+on+demand.">Perspectives on whole-organ assembly: moving toward transplantation on demand.</a> J Clin Invest. 122, 3817-3823 (2012).</li><li>Soto-Gutierrez, 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=A+whole-organ+regenerative+medicine+approach+for+liver+replacement.">A whole-organ regenerative medicine approach for liver replacement.</a> Tissue engineering. Part C, Methods. 17, 677-686 (2011).</li><li>De Kock, 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=Simple+and+quick+method+for+whole-liver+decellularization:+a+novel+in+vitro+three-dimensional+bioengineering+tool.">Simple and quick method for whole-liver decellularization: a novel in vitro three-dimensional bioengineering tool.</a> Archives of toxicology. 85, 607-612 (2011).</li><li>Park, K. M., Woo, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+decellularization+for+multi-organ+scaffolds+in+rats.">Systemic decellularization for multi-organ scaffolds in rats.</a> Transplantation proceedings. 44, 1151-1154 (2012).</li><li>Shirakigawa, N., Ijima, H., Takei, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+liver+as+a+practical+scaffold+with+a+vascular+network+template+for+liver+tissue+engineering.">Decellularized liver as a practical scaffold with a vascular network template for liver tissue engineering.</a> J Biosci Bioeng. , (2012).</li><li>Ren, 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=Evaluation+of+two+decellularization+methods+in+the+development+of+a+whole-organ+decellularized+rat+liver+scaffold.">Evaluation of two decellularization methods in the development of a whole-organ decellularized rat liver scaffold.</a> Liver Int. 33, 448-458 (2013).</li><li>Struecker, B., 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=Improved+rat+liver+decellularization+by+arterial+perfusion+under+oscillating+pressure+conditions.">Improved rat liver decellularization by arterial perfusion under oscillating pressure conditions.</a> J Tissue Eng Regen Med. , (2014).</li><li>Struecker, B., 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=Porcine+Liver+Decellularization+Under+Oscillating+Pressure+Conditions:+A+Technical+Refinement+to+Improve+the+Homogeneity+of+the+Decellularization+Process.">Porcine Liver Decellularization Under Oscillating Pressure Conditions: A Technical Refinement to Improve the Homogeneity of the Decellularization Process.</a> Tissue engineering. Part C, Methods. , (2014).</li></ol>

The authors have nothing to disclose.

ラット肝収穫と脱細胞化のための提示の技術を容易に再現可能であるが、考慮すべき特定の重要なステップがあります： それは血液凝固を活性化し、肝臓内の血栓形成につながる可能性があるため、肝臓収穫の準備中に、重度の出血を回避することが重要です。私どもの意見では、門脈を介して灌流の間、肝動脈を介して血液流入を回避するために、門脈のカニューレ?...

脱細胞化および再細胞化は、in vitro 1 における機能性、移植臓器の生成を可能にすることができます。器官からの細胞および抗原性物質（ 例えば 、DNA、α-GALエピトープ）を除去することにより、以下の非免疫原性細胞外マトリックス（ECM）を得ることができます。この行列は、臓器の三次元ミクロ解剖学を節約し、別の、おそらく異種起源2の細胞を用いた再増殖のための理想的なバイオマトリックスとして機能することができます。このように、脱細胞化ラット肝臓マトリックスは、ヒト肝細胞で再増殖することができます。このヒト化マイクロ肝臓疾患の研究のために（ 例えば 、先天性代謝性疾患、ウイルス性疾患または悪性腫瘍）または前臨床医薬品試験3のためのex vivoでのモデルとして役立つことができます。 ラット肝灌流脱細胞化するためのいくつかの異なるプロトコルは、すでに4-13を公開されています。すべてのプロトコルでは、decellular化は、カニューレを挿入し、門脈を介してアルカリイオン性または非イオン性界面活性剤の灌流によって達成されました。我々の知る限り、我々は門脈および/ ​​またはラットの肝動脈14を介して選択的灌流によりラット肝脱細胞化を報告する最初のグループでした。肝臓内の異なる血管系の選択的灌流を有効にすると、よりよい脱細胞化した結果を可能にして、さらに、細胞再生に重要な役割を果たしている可能性があります。 ここで詳細な研究では、肝臓は、振動圧力条件下で灌流を可能にする、カスタムメイドの独自の灌流装置で灌流しました。これらの圧力条件は、肝臓の生理的な呼吸器依存灌流を模倣する： その場で 、肝臓は動き、呼吸時の肝灌流に直接影響を与え、振動板のコピュラの下でハングアップします。インスピレーションは、具体的に最適化し、肝臓の振動板と絞りの低下につながります肝静脈流出が、一方、有効期限は、肝臓の上昇とポータル静脈流入15を最適化するために、腹腔内圧の低下につながります。 私たちの目的は、振動圧力条件は、ex vivoでの腹腔内条件を模倣することによって、ラット肝灌流脱細胞化の均一性に影響を与えるかどうかを評価することでした。脱細胞化プロセスの均一性は灌流脱細胞化で過小評価要因になることがあります。 ECMに肝臓の脱細胞化の原因の変更のために使用されるすべての知られている薬剤。他の領域がすでに完全に脱細胞化されているのに対し、乏しい灌流領域の細胞は、ECM内に残ります。残りの細胞を溶解するために、灌流期間または圧力が十分に灌流領域に複数の変化を引き起こし、上昇する必要があります。このように、脱細胞化のための界面活性剤は、臓器内に均一に分布されるべきです。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Self built arterial cannula</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/100</td>
			<td>0,28mm ID 0,61mm OD</td>
		</tr>
		<tr>
			<td>&#160;Portex Non Sterile Polyethene Tubing</td>
			<td>SIMS Portex</td>
			<td>REF 800/110/200</td>
			<td>0,58mm ID 0,96m OD</td>
		</tr>
		<tr>
			<td>Venodrop Safe butterfly catheter</td>
			<td>Fresenius Kabi</td>
			<td>3275851</td>
			<td>21 G</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>portal vein cannula</td>
		</tr>
		<tr>
			<td>Periphereal Venous Catheter</td>
			<td>BD</td>
			<td>393224</td>
			<td>BD Venflon Pro 20G</td>
		</tr>
		<tr>
			<td>three-way stopcock</td>
			<td>smiths medical</td>
			<td>888-101RE</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>surgery</td>
		</tr>
		<tr>
			<td>Cotton Sticks</td>
			<td>Hecht-Assistent</td>
			<td>4302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Pads&#160;</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>13H118-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Bandage</td>
			<td>Hubei Haige&#160; Medical Instruments</td>
			<td>14388</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Ringer Solution</td>
			<td>Fresenius Kabi</td>
			<td>13 HKP022</td>
			<td>1000ml</td>
		</tr>
		<tr>
			<td>10ml Syringe</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td>10ml/ Luer Solo</td>
		</tr>
		<tr>
			<td>5ml Syringe</td>
			<td>Braun</td>
			<td>4606061V</td>
			<td>5ml /Luer Solo</td>
		</tr>
		<tr>
			<td>Suture (Silk 6/0)</td>
			<td>Resorba</td>
			<td>H1F</td>
			<td>LOT 105001.81</td>
		</tr>
		<tr>
			<td>medical drape</td>
			<td>Shaoxing Zhengde Surgical dressing</td>
			<td>D0613011</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>surgical instruments</td>
		</tr>
		<tr>
			<td>needle holder</td>
			<td>Geuder</td>
			<td>17570</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-forceps</td>
			<td>Inox-Electronic</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-scissors</td>
			<td>Martin</td>
			<td>11-740-11</td>
			<td></td>
		</tr>
		<tr>
			<td>micro-forceps</td>
			<td>S&#38;T&#160;</td>
			<td>112314</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp</td>
			<td>Aesculap</td>
			<td>BH111R</td>
			<td></td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>F S T&#160;</td>
			<td>14501-14</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps</td>
			<td>Aesculap</td>
			<td>BD 557</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Decellularisation</td>
		</tr>
		<tr>
			<td>Respirator</td>
			<td>Resmed</td>
			<td>14.24.11.0004</td>
			<td>SmartAIR ST</td>
		</tr>
		<tr>
			<td>Perfusion Device</td>
			<td>Charite, medical engineering laboratory</td>
			<td>custome-made device</td>
			<td>decellularisation device</td>
		</tr>
		<tr>
			<td>peristaltic pump ismatec reglo ICC</td>
			<td>IDEX</td>
			<td>ISM4408&#160;</td>
			<td>4-channel</td>
		</tr>
		<tr>
			<td>heidelberger extension 75 cm&#160;</td>
			<td>Fresenius Kabi</td>
			<td>2873</td>
			<td>75 cm</td>
		</tr>
		<tr>
			<td>MS/CA pump-segment</td>
			<td>IDEX</td>
			<td>IS 3510&#160;</td>
			<td>MS/CA/click&#39;n&#39;go/POM-C</td>
		</tr>
		<tr>
			<td>CA 2-stopper tube</td>
			<td>Pharmed</td>
			<td>BPT NSF-51</td>
			<td></td>
		</tr>
		<tr>
			<td>bubble trap&#160;</td>
			<td></td>
			<td>custome-made item</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock hose connector</td>
			<td>Neolab</td>
			<td>No. 02-1887</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Detergents</td>
		</tr>
		<tr>
			<td>SDS pellets</td>
			<td>Carl Roth&#160;</td>
			<td>CN30.4&#160;</td>
			<td>2,5 kg</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth&#160;</td>
			<td>3051.1&#160;</td>
			<td>10l</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>DPBS</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>staining</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Morphisto</td>
			<td>10177</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer hematoxylin</td>
			<td>AppliChem</td>
			<td>A4840</td>
			<td></td>
		</tr>
		<tr>
			<td>gomori staining</td>
			<td>Morphisto</td>
			<td>11104</td>
			<td></td>
		</tr>
		<tr>
			<td>AlcainBlue-PAS staining</td>
			<td>Morphisto</td>
			<td>11388</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct Red 80&#160;</td>
			<td>Sigma Aldrich</td>
			<td>365548</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

均一性、したがって異なる脱細胞化プロトコルの有効性を肉眼観察し、組織学的分析、及び脱細胞化肝臓行列内の残りのDNA含量の分析によって評価しました。また、腐食鋳造は脱細胞化後の肝臓の無傷のミクロ解剖学を視覚化するために実施しました。 肉眼検査脱細胞化の間に、肝臓は、携帯コンテンツの削除を示す、ルーセントとなります。門脈?...

Watch this Scientific Journal Video about Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device at JoVE.com

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

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 ...

procedure-for-decellularization-rat-livers-an-oscillating-pressure

Research

JoVE Journal

Bioengineering

10.2K ビュー.  Charité – Universitätsmedizin Berlin. 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.

ビデオ: Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

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 ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

灌流固定標本のコントラストコンピュータ断層撮影を用いた人間の心臓静脈系の解剖学的再建

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

糖尿病モデルラットにおける神経伝導の超音波ツール ブロックします。

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

食品由来病原体を検出する電気化学的 DNA のバイオ センサーの開発

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

組織工学用植物組織の Decellularization の 2 つの方法

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

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 ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

ラットでの肝部分工学生体内での基盤として新しい手術手技

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

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 ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

ハイスループットマイクロ流体装置を用いた動的環境条件の生成

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...