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。

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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><tbody><tr><td colspan="4">Self built arterial cannula</td></tr><tr><td>&amp;nbsp;Portex Non Sterile Polyethene Tubing</td><td>SIMS Portex</td><td>REF 800/110/100</td><td>0.28 mm ID 0.61 mm OD</td></tr><tr><td>&amp;nbsp;Portex Non Sterile Polyethene Tubing</td><td>SIMS Portex</td><td>REF 800/110/200</td><td>0.58 mm ID 0.96 mm OD</td></tr><tr><td>Venodrop Safe butterfly catheter</td><td>Fresenius Kabi</td><td>3275851</td><td>21 G</td></tr><tr><td colspan="4">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 colspan="4">surgery</td></tr><tr><td>Cotton Sticks</td><td>Hecht-Assistent</td><td>4302</td><td></td></tr><tr><td>Cotton Pads&amp;nbsp;</td><td>Shaoxing Zhengde Surgical dressing</td><td>13H118-03</td><td></td></tr><tr><td>Gauze Bandage</td><td>Hubei Haige&amp;nbsp; Medical Instruments</td><td>14388</td><td></td></tr><tr><td>&amp;nbsp;Ringer Solution</td><td>Fresenius Kabi</td><td>13 HKP022</td><td>1000 ml</td></tr><tr><td>10 ml Syringe</td><td>Braun</td><td>4606108V</td><td>10 ml/ Luer Solo</td></tr><tr><td>5 ml Syringe</td><td>Braun</td><td>4606061V</td><td>5 ml /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 colspan="4">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&amp;amp;T&amp;nbsp;</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&amp;nbsp;</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 colspan="4">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&amp;nbsp;</td><td>4-channel</td></tr><tr><td>heidelberger extension 75 cm&amp;nbsp;</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&amp;nbsp;</td><td>MS/CA/click&#039;n&#039;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&amp;nbsp;</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 colspan="4">Detergents</td></tr><tr><td>SDS pellets</td><td>Carl Roth&amp;nbsp;</td><td>CN30.4&amp;nbsp;</td><td>2.5 kg</td></tr><tr><td>Triton X-100</td><td>Carl Roth&amp;nbsp;</td><td>3051.1&amp;nbsp;</td><td>10 L</td></tr><tr><td>PBS&amp;nbsp;</td><td>Gibco</td><td>14190-094</td><td>DPBS</td></tr><tr><td colspan="4">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&amp;nbsp;</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

Universidad de Cartagena UDEC

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

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

精製マウス肝類洞内皮細胞と肝細胞の灌流

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

生化学

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 &#176;C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material&#39;s enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

マウスにおけるノルマザーミック Ex vivo 肝臓機械灌流

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing ...

工学

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 ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

ラットポータル線維芽細胞の分離により、<em&gt;その場</em&gt;肝灌流

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

生物学

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

ラット肝細胞の単離及び初代培養

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

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 ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

Ex Vivo常温肝灌流の小さな動物モデル

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

医学

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

多パラメータ灌流制御による原発性ラット肝細胞の単離

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

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

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この動画の章

全肝臓の細胞化とRecellularization

精製マウス肝類洞内皮細胞と肝細胞の灌流

マウスにおけるノルマザーミック Ex vivo 肝臓機械灌流

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

ラットポータル線維芽細胞の分離により、

ラット肝細胞の単離及び初代培養

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

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

Ex Vivo常温肝灌流の小さな動物モデル

多パラメータ灌流制御による原発性ラット肝細胞の単離

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

要約

さらに動画を探す

この動画の章

全肝臓の細胞化とRecellularization

精製マウス肝類洞内皮細胞と肝細胞の灌流

マウスにおけるノルマザーミック Ex vivo 肝臓機械灌流

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

ラットポータル線維芽細胞の分離により、

ラット肝細胞の単離及び初代培養

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

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

Ex Vivo常温肝灌流の小さな動物モデル

多パラメータ灌流制御による原発性ラット肝細胞の単離

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