著者らは 、in vitro 潅流チャンバーでの設計のためのジャックMilwidに感謝します。この作品は、KU、米国NSF KUとBEUへのシュライナーズ子ども病院へCBET - 0853569への私の、R00DK080942に米国NIH、R01DK59766とR01DK084053からの助成金（助成金なし。8503）によってサポートされていました。我々はまた、サポートやシュライナーズ子ども病院を認める。

1。肝臓の細胞化<ol><li>門脈の使用カニュレーションと18ゲージのカテーテルを用いてラットの肝臓を収穫。劣ると上大静脈開いたままにしておきます。リン酸塩で臓器が水分を保つ10cmのペトリ皿に（PBS）緩衝生理食塩水。 </li><li> 8リットルのタンク、蠕動ポンプとバブルトラップで構成されて灌流システムを設定します。 </li><li>リン酸塩で灌流系を埋める緩衝生理食塩水、それが10分間実行し続ける。 10cmのシャーレにPBSを満たし、リン酸の流量を減らすには、1mL /分に緩衝生理食塩水。 </li><li>慎重にリン酸塩に収穫された肝臓を転送すると、生理食塩水充填した10cmのペトリ皿をバッファ。 </li><li>一晩PBSで灌流に進みます。 </li><li> 5分間蒸留水で0.01％（w / v）のドデシル硫酸ナトリウム（SDS）で灌流を開始します。 </li><li> 1時間PBSで灌流。 </li><li>各時点で10、15および20分にSDSの灌流時間を増加させるステップ1.6および1.7を3回繰り返します。 </li><li> 24時間では0.01％（w / v）のSDSで灌流を続ける。 </li><li> 24時間0.1％（w / v）のSDSで灌流を続ける。 </li><li> 3時間で0.2％（w / v）のSDSを灌流。 </li><li> 3時間0.5％（w / v）のSDSを灌流。 </li><li> 15分間蒸留水で灌流。 </li><li>バインドされた核酸を除去するために30分間1％（w / v）のTriton X - 100を蒸留水にして灌流。 </li><li>脱細胞肝行列（DLM）、2時間PBSで浸み込ませるを洗う。 </li><li>オプション：中葉を除くすべての葉を切除。 </li><li> （図1）を使用する準備ができるまで4℃でPBSに浸した清潔な密閉シャーレにDLMを保管してください。 </li><li> DLMを殺菌するには：1）滅菌PBSで4℃で0.1％（v / v）の過酢酸及び4％（v / v）の3時間のためのエタノールとインキュベートを含むでDLMをフラッシュ2）滅菌PBSで2回洗浄する。 3）滅菌PBS、2％ペニシリン-ストレプトマイシン、10ug / mLのゲンタマイシン、および2.5 ug / mlとアムホテリシンBストアrecellularizationの実験に使用するまで4℃で同じ溶液中で脱細胞肝臓を含むで洗浄する。 </li></ol> 2。脱細胞肝マトリックスのRecellularization <ol><li>灌流チャンバー、滅菌条件下で蠕動ポンプとバブルトラップで構成されて灌流システムを設定します。培地、例えば、高グルコースDMEM（Sigma社）、10％ウシ胎児血清（Hyclone、）、100 U mL -1のペニシリン、および100μg/mlのmL -1のストレプトマイシン（Invitrogen社製）200mlで灌流系を埋める。 </li><li>灌流チャンバ内の脱細胞肝臓の行列を置き、ポンプは、気泡の形成を避けるために、5 mL / minの時に実行中に門脈カニューレを介して灌流システムにDLMを接続する。 </li><li> 30分間DLMによって培地の灌流を許可する。 </li><li>少なくとも90％の生存率と成熟ラットからの初代肝細胞を分離する。 </li><li>灌流系で流れを止め、徐々に気泡トラップを介して灌流システムに5000万肝細胞を（培地1-3 mLの）注入する。 </li><li> 10mL /分でフローを開始し、10分のための媒体を再循環させる。 </li><li> 2億セルの合計がDLM（図2）6に導入されるまで、手順2.5と2.6を繰り返します。 </li><li>一度、すべての細胞がDLMに灌流される、10分間600rpmで4つの50 mlの遠心管と遠心分離機に灌流液を収集する。上清を捨て、一つのチューブにペレットを収集する。播種の効率を決定するためにトリパンブルー排除を経由して細胞と生存率の数を決定します。 </li></ol> 3 Recellularized肝臓移植片のin vitro培養 <ol><li>灌流チャンバーから構成される灌流系、蠕動ポンプ、人工肺と無菌条件下での泡のトラップ（図3）を設定します。培地50 mLで灌流系を埋める、など、ウィリアムズ&#39;E（シグマ）、5％ウシ胎児血清（Hycloneは、）、0.5 U mL -1のインスリン （イーライリリー）、20ngのmL -1の EGF（インビトロジェン社） 、14 ngのmL -1のグルカゴン（ベッドフォード研究所）、7.5μgのmL -1のヒドロコルチゾン（ファルマシア）、100 U mL -1のペニシリン、および100μg/mlのmL -1のストレプトマイシン（Invitrogen社）。 </li><li>灌流チャンバ内のrecellularized肝臓移植片を配置し、ポンプは、気泡の形成を避けるために、5 mL / minの時に実行中に門脈カニューレを介して灌流システムにDLMを接続する。 </li><li>無菌的に灌流チャンバーを閉じて、培養中に任意の漏洩を避けるために、しっかりと密封する。 </li><li> 37℃であり、10％のCOを持っているインキュベーターに灌流システムを転送する2〜15 mL / minに灌流流量を増加させる。 </li><li> 95％O 2および5％CO 2混合ガスのタンクに人工肺を接続し、ガス流量0.5リットル/分に設定します。これは、近似の酸素分圧を達成する必要がありますtely 400 mmHgの。 </li><li>文化は、培地の日変化と10日前まで続けることができます。培地は、アルブミン、尿素および総胆汁酸の分泌のような移植片の肝機能のモニタリングのために毎日サンプリングされることがあります。培養期間の終了時に、recellularizedの肝臓移植は、分子と組織学的分析のためにサンプリングされることがあります。 </li></ol> 4。代表的な結果： ラットの肝臓の完全な細胞化は、記述されたプロトコルを使用して約72時間かかります。結果の行列は、繊維状のコラーゲンの100％、グリコサミノグリカンの50％、ネイティブの肝臓（ 表1）6のDNAのわずか5％を保持。行列の血管構造は電子顕微鏡の解析（図4）6をキャストしてスキャンするように腐食によって明らかに保持されます。 DLM内の血管のマイクロアーキテクチャーの存在は96％の効率とin vitro培養のためのその後の血流と細胞との再投入を容易にします。 recellularized肝移植は、in vitroで 10日間まで培養し、のようなアルブミン、尿素および総胆汁酸の分泌（図5）6を介して確認された適切な肝臓機能を表示することができます。 <img src="/files/ftp_upload/2394/2394fig1.jpg" alt="図1" /> 図1。細胞化の過程の最後に脱細胞の肝臓の行列。切除後の（A）全体の肝臓は、（b）の中葉。 <img src="/files/ftp_upload/2394/2394fig2.jpg" alt="図2" /> 図2 DLMのrecellularizationの模式図。 <img src="/files/ftp_upload/2394/2394fig3.jpg" alt="図3" /> 図3。recellularizedの肝移植のin vitro培養用灌流システムのセットアップ。 <img src="/files/ftp_upload/2394/2394fig4.jpg" alt="図4" /> 図4微小血管の構造は、脱細胞の肝臓のマトリックスに保持されます。 ）正常な肝臓は、b）脱細胞の肝臓、ポータル（赤）と静脈（青）血管系の腐食キャストのイメージ。 DLMの電子顕微鏡像胆管のような細い血管（矢印）、スケールバー（A、B）5ミリメートル（C、D）20μmをフィーチャリングC）容器、d）のセクションをスキャン。 <img src="/files/ftp_upload/2394/2394fig5.jpg" alt="図5" /> 図5肝in vitroでの灌流培養中recellularized肝移植の特定の機能。 ）アルブミン分泌した（p = 0.5249）、B）尿素製造した（p = 0.5271）およびc）の総胆汁酸の分泌した（p = 0.0114）。実験と対照との差の統計的分析は、= 0.01でフリードマン検定により10日間の培養期間にわたって行われた。エラーバーは標準誤差（n = 3の）を表します。 <table border="1"><tbody><tr><td></td><td> 新鮮なlivera </td><td> 脱細胞肝行列 </td><td rowspan="2"> p値 </td><td rowspan="2"> 新鮮な肝臓の％ </td></tr><tr><td></td><td> n = 4の</td><td> N = 8 </td></tr><tr><td> コラーゲン </td><td rowspan="2"> 0.07 ± 0.01 </td><td rowspan="2"> 0.08 ± 0.03 </td><td rowspan="2"> 0.56 </td><td rowspan="2"> 114パーセント</td></tr><tr><td> （gの肝臓あたりのミリグラム） </td></tr><tr><td> グリコサミノグリカン </td><td rowspan="2"> 73.1 ± 6.7 </td><td rowspan="2"> 34.2 ± 2.9 </td><td rowspan="2"> 0.004 </td><td rowspan="2"> 47パーセント</td></tr><tr><td> （gの肝臓あたりのミリグラム） </td></tr><tr><td> DNA </td><td rowspan="2"> 14.9 ± 5.6 </td><td rowspan="2"> 0.44 ± 0.08 </td><td rowspan="2"> 3.3 10 -5 </td><td rowspan="2"> 2.9パーセント</td></tr><tr><td> （gの肝臓あたりのミリグラム） </td></tr></tbody></table> 表1。脱細胞の肝臓のマトリックスの生化学的組成は、ネイティブの肝臓と比較して。 値は平均± SEMとして表されます。

<ol>
	<li>Kulig, K. M., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+tissue+engineering.">Hepatic tissue engineering.</a> Transpl Immunol. 12, 303-310 (2004).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nature biotechnology. 23, 47-55 (2005).</li><li>Dahl, S. L., Koh, J., Prabhakar, V., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+native+and+engineered+arterial+scaffolds+for+transplantation.">Decellularized native and engineered arterial scaffolds for transplantation.</a> Cell Transplant. 12, 659-666 (2003).</li><li>Schechner, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+a+vascularized+human+skin+equivalent.">Engraftment of a vascularized human skin equivalent.</a> FASEB J. 17, 2250-2256 (2003).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F., F, S. <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>Uygun, 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=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></ol>

利害の衝突は宣言されません。

ここで説明灌流細胞化法は、同一の全体構造とネイティブの肝臓の血管のマイクロアーキテクチャを持っている全体の肝臓足場を生成します。足場は、ネイティブの肝臓と同様の細胞外マトリックスの組成を有する。 recellularizationの方法は、試験のin vitro培養期間中に実行可能と機能性を維持高効率と細胞での細胞と足場の再投入を実現しています。 recellularizedの肝臓の移植への非実?...

A correction was made to <a href="http://www.jove.com/Details.php?ID=2394">Decellularization and Recellularization of Whole Livers</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Nima Saeidi
instead of:
Nima Saedi.

Erratum: Decellularization and Recellularization of Whole Livers

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<td>Sodium dodecyl sulfate</td>
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<td>Triton X-100</td>
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<td>Sigma-Aldrich</td>
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<td>Masterflex L/S Digital Drive</td>
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decellularization and recellularization of whole livers

肝臓は1を生き残るために栄養と酸素と廃棄物の除去の配信のための一定の灌流を必要とする複雑な器官です。組織工学と微細加工技術を用いてアプローチ上の理由で肝臓の微細構造を再作成したり、模倣するための努力は、この設計上の課題のためにこれまでのところ成功していない。さらに、肝組織工学のアプリケーションのために足場を作成するために使用される合成生体材料は、適切な細胞機能2を誘導する特異的細胞結合モチーフの不足のため大部分の組織の再生と修復を誘導することに限定されている。一方、脱細胞ネイティブ組織など血管3と皮膚4は、組織工学における多くのアプリケーションを発見した、との課題のいくつかの実用的なソリューションを提供している。脱細胞ネイティブマトリックスの利点は、それ故に細胞接着と組織再編5を高め、ある程度、元の組成、および微細構造に、保持されることが。 本研究では、主要な血管の構造を保持する無傷の肝臓bioscaffoldが得られるようなことが肝臓の灌流-細胞化を実行するメソッドを記述します。さらに、我々はin vitroで機能的な肝臓の移植片を作成し、成人原発性肝細胞でこれらのbioscaffoldsをrecellularizeする方法を説明し、in vivoでの移植のために必要な血管アクセスを持っています。

Watch this Scientific Journal Video about Decellularization and Recellularization of Whole Livers at JoVE.com

Decellularization and Recellularization of Whole Livers

灌流細胞化は、臓器の細胞外マトリックスの組成とマイクロアーキテクチャを保持する全体の肝臓の足場を生成する新規な手法です。ここで、肝細胞で灌流細胞化およびその後の再投入を使って臓器全体の足場を準備する方法について説明する。機能性と移植肝移植は、このテクニックを使用して生成することができます。

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 ...

decellularization-and-recellularization-of-whole-livers

Research

JoVE Journal

Bioengineering

21.9K ビュー.  Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children. 灌流細胞化は、臓器の細胞外マトリックスの組成とマイクロアーキテクチャを保持する全体の肝臓の足場を生成する新規な手法です。ここで、肝細胞で灌流細胞化およびその後の再投入を使って臓器全体の足場を準備する方法について説明する。機能性と移植肝移植は、このテクニックを使用して生成することができます。

ビデオ: Decellularization and Recellularization of Whole Livers

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 ...

生物工学

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

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

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

ナチュラル無細胞腸基質の産生のための脱細胞化の方法論

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

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

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 ...

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

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

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

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

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

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 ...

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 ...

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 ...