この作品は、SNF [31003A-138528]、OctavとマルセラBotnar財団、ノバルティス財団とエママスチャンプ財団からの補助金によってサポートされていました。私たちは、その優れた技術支援のためにマルティーヌLambelet、ジャン=クリストフシュテーレに感謝します。

ローザンヌ大学の倫理委員会は、ヒト組織の使用のために1983年に改訂され、1975年のヘルシンキ宣言で概説した原理に従っている実験を、承認した。 1人間の大伏在静脈の収穫<ol><li>虚血下肢バイパス手術を受けた患者から非静脈瘤人間伏在静脈の余剰部分を取得します。手術室では、ヨウ素溶液で脚全体を消毒し、足に脚の付け根から足を露出するために患者をドレープ。 </li><li>膝への鼠径部から正中切開を行います（中断された皮膚の部分を残して）。 </li><li>周囲の組織（ノータッチ技術）の椎弓と大伏在静脈を収穫。 4-0シルクネクタイと静脈のセキュアな側枝。すぐにRPMI-1640、G、4℃で9センチメートル2.5〜4ミリメートルの外径と大伏在静脈の長い余剰セグメントの最小値を格納するlutamax培地、12.5％ウシ胎児血清を補充した、実験室に持ってきて。 </li></ol> 2 EVPSデザイン<ol><li> 図1に示されている一般的な機器を組み立てます。すべての機器をオートクレーブ滅菌条件下ですべてのコンポーネントを保つ。また、システムは、防水であり、培地中に化学物質を漏洩しないことを確認してください。表紙にポリメチルメタクリレート（PMMA-GS）を使用します。スチール（X5クロムニッケル18 10）と静脈のサポートなどポリオキシメチレンプラスチック（POM）。 </li><li>静脈との接続の配置を可能にするために所望の形状に灌流チャンバーを設計します。 （円筒構造を使用している場合、または半径）の深さを確認してください、それが培養培地により一定報道（ 図1）と一緒に容器の最小限の屈曲および拡張を可能にするように少なくとも2.5 cmです。シールは、主要な問題であり、長方形のPMMA-GS建設が使用されている理由である。 </li><li>希望geometrに静脈のサポートをデザインyを。静脈よじれや膨満の上を回避するには、（静脈がねじで一緒にねじられるようねじは、その目的のために使用することはできません）押したり引っ張って長さ調節が可能です。 NOTE：L字型（血管に適合するように直径5mm）2静脈シリンダをサポートピース及び静脈（ 図1Bおよび図2）スライド2によって接続されたフルスチールロッドは、ここで使用されている。 </li><li>のp流体柱の= 0-10 = hxをρ×gではp =圧力（N / m 2であり、ペンシルベニア州）は、h =高さ（m）ρ：システムに適用される「静止圧」であることなど、低圧塔を設計する=液体の密度（㎏/ m 3）であり、g =重力定数（9.81メートル/秒2）。上から下へのデザイン4つの接続ダクト、：（静脈へ）、（静脈）からの流出のために、流入圧力を加えると培地交換を可能にするために。 </li><li>メディアを準備します。これまでの研究5,11-14に基づき、12.5グルタマックスを補充した、RPMI-1640]を選択します％ウシ胎児血清および1％抗生物質 - 抗真菌溶液（10,000 U / mlのペニシリンG、プラス10 mg / mlの硫酸ストレプトマイシンを加えた25 mg / mlのアムホテリシンB、プラス0.5 / mlの：ゲンタマイシン）。剪断応力（SS）は3 Qは流速（ml /秒）であるSS = 4μQ/π* rで与えられる、静脈セグメントの半径（cm）のrは、μは灌流媒体の粘度である。 <ol><li> 70kDaのデキストランを添加して粘度を調整することでSSを調節する。粘度計粘度を測定します。ここでは、9〜15ダイン/ cm 2とSSを設定するために、8％の70kDaデキストランを追加します。 </li></ol></li><li> 60パルス/分、圧力システムに適用され、コンピュータによって制御から独立した150±15ml /分の一方向の流れを生成する一定振幅の拍カージオイド信号を誘導するために歯車ポンプを設定する。運転ソフトウェアは一定の取得と監視圧力、流速、脈​​拍数、及び信号を積分いることを確認してください。必要に応じて、第二のポンプを使用します（非同期hronized）非層流、乱流を生成する。 </li></ol> 3 EVPSアセンブリ（図1） <ol><li>開始する前に、すべての機器が滅菌されていることを確認してください。層流フード内で無菌状態の下で、以下のすべての手順を実行します。 </li><li>媒質で満たされたペトリ皿に静脈を置きます。外科用メスを使用して、3等分静脈を分割。 </li><li>すぐに、PBS中の1セグメントをすすぐ。 3部にセグメントを分割し、形態計測のためにホルマリンに1を修正。定量的な転写産物（RT-PCR）とタンパク質（ウェスタンブロット）分析のために他の二つをフリーズします。コントロールとして、これらのセグメントを考慮して、非灌流静脈。 </li><li>灌流2残りのセグメントを使用してください。 <ol><li>非常に穏やかに静脈内に媒体を注入し、通常の流れ方向を決定する;弁の存在下で、静脈が反転される。 </li><li>静脈をシーリング実験成功に最も重要である。担保を通して漏れがないか確認してください。 6-0絹縫合糸を持つ任意のリークを固定します。 </li> &lt;/オール&gt; </li><li>時間（2.3、 図1）での静脈2つの金属シリンダー間のセグメント、一方の端を接続します。くぼみの周りEthibon 3-0（ 図1AおよびB）でシリンダーを固定します。 <ol><li>以前媒質で満たされた灌流チャンバー内に全体を静脈セグメントを配置します。第二のセグメントに対して同じ手順を繰り返します。 注記：正しくシリンダに静脈をシールしないと、漏れの原因となる再介入を必要とし、大幅に感染し、実験失敗のリスクが増加します。 </li></ol></li><li> （メッシュ）第二のセグメントを強化するには、L字型のピース（2.3及び図1）から（添付静脈で）2シリンダーを離します。 <ol><li>やわらかに、任意の楽器と静脈を触れないでください。静脈へのその後のシリンダの最初のメッシュをスライドさせます。プッシュ/プル押し合いは、静脈上のメッシュを取得します。 </li><li>メッシュはJを確保静脈の全面を覆うたらEthibon 3-0とシリンダにacketed静脈。 </li><li> L字型の支持体に静脈/シリンダ化合物を再構成し、以前媒質で満たされた灌流チャンバーに移す。 </li></ol></li><li> 3.2ミリメートルの内径過酸化物で処理したシリコンチューブを使用して、Yスプリッタにそれぞれ金属製の円筒（インおよびアウトフロー）を接続します。 </li><li>チューブの同じタイプを使用して第2のYスプリッタに流出スプリッタを接続します。このYスプリッタからは、両方の血管を通る灌流圧を測定するために一本のチューブを使用しています。閉ループ系（ 図2）を形成するために戻って列に、もう一方を接続します。 </li><li>インキュベーターの内部では、ポンプヘッドに低圧塔を接続するために長い（1メートルの長さ）のチューブを使用しています。 </li><li>他の長尺管を流入Yスプリッタにポンプヘッドを接続することにより設定します（ 図1）完了します。 </li></ol> 4。静脈灌流<ol><li> EVPSアセンブリは、同時された後mpleted、（補充できるように、静脈流出ダクトの下に留まる）培地でカラムを埋める。システムが一杯になるまでカラムに多くの媒体を追加します。 pHが37±0.1℃に維持インキュベーターにすべてのシステムを移動（ヘンダーソン·ハッセルバッハ式に基づいてCO 2 / pHのアルゴリズムを使用して）7.40±0.01で一定に保った。 </li><li>インキュベーターの外歯車ポンプヘッドを持参し、ギアポンプドライブに接続します。アセンブリを固定する棒をねじ込み。 </li><li>ポンプパワーのスイッチを入れ、それが駆動ソフトウェア上で起動しても同様に、すべての区画内に分配される媒体5分を許可していることを確認してください。 </li><li>圧力を監視するには、動脈ラインの監視を使用しています。コンピュータにリンクされた圧力変換器にEVPS圧力出力を（それが動脈カテーテルに相当）を接続します。 <ol><li>チューブが完全に媒体で充填され、任意の気泡を含んでいないことを確認してください。 「動脈リチウムを通じて脱泡培養システムね&quot;チューブ（ 図2）。ディスプレイに注意を払い、60パルス/一定の振幅の分の拍動カーディオイド信号を探します。この時点で、平均圧力0〜10 mmHgでの間である。圧力が&lt;0で、列には、漸進的に（静脈とチューブとの間の静脈担保または不十分なシール）漏れを探して空になります。 </li></ol></li><li>静脈試験用または動脈試験のための90ミリメートルHgで6ミリメートルHgのに最小限の圧力を設定してください。これらの条件下で、空気インジェクタ、カラムおよびシステムに必要な圧力を加える。 </li><li>高圧塔に接続されたチューブを使用して、2日ごとに培地を変更します。圧力変化の損傷を防止するために、最初の列プラグを開く。 </li></ol>灌流の5。完了<ol><li>灌流の3または7日後：インキュベーターからEVPSを取ると静脈をディスマウント。捨てる5mmの近位および遠位静脈が機器に付着し終了します。中央、厚さ5mm厘をカット残りのセグメントからのGSとホルマリンに（形態計測）を固定。残りのフラグメントを凍​​結し、さらなる分子解析のための粉末に減らす。 </li></ol>

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著者らは、開示することは何もない。

この研究は、ヒトの静脈に広範囲血行動態の研究を実行するためのex vivoで静脈灌流システム（EVPS）をuncovers。このシステムは、 生体内で細胞を循環させることによって放出された炎症性悪化や成長因子の非存在下で定義された血行動態パラメータの下に伏在静脈灌流を可能にします。このように、人間の静脈グラフト5,11,12,15におけるIHの制御に関与根底にある経路のよ...

心血管疾患は、欧米諸国1における罹患率および死亡率の主要な原因である。血管内治療の進歩にもかかわらず、バイパス手術は、このように万人以上の静脈グラフトは、米国で毎年行われている、現代的な治療法の柱のまま。しかし、研究の数十年にもかかわらず、下肢静脈グラフト30〜60％が内膜肥厚（IH）2による第一年以内に失敗する。機械的な力、特に低せん断応力（SS）と高い壁張力が、この過形成応答3,4の開始と発展に極めて重要である。この問題に対処するために、ex vivoで静脈灌流システム（EVPS）は厳密に制御され、血行動態条件（圧力およびせん断応力）、ヒト伏在静脈の行動の下で、研究し作成した。本研究では、動脈のような循環への挿入に続いて、高圧（= 100mmHgの平均）prolifを刺激するのに十分であったや機能の内膜層（IH）5への平滑筋細胞の遊走。 哺乳類の研究は、「動脈血静脈」をサポートし、急性の血行動態は静脈が一度動脈環境に移植の顔に変化に対抗するための効率的な方法として、外部の補強を使用することを示唆している。過膨張を防止メッシュは、せん断応力が増加し、壁張力、その結果、IH 6-10を減少させた。しかし、根底にある機序およびバイパス開通性を向上させる上で、人間の静脈への適用は十分に特徴付けられていない。私たちのEVPS静脈を一旦動脈レジメン（高いせん断応力および圧力）、外部マクロ多孔質ポリエステル管状メッシュの非存在下および存在下でのヒト伏在静脈の挙動に挿入面する変化を模倣する条件において、比較した。病理学的リモデリングおよびIHを防止することにより、メッシュは、その潜在的な臨床効率11の証拠を提供</sup&gt;。 この研究は1）は、外部マクロ多孔質ポリエステルメッシュはIHを減少させ、その潜在的な臨床応用するために重要な情報を提供することを実証制御された圧力とせん断応力の2）の下でex vivoでのヒト伏在静脈灌流のモデルを紹介します。

<table border="0" cellpadding="0" cellspacing="0" width="1113">
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			<td height="21" style="height:21px;width:453px;">Name</td>
			<td style="width:371px;">Company</td>
			<td style="width:145px;">Catalog Number</td>
			<td style="width:144px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 - Glutamax</td>
			<td>Life Technologies</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"><a name="RANGE!A3">Penicilline/Streptomycine/Fungizone</a></td>
			<td>Bioconcept</td>
			<td>4-02F00-H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran from Leuconostoc spp. 500 gr.</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tampon PBS CHUV pH 7.1-7.3 1 lt.</td>
			<td>Laboratorium und Grosse Apotheke Dr. G. Bichsel AG</td>
			<td>100 0 324 00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryosectionning embedding medium - Tissue-Tek OCT Compound</td>
			<td>Fisher Scientific</td>
			<td>14-373-65</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon Tubing (Peroxide) L/S 16 (96400-16 ) - 7.5m</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>MF0037ST</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-splitter&#160;</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>Y-connector</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm Culture dish</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430165-100EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml Falcon tube</td>
			<td>BD Bioscence</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml Falcon tube</td>
			<td>BD Bioscence</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gearing pump - Reglo-Z</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>SM 895&#160;</td>
			<td>&#160;App-Nr 03736-00194</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pump Head</td>
			<td>Idex Health &#38; Science GMBH</td>
			<td>MI0008&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monitoring Kit TRANSPAC IV</td>
			<td>icumedical</td>
			<td>011-0J736-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 mL Syringes</td>
			<td>B. Braun Medical SA</td>
			<td>4612041-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Etibon 3-0 FS-2</td>
			<td>Ethicon- Johnson&#38;Johnson</td>
			<td>EH7346H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mesh ProVena 6-8mm</td>
			<td>B. Braun Medical SA</td>
			<td>1105012-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl: Sodium Chlorure Solution perfusion 0.9% (100 ml)</td>
			<td>B. Braun Medical SA</td>
			<td>534534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Masterflex L/S Standard Drive</td>
			<td>Cole-Parmer Instrument Co</td>
			<td>7521-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acquisition card</td>
			<td>National Instruments</td>
			<td>PCI-6024 E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowmeter module</td>
			<td>Transonic Systems Inc.</td>
			<td>TS410 and T402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stopcock with 3-ways</td>
			<td>BD Connexta Luerlock</td>
			<td>394600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex Filter</td>
			<td>Milian</td>
			<td>SE2M229I04</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for human saphenous veins ex vivo perfusion and external reinforcement

大規模な閉塞性動脈疾患のための現代的な治療の主力は、静脈バイパス移植である。しかし、その耐久性は、最終的には血管閉塞及び移植の失敗につながる内膜肥厚（IH）の脅威にさらされている。機械的な力、特に低せん断応力と高い壁の張力は、開始するために、これらの細胞および分子の変化を維持すると考えられているが、それらの正確な寄与は解明されていない。選択的にIHの生物学上の圧力とせん断応力の役割を評価するために、ex vivoで灌流システム（EVPS）は、動脈レジメン（高せん断応力および高圧）の下で人間の伏在静脈のセグメントを灌流するために作成されました。さらなる技術革新は、同じ静脈、外部メッシュで補強された1から2のセグメントの同時灌流を可能にした。静脈は、非接触技術を使用して採取し、直ちにEVPSアセンブリ実験室に移した。たてISOLの一つのセグメントated静脈（コントロール、0日目）に灌流されていませんでした。他の二つのセグメントが最大7日間灌流し、1は完全に4ミリメートル（直径）外部メッシュで保護されている。圧力、流速、および脈拍数を連続的にモニターし、大腿動脈内で優勢な血行力学的条件を模倣するように調整した。潅流が完了すると、静脈がディスマウントされ、組織学的および分子分析のために使用される。 エキソビボ条件下では、高圧灌流（動脈、平均値= 100 mmHgで）がIHおよびヒト静脈のリモデリングを生成するのに十分である。これらの変化は、外部ポリエステルメッシュの存在下で還元されている。

EVPSは、人間の伏在静脈の再構築およびIHをグラフト上で独立して血流力を評価するための貴重なツールを提供します。 図1は、灌流チャンバーと静脈のサポートを示しています。 図1AおよびBにおいて、前静脈サポート（ 図1A）および後（ 図1B）アセンブリは、それぞれ描かれている。これは、簡単に（左か...

Watch this Scientific Journal Video about Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement at JoVE.com

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

内膜過形成（IH）及び静脈グラフト不全の発症を導くメカニズムはまだよく理解されていない。本研究では、制御された流れと圧力の下で、人間の静脈を灌流するためにex vivoでシステムが記載されている。さらにIHの開発を制限する外部メッシュ補強の効率を評価した。

人間の伏在静脈のための手順<em&gt;生体外</em&gt;灌流および外部補強

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

procedure-for-human-saphenous-veins-ex-vivo-perfusion-external

Research

JoVE Journal

Medicine

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

11.1K ビュー.  Brigham and Women's Hospital/Harvard Medical School. 内膜過形成（IH）及び静脈グラフト不全の発症を導くメカニズムはまだよく理解されていない。本研究では、制御された流れと圧力の下で、人間の静脈を灌流するためにex vivoでシステムが記載されている。さらにIHの開発を制限する外部メッシュ補強の効率を評価した。 

ビデオ: Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Subnormothermicのテクニック<em&gt;生体外</em&gt;肝臓ストレージ、評価のための灌流、およびマージナル肝移植片の修復

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

医学

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

孤立の方法<em&gt;エクスビボ</emラットモデルにおける&gt;肺灌流：ラットEVLPプログラムの開発の教訓

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

アン<em&gt;エクスビボ</emヒト乳癌でホルモンの作用を研究するために&gt;モデル

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

人間デュピュイトラン<em&gt;エクスビボ</em筋線維芽細胞と細胞外マトリックス相互作用の研究のために&gt;文化

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

正常温度の<em&gt;エクスビボ</em&gt;腎臓の保全のための腎臓の灌流は移植前の移植片

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Kinomicプロファイリングと薬物反応検査のための3D人間バイオゲル培養システムと患者由来の神経膠芽腫細胞を用いた微小腫瘍の発生

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

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, ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

元ヴィヴォ・ランゲンドルフ・パーフュード・ハーツの眼電図と電気生理学研究

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Determining and Controlling External Power Output During Regular Handrim Wheelchair Propulsion

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which has led to improved research techniques becoming available over the last decades. To increase the understanding of wheeled mobility performance, monitoring, training, skill acquisition, and optimization of the wheelchair-user interface in rehabilitation, daily life, and sports, further standardization of measurement set-ups and analyses is required. A crucial stepping-stone is the accurate measurement and standardization of external power output (measured in Watts), which is pivotal for the interpretation and comparison of experiments aiming to improve rehabilitation practice, activities of daily living, and adaptive sports. The different methodologies and advantages of accurate power output determination during overground, treadmill, and ergometer-based testing are presented and discussed in detail. Overground propulsion provides the most externally valid mode for testing, but standardization can be troublesome. Treadmill propulsion is mechanically similar to overground propulsion, but turning and accelerating is not possible. An ergometer is the most constrained and standardization is relatively easy. The goal is to stimulate good practice and standardization to facilitate the further development of theory and its application among research facilities and applied clinical and sports sciences around the world.

Accurate and standardized assessment of external power output is crucial in the evaluation of physiological, biomechanical, and perceived stress, strain, and capacity in manual wheelchair propulsion. The current article presents various methods to determine and control power output during wheelchair propulsion studies in the laboratory and beyond.

通常のハンドリム車いす推進時の外部電力出力の決定と制御

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which ...

人間の伏在静脈のための手順

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Subnormothermicのテクニック

孤立の方法

アン

人間デュピュイトラン

Ex Situ Normothermic Machine Perfusion of Donor Livers

正常温度の

Kinomicプロファイリングと薬物反応検査のための3D人間バイオゲル培養システムと患者由来の神経膠芽腫細胞を用いた微小腫瘍の発生

Ex Vivo常温肝灌流の小さな動物モデル

元ヴィヴォ・ランゲンドルフ・パーフュード・ハーツの眼電図と電気生理学研究

通常のハンドリム車いす推進時の外部電力出力の決定と制御