recCD47 1.変更高分子表面注：プロトコルは、図1に概略的に要約されているが、 図1Aは、チオール反応性ポリマー表面の生成を示す図1Bは、チオール反応性recCD47の生成を示す。 <ol><li> 1日目<ol><li>滅菌水中PDT-BzPhの溶液（1 mg / ml）で、および重炭酸カリウム（KHCO 3）（0.7 mg / mlで）を調製。 4℃（ 光から保護する ）で一晩撹拌する。 </li></ol></li><li> 2日目<ol><li> （回転車輪の周りにフィットするのに十分な長）長さ40cmの片に、ポリマーチューブをカットします。 </li><li>シェーカー上またはチャンドラーループ装置上で室温で90分間hexacylpyridiniumの0.1％水溶液中でチューブを浸す。 90分浸した後、滅菌水で3回でチューブを洗浄します。 </li><li> （15％水性リン酸二水素カリウムを添加することにより、1日目からPDT-BzPhの溶液を酸性KH 2 </suB&gt; PO 4）。曇ったミセル溶液を形成し、PDT-BzPhの1ml当たり50μlのKH 2 PO 4を追加します。 </li><li>シェーカー上又は装置上で室温で40分間、酸性化PDT-BzPh溶液中にチューブを浸す。 </li><li>一度：希酢酸（千1）で管を洗浄します。 </li><li> 60分の合計時間のための紫外線照射にチューブを公開します。チューブを回転させて¼表面領域全体を照射するために15分毎に回す。 </li><li>シェーカー上又は装置上で室温で20分間、20 mg / mlの炭酸水素カリウム（KHCO 3）の溶液中にチューブを浸す。 </li><li>滅菌水で4℃で滅菌水の3倍と店舗との管を洗浄します。 </li></ol></li><li> 3日目<ol><li> （：ドラフト内で実行します。注意 ）200μlのジメチルホルムアミド（DMF）中に5mgのスルホSMCCを溶かす。 </li><li> recCD47のポリリジン溶液をステップ1から0.1 mg / mlの中で調製スルホSMCC溶液50μlを追加し、ROで60分間攪拌オム温度。 </li><li> 60分攪拌中に、ドガはダルベッコのリン酸緩衝生理食塩水（DPBS）を無菌で、取っておく。 </li><li>浄化スルホ-SMCCは、製造業者の説明書に従って7K分子量カットオフスピン脱塩カラムを使用してrecCD47を反応させた。最終的なフロースルー（高品質のチオール反応recCD47）を収集し、コートDPBSでチューブの内部に必要な容量まで希釈する。 </li><li>無菌で2日目からのチューブを洗浄し、DPBS 3倍を脱気。 </li><li>以下で2​​分間、脱気DPBS中の20 mg / mlのとトリス（2 - カルボキシエチル）ホスフィン塩酸塩（TCEP）の溶液をチューブの表面修飾反応する。無菌脱気DPBS 4倍で管を洗浄します。 </li><li>チューブにスルホSMCC反応recCD47を追加し、シェーカー上または装置上で4℃で一晩インキュベートする。 </li></ol></li></ol>修飾された表面上のrecCD47 0002イムノアッセイ定量<ol><li> 3日目ウィットから定量化するように変更さのチューブをすすぐ時間DPBS 3倍。店舗チューブは4℃で、DPBSで定量化のために使用されていません。 </li><li>重複チューブサンプルを作成し、96ウェルプレートのウェル中の生検パンチを配置し4mmの生検パンチを使用する。 </li><li>検出抗体で処理されていない未修正のチューブサンプルからなるネガティブコントロールを準備します。検出抗体で処理された未修正のチューブサンプルからなる抗体コントロールを準備します。検出抗体で処理された修飾されたチューブ試験サンプルを調製する。 </li><li>シェーカー上で室温で60分間、DPBS中の0.4％ウシ血清アルブミン（BSA）でブロック·サンプル。 </li><li> 、吸引BSAをブロッキングした後、トリス緩衝生理食塩水を加えた1％のTween-20（TBST）シェーカー上で3倍、10分ごとに洗い流してください。 </li><li>免疫アッセイのための製造業者の推奨希釈率に応じて、0.4％BSA中での抗体の使用希釈を準備します。ヒトCD47抗体B6H12-FITC 1に希釈する：0.4％BSA中100。 </li><li>適切なウェルに抗体希釈液200μlを追加します。 Incubaわずか0.4％BSAを含むTEネガティブコントロール。 （ 光から保護する ）シェーカー上で60分間室温でインキュベートする。 </li><li> TBSTで3回、シェーカー上で10分間ずつで洗い流してください。最後TBSTリンスを吸引し、チューブのサンプルウェルに200μlのDPBSを追加。 </li><li>最終的なフロースルー、高品質なチオール反応性の組換えCD47をある収集し、コートDPBSでチューブの内部に必要な容量まで希釈する。 </li><li> FITC励起（485 nm）を発光（538 nm）の設定で、マイクロプレートリーダーを用いて、FITCシグナル強度をお読みください。 </li><li>計算recCD47は、自己蛍光および非特異的抗体対照試料からの結合を占め、標準値に基づいて、ポリマー表面に結合した。 </li></ol> 3チャンドラーループ装置プロトコル採血プロトコル前ヒト血液サンプルの収集の開始にインフォームドコンセントの書類の治験審査委員会（IRB）の承認を受けてください。情報を入手rmedヒト血液ドナーの同意。 注：装置の説明図が図2に示されている。 <ol><li>約1/2車輪が水没している直径になるまで蒸留水で水浴を埋める。 10％の漂白剤溶液を作るために、水浴に十分な漂白剤を追加します。 37℃に水浴を設定し、温度が平衡化させる。 </li><li>金属アダプタ（ 図1Bに示されている）、非修飾および修飾チューブチューブを収集し、 図1Cに示すように、装置のホイールの周りに組み立てる。チューブが所定の位置に金属製のアダプタを備えている車輪の周りにぴったりとフィットしていることを確認してください。 </li><li>クエン酸塩、または他の抗凝固剤の2ミリリットルを予め充填したバイアル中、IRBが承認したプロトコルを使用して、30ミリリットルの血液サンプルを入手し、検体が収集された後、凝固防止するために、転倒混和。 </li><li>チューブ内のいくつかの空気を残して、金属バルブとシリンジを使用して、各チューブへの血液の約10ミリリットルを追加します。バルブキャップとrを固定します3時間otate。 </li><li> 10％の漂白剤溶液で処理された廃棄物のビーカーに血液を排出（または施設の方針によって必要とされるような）。 </li><li>静かに、血液の痕跡をすべて削除するDPBSで配管内部を洗浄します。フロースルー廃棄物のビーカー内を収集します。制度的方針に基づき、血液廃棄してください。 </li><li>装置および任意の血液の10％漂白剤溶液を用いて、接触面または施設のポリシーに応じて駆除。 </li><li>蛍光顕微鏡または走査型電子顕微鏡のための処理サンプルは、下記のように。 </li></ol> 4。蛍光顕微鏡および細胞計数<ol><li> 4％パラホルムアルデヒド（PFA）溶液（ 注意：ドラフト内で実行します ）を準備します。 </li><li> 4℃で一晩4％PFA溶液中でチューブをインキュベートする。 </li><li>一晩インキュベートした後、4％PFAを削除し、DPBSで非常に優しくフィルムをすすぐ。 </li><li>染色のために内腔面を露出するセクションにチューブをカットします。 </li><li>染み室温で30分間、4 &#39;、6 -ジアミジノ-2 -フェニルインドール（DAPI）でメディアを装着する数滴のチューブ部（ 光から保護 ）。染色した後、バックグラウンドDAPIシグナルを減らすためにDPBSで非常に優しくチューブをすすいでください。 </li><li> 200X倍率の下で見ると9盲目的選択されたフィールド内のセルの数をカウントするためにDAPIフィルタとデジタルカメラを装備した蛍光顕微鏡を用いた画像。 </li><li>レコードのセルは、視野当たりのカウントし、結果の統計的有意性を決定するために、適切な統計分析を行う。 </li></ol> 5。走査型電子顕微鏡<ol><li> 2％グルタルアルデヒド溶液（ 注意：ドラフト内で実行します ）を準備します。 </li><li> 4℃で一晩、2％グルタルアルデヒド溶液中でチューブをインキュベートする。 </li><li>一晩のインキュベーションの後、静かにDPBSでチューブ3回すすいでください。 </li><li>四酸化オスミウムの1％溶液を準備します（ 注意：重度吸入の危険性！ ）ドラフト内で使用してください。 </li><li>室温で15分間、四酸化オスミウムの1％溶液中でチューブをインキュベートする。 </li><li>静かにDPBSでチューブ3回すすいでください。 </li><li>エタノール濃度のシリーズ（25％、50％、75％、95％、および100％エタノール）を調製。 </li><li>エタノール濃度の一連のインキュベーションによりチューブを脱水。 25％、50％、75％、20分ごとに、95％エタノール中でインキュベートする。 30分間、100％エタノール中でインキュベートする。 </li><li>臨界点は、すべての水分を除去するために45分間CO 2を有するチューブを乾燥させる。 </li><li>銀ペーストまたはグラファイトで試料スタブ上のマウントチューブセクション。 </li><li>金 - パラジウムの12.5 nmのとコートチューブセクション。 </li><li>走査型電子顕微鏡で調べます。 </li></ol>

<ol>
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この刊行物で報告された研究は、ナショナルの、受賞番号T32 HL007915（JBSとRJL）の下で、受賞番号R21 EB015612（SJS）、国立心肺血液研究所の下で医学と生物工学、総合研究所によってサポートされていました保健研究所。

（ 図1に要約する）光活性化化学は、光活性化PDT-BzPhをするPDT-BzPhの付着およびそれに続くUV照射を容易にするために十分な炭化水素を有する実質的に任意のポリマー表面の改質を可能にする。反応性チオール基を有するポリマー表面を官能することは興味のある検証可能な分子の範囲のその後の取り付けを可能にします。私たちの特定の研究において、本発明者らは、組換えCD47 ...

そのような人工心肺や腎臓透析などの多くの臨床手順は、ポリマー血液導管の使用を必要とし、多くの場合、術後の合併症1に関連している。血液で灌流すると、これらのポリマーは血液タンパク質および血小板、単球/マクロファージ付着の吸着、その結果、異物反応（FBR）を誘発（illicit）、および、術後の合併症に寄与する全てが炎症誘発性サイトカインの放出/またはデバイスの故障2,3。したがって、この問題に対処するための戦略は、生体材料研究の重要かつ継続的な地域のまま。研究者らは、生体活性または生体不活性分子を4-6と表面を接触させること、血液を変更することによってこの問題に対処しようと試みてきた。私たちの研究室での研究は、FBRを軽減し、これらの材料の有効性を高めるための戦略として、高分子生体材料への組換えCD47（recCD47）を追加に焦点を当てている。 CD47は遍在的に発現transmembrです細胞7-10やショーを表現する際の免疫回避の既知の役割、付与「自己」ステータスを持つメタンタンパク質がポリマー表面11-13に付加するとき、生体適合性を付与するに約束します。シグナル調節タンパク質α（SIRPα）、CD47のための同族受容体、および膜貫通タンパク質のファミリーを含有免疫受容体チロシンベースの阻害モチーフ（ITIM）のメンバーは、骨髄起源14の細胞に発現しています。私たちは、以前にCD47は、SIRPαを介した細胞シグナリングを介して、 インビトロ 、 エキソビボでポリウレタン（PU）、およびポリ塩化ビニル（PVC）に対する免疫応答を下方制御、およびin vivoモデル 11-13 にすることを実証した。 私たちの研究の中心は化学的に反応性のチオール基が共有結合多官能性ポリマー（PDT-BZでチューブを反応させることによって、ポリマーのチューブに追加された、本明細書に記載され、比較的小説光活性化化学で2 -ピリジルジチオ（PDT）から成る）Phは、光反応性ベンゾフェノン（BzPh）とカルボキシ変性ポリアリルアミン11-13。トリス（2 -カルボキシエチル）ホスフィン塩酸塩（TCEP）11と共有結合的に付加さPDT基を還元すると、続いて治療的部分と反応させることができるチオール化表面が得られる。本明細書に詳述以前12,13、さらなるC末端のポリ-リジン尾部12,13の添加によって修飾されrecCD47は、スルホスクシンイミジル-4 - [N -maleimidomethyl]シクロヘキサン-1カルボキシレート（スルホ-SMCC）と反応させて1時間チューブとrecCD47 11との間のモノスルフィド結合形成を可能にする、チオール反応性基を生成する。 CD47官能化された表面の抗炎症能力がもともと血栓凝固15 のin vitroモデルとして1958年に記載されたヒト全血、チャンドラーループ装置を使用して、 元VIV O、試験した。この装置は、に依存していますチューブ部分的に空気を充填システムとチューブ15に血液を循環させる回転モータを閉じた。この実験モデルは、修飾および非修飾表面の際に血液暴露の影響、ならびに血液の細胞の生理時にこれらの表面修飾の効果を検討する機会を提供する。 recCD47は、この光活性化化学を用いてポリマー表面の多様に付加することができ、その抗炎症能力は、ポリマー表面11,12の上に血液灌流を模倣する臨床的に関連エキソビボモデルを利用することによって評価することができる。 recCD47で修飾された臨床グレードの血液導管は、装置内のヒト血液にさらされたときに、未修飾ポリマーと比較して有意に低い血小板および炎症細胞の付着を示す。この変形プロセスのステップバイステップの説明は、以下に詳述する。

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:107px;">Catalog Number</td>
			<td style="width:251px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">16% Paraformaldehyde (PFA)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">58906</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">25% Glutaraldehyde</td>
			<td style="width:175px;">VWR</td>
			<td style="width:107px;">AAA17876-AP&#160;</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">2-pyridyldithio,benzophenone (PDT-BzPH)</td>
			<td style="width:175px;">Synthesized in lab</td>
			<td style="width:107px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">A3059-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Citrate</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">S5770-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Digital Camera</td>
			<td style="width:175px;">Leica</td>
			<td style="width:107px;">DC500</td>
			<td>Out of production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dimethylformamide (DMF)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">270547-100ML</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:175px;">Gibco/Life Technologies</td>
			<td style="width:107px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fluorescent Microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:107px;">TE300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glacial Acetic Acid</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">A38-212</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Human CD47 (B6H12) &#8211; FITC Antibody</td>
			<td style="width:175px;">Santa Cruz Biotechnology</td>
			<td style="width:107px;">SC-12730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Osmium Tetroxide</td>
			<td style="width:175px;">Acros Organics</td>
			<td style="width:107px;">197450050</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Bicarbonate (KHCO3)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">237205-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Phosphate Monobasic (KH2PO4)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">P5655-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PVC Tubing (Cardiovascular Procedure Kit)</td>
			<td style="width:175px;">Terumo Cardiovascular Systems</td>
			<td style="width:107px;">60050</td>
			<td>Most clinical-grade tubing will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>JSM-T330A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Sodium Chloride (NaCl)</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:269px;">Microplate Reader</td>
			<td style="width:175px;">Molecular Devices</td>
			<td style="width:107px;">Spectramax Gemini EM&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sulfo-SMCC</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">M6035-10MG</td>
			<td>Moisture Sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">tris (2-carboxyethyl) phosphine (TCEP-HCl)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">20491</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tris Base</td>
			<td style="width:175px;">Sigma</td>
			<td>T1503-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tween-20</td>
			<td>Bio-Rad</td>
			<td>170-6531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Vectashield with DAPI</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">H-1200</td>
			<td>Light sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Zeba Spin Desalt Columns &#8211; 7K MWCO</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">89891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of the ex vivo chandler loop apparatus to assess the biocompatibility of modified polymeric blood conduits

合成表面は、身体に導入されたときに異物反応が起こる。これは、術後の合併症につながる、血液タンパク質の吸着および血小板の付着およびそれに続く活性化、単球/マクロファージの接着性、および炎症性細胞シグナル伝達事象によって特徴付けられる。チャンドラーループ装置は、研究者が、血液の大量のは、ポリマーの導管を介して灌流されているときに発生する分子や細胞間相互作用を研究することを可能にする実験的なシステムです。そのために、この装置は、各種高分子表面修飾の抗炎症特性の評価を可能にex vivoでのモデルとして使用されてきた。当研究室では、共有結合した組換えCD47と光活性化化学により修正された血液導管は、ポリマー表面に生体適合性を付与することができることが示されている。ポリマー表面にCD47を付加することは、ポリマーの血液導管の有効性を促進するための有効な手段である可能性があります。彼女のEINはCD47修正されたと制御導管と血液の相互作用を調べるために臨床的に関連性ポリマー血液導管および ex vivo実験モデルとしてチャンドラーループの使用に組換えCD47を追加するために使用される光活性化化学を詳細に方法論である。

SMCCを用いて、チオール反応recCD47のポリリジンと一緒にPDT-BzPhおよびTCEPを使用することにより、チオール反応性高分子表面の生成は、ポリマー表面にrecCD47の取り付けが可能になります。修正処理は、 図1に概略的にまとめられている。この変更処理の利便性タンパク質は、アミン含有リジンのような十分な化学的反応基で修飾することができると仮定すると、それは多くの異な?...

Watch this Scientific Journal Video about The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits at JoVE.com

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

の使用<em&gt;生体外</em&gt;チャンドラーループ装置は、改質ポリマー血液導管の生体適合性を評価するための

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

the-use-ex-vivo-chandler-loop-apparatus-to-assess-biocompatibility

Research

JoVE Journal

Bioengineering

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

11.5K ビュー.  The Children's Hospital of Philadelphia. Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits. 

ビデオ: The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

生物工学

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

使用プラ​​センタアクロス異物およびナノ材料の輸送速度の決定<em&gt; ex vivoで</em&gt;ヒト胎盤灌流モデル

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

人間の細胞に基づく、血液脳関門の接触モデルの作製のための改善された方法

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

遺伝的に改変された細胞移植のためのマイクロパターン培養プレート上スフェロイド細胞に対する遺伝子導入

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

内視鏡的粘膜下注入材料の性能の評価のための新しいEx Vivoモデル

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

浸透哺乳動物の心臓のトランスムラル吸光分光法を用いた細胞代謝モニタリングのための心臓内側発射光カテーテル

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

超高速ドップラーによる血流イメージング

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

&quot;Avatar&quot;- Optimized Ex Vivo Work Loop Experiment to Emulate In Vivo Muscle Force

"Avatar", a Modified Ex vivo Work Loop Experiments Using In vivo Strain and Activation

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and mechanical systems occurs at all levels of biological organization concurrently, from the tuning of leg muscle properties while running to the dynamics of the limbs interacting with the ground. Understanding the conditions under which animals shift their neural control strategies toward intrinsic muscle mechanics (&#39;preflexes&#39;) in the control hierarchy would allow muscle models to predict in vivo muscle force and work more accurately. To understand in vivo muscle mechanics, ex vivo investigation of muscle force and work under dynamically varying strain and loading conditions similar to in vivo locomotion is required. In vivo strain trajectories typically exhibit abrupt changes (i.e., strain and velocity transients) that arise from interactions among neural activation, musculoskeletal kinematics, and loads applied by the environment. The principal goal of our &#34;avatar&#34; technique is to investigate how muscles function during abrupt changes in strain rate and loading when the contribution of intrinsic mechanical properties to muscle force production may be highest. In the &#34;avatar&#34; technique, the traditional work-loop approach is modified using measured in vivo strain trajectories and electromyographic (EMG) signals from animals during dynamic movements to drive ex vivo muscles through multiple stretch-shortening cycles. This approach is similar to the work-loop technique, except that in vivo strain trajectories are scaled appropriately and imposed on ex vivo mouse muscles attached to a servo motor. This technique allows one to: (1) emulate in vivo strain, activation, stride frequency, and work-loop patterns; (2) vary these patterns to match in vivo force responses most accurately; and (3) vary specific features of strain and/or activation in controlled combinations to test mechanistic hypotheses.

Author Spotlight: Bridging the Gap Between In Vivo and Ex Vivo Studies with the &quot;Avatar&quot; Technique to Advance Muscle Mechanics Research 

This article details the methodology for emulating in vivo muscle force production during ex vivo work loop experiments using an "avatar" muscle from a laboratory rodent to assess the contributions of strain transients and activation to the muscle force response.

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and ...

Preparation of Cardiomyocytes and Setting-Up the Apparatus for the Slow Injection Method

Prepare the 10%gelatin stock solution by weighing and dissolving the gelatin in the ADS buffer, and autoclaving the solution. Rewarm the 10%gelatin stock at 37 degrees Celsius, until it becomes liquid. Then dilute the gelatin stock, using a prewarmed ADS buffer, to obtain the final injectable 5%gelatin solution.Suspend 96 cardiomyocyte balls in 100 microliters of prewarmed injection solution. Load the cardiomyocyte ball suspension into the cell injection syringe without aspirating the excess air. Next, hold the syringe vertically, with the needle facing upward, and tap the syringe to collect the bubbles at the upper ridge of the syringe.Also, observe the cardiomyocyte balls gradually settling onto the rubber seal of the plunger. Maintain the vertical position of the syringe, and slowly push the plunger to discard the bubbles and excess cell suspension solution, until 20 microliters of the cell suspension remain in the syringe, and the cardiomyocyte ball remains resting on the rubber seal of the plunger. Once done, immerse the capped syringe in an ice bath for five minutes, and fix the injection syringe apparatus on the animal stage before activating the main syringe pump during percutaneous cell transplantation in an anesthetized rat.

心筋細胞の調製とスローインジェクション法のための装置のセットアップ

免疫蛍光染色による血液脳関門モデルの解析

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

著者スポットライト:ヒト組織研究のための組織チップ技術の使用の促進 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

血液脳関門のモデル化のためのMicroSiM(μSiM)バリア組織プラットフォームの使用

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...