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1。灌流システムの準備<ol><li>水浴、灌流チャンバー、酸素化のための2つの列が、2蠕動ポンプ、2バブルトラップ、2つの流ヒーターやつの圧力センサ（ 図1）で構成される灌流システムをセットアップします。 図2のスキームに従ってシリコーン及びポリ塩化ビニル材料から成るチューブセクションで、これらのコンポーネントを接続します。最後にそれぞれ、胎児と母体の回路を表す2つの回路があります。 </li><li>水浴、フローヒーターや灌流チャンバーの加熱をオンにします。温度は37℃であるべき</li><li>灌流媒体を暖める（NCTC-135組織培養培地は、アールのバッファ（6.8グラム/ L塩化ナトリウム、0.4グラム/ L塩化カリウム、0.14グラム/ Lリン酸一ナトリウム、0.2グラム/ L硫酸マグネシウム、0.2グラムで1:2に希釈したグルコース（1g / Lの）を補充/ Lの塩化カルシウム1,2グラム/ Lグルコース）、デキストラン40（は10g / L）、（10グラムウシ血清アルブミン/ L）、ヘパリンナトリウム（2,500 IU / L）、amoxicilline（250ミリグラム/ L）および重炭酸ナトリウム（2.2グラム/ L）、水浴のpH 7.4）。 </li><li> - 20ミリリットル15：連続して再び200ミリリットルの蒸留水（流量）を200ミリリットルの蒸留水を用いて胎児および母体回路の動脈系、b）は、50ミリリットルの1％水酸化ナトリウム、c）が1％のリン酸と、d）リンス/分）。 </li><li>胎児の動脈管に、胎児のカニューレ（鈍針が修正翼針輸液セットに接続されるべきであるØ1.2ミリメートル）を接続します。 </li><li>すべてのチューブが培地（:15-20 ml /分の流量）が含まれているまで、灌流媒体と胎児と母体の回路の動脈システムをすすぐ。このステップの間にバブルトラップを埋めると、トラップのすべての泡下流を削除します。その後、ポンプを停止します。これは、求心性動脈管は常に気泡の自由であることが本当に重要である、そうでない場合は挿管後、特に細かい胎児の血管が破裂することができます。 </li><li>ガス流量をオンにします。母性回路が無線酸素化され第5％の二酸化炭素および95％合成空気および5％の二酸化炭素および95％の窒素と胎児の回路。 </li><li>圧力センサの記録を開始します。 </li></ol> 2。プラセンタをカニューレ挿入<ol><li>プライマリー帝王切開後の合併症のない長期の妊娠から無傷の胎盤を入手。書面による同意は、配信前に母親（私たちの研究の場合に得られた）が与えられなければならず、研究は（我々の研究でそうだった）地元の倫理委員会によって承認されなければならない。最初のビジュアルコントロールは、健康と無傷胎盤を保証するために助産師が行ってください。 </li><li>胎盤のカニュレーションは重要なステップです！灌流時に組織内のすべての小さな混乱は母体と胎児の循環間のリークにつながることができます。胎盤は分娩後30分以内に取得する必要があります。 </li><li>母親の側に見える混乱することなく、胎盤の辺縁帯に無傷子葉を選択します。絨毛膜板で、臍動脈および外科縫合材料を使用して後でカニューレ側（へその緒に向かって）に上流の静脈の両方に関連する枝を縛る​​。常に2つの結び目を作る。 </li><li>最初の胎児の動脈カニューレを挿入。胎児胎盤動脈は常に静脈より小さく、薄い。 </li><li>胎児の動脈の周りに縫合糸を作ることが、それをすぐにタイアップされません。鉗子で容器を持ち、慎重に容器をカットし、動脈に小さなカニューレ（Ø1.2ミリメートル）を置く。次に（2ノット）縫合糸を結ぶ。 </li><li>同じように胎児の静脈に進みますが、大きめのカニューレ（ミリメートルØ1.5から1.8、鈍針が修正翼針輸液セットに接続する必要があります）を使用します。 </li><li>胎児のポンプ（2 ml /分）をオンにします。そこには目に見える漏れはありませんと、血液が胎児の静脈カニューレの外に発する場合は、徐々に4ミリリットル/分まで流量を増加。胎児の動脈内の圧力を守って、それが70 mmHgのを超えてはならない。胎児またはメイトで流体が漏れる場合外部カニューレは別の縫合糸で固定します。 </li><li>胎児側にティッシュホルダーに胎盤を置き、スパイク以上胎盤膜および組織を引っ張る。最後に灌流子葉はティッシュホルダーの穴の真ん中にする必要があります。 </li><li>唯一の膜はシリコン膜（Ø1ミリメートル）あるいは、2パラフィルムの部分と胎盤を保持している部分を安定させる。 </li><li>完全なティッシュホルダーを組み立て、ネジを締めると張り出した組織を切断する。静脈と動脈カニューレが挟まれていないが、代わりにティッシュホルダーの小さなチャネルにあったことに注意してください。 </li><li> 、逆さまにティッシュホルダーを回し灌流チャンバーに入れ、カバーを追加します。さて、母親の側が一番上にある必要があります。胎児の回路はまだそのままであると媒体が胎児の静脈管から流出されている場合は、必ず確認してください。 </li><li>母性ポンプ（12 ml /分）をオンにします。番目で3鈍カニューレ（Ø0.8ミリメートル）を導入脱落膜プレートを貫通することにより絨毛空間に母親の動脈管の電子終わり。母性回路に灌流液を戻すにしても灌流チャンバー上部の最も低い位置に母親のポンプに接続されている静脈ドレインとして一本のチューブを入れた。 </li><li>胎児の静脈管に胎児の静脈カニューレを接続します。 </li></ol> 3。灌流の前および実験フェーズを実行する<ol><li>組織は、出産後虚血期から回復すると絨毛空間で血を洗い流すようにするには、20分のプレオープン段階が必要である。母体と胎児の静脈を意味する灌流媒体を含む動脈リザーバに戻ってつながるされていない。瓶の中に胎児と母体の静脈流出を収集し、事前フェーズの後に、それを捨てる。 </li><li>灌流の整合性を評価する20分の別のプレフェーズを実行しますが閉回路にする。灌流培地で2つの別々の貯水池を使用胎児と母体の回路用と胎児の貯水池と母性貯水池における母性静脈流出裏に戻って胎児の静脈流出を導くことによって回路を閉じます。 </li><li>メイン灌流実験では120ミリリットル灌流培地（妊産婦のためのものと胎児の貯水池のためのもの）を持つ2つのフラスコを準備します。 1つは母親のリザーバーに分析したいと考えている蛍光標識生体異物またはナノ粒子：放射性標識された14 C-アンチピリン（放射性物質;、陽性対照として注意4 NCI / ml）を追加します。よく母親の灌流液を混ぜる。 </li><li> 2準備フラスコ（胎児と母体の貯水池）で純粋な灌流培地を交換することによって、実験を開始します。胎児の貯水池とバック母性貯水池における母性静脈流出に戻って胎児の静脈流出を導くことによって回路を閉じます。 </li><li> 6時間灌流を継続し、定期的にサンプルを取る。常に胎児と母体の中の培地を再懸濁し撤退前の貯水池。 </li><li>灌流時の胎児の動脈（70 mmHgのを超えてはならない）、両方の回路内のpH（生理的範囲7.2から7.4にする必要があります）や貯水池の両方のボリューム（胎児のボリュームの損失は4ミリリットル/時を超えてはならない）内の圧力を制御。必要ならば、塩酸または水酸化ナトリウムのいずれかを使用してpH値を調節​​する。 </li><li>胎児のリザーバ内容積損失が4を超える場合ミリリットル/時そこに、組織内のリークであり、1つは、灌流を停止する必要があります。漏れなしで6時間灌流の成功率は15〜20％程度である。 </li><li> 6時間後の血流を停止します。ポンプ、水浴、フローヒーターやガスの流れをひっくり返す。 </li><li>ティッシュホルダーから胎盤を外し、灌流子葉をカット（unperfused組織よりも明るい）、それを重量を量る。 </li><li> unperfused（初めにカットされた胎盤の一部、すでにプレフェーズ中に取ることができる）からサンプルを取り出し、灌流組織（各約1g）、-20℃で保管し均質化されるまで以降の分析のために液体窒素である。組織病理学的評価のための4％ホルマリンで別の組織サンプルを修正。サンプルは胎盤のすべての層を含める必要があります。 </li><li> ）50mlの1％水酸化ナトリウム、cが再び））を順次）200ミリリットルの蒸留水、bを加え胎児および母体回路の動脈系をすすぐことによって灌流した後、200ミリリットルの蒸留水を50ミリリットルの1％リン酸と、dチューブをきれい（流量:15-20 ml /分）。 </li></ol>胎盤灌流実験の全体の作業手順を図3に示されている。 4。サンプルの分析<ol><li>残留赤血球を除去するために分析の前に800×gで10分間灌流液のサンプルを遠心分離します。さらに分析するために、上清を取る。サンプルは4℃で一晩放置することができますレプチンとhCGの生産の分析のための試料を-20℃で保存することができます</li><li>の透過性を評価するために、胎盤は、液体シンチレーションによって14 C-アンチピリンを分析。ベータカウンターで5分間3ミリリットルのシンチレーションカクテルとメジャーで胎児と母体のサンプル300μlのを混ぜる。 </li><li>蛍光ナノ粒子の移転や、興味のある異物がマイクロプレートリーダー（示された波長は、我々は、ナノ粒子に使用黄緑色のラベルの分析のためのもの）で485 nmの励起と528 nmの放射で蛍光を読み取るを評価する。 </li><li>灌流時の胎盤組織の実行可能性を判断するには、自動化された血液ガスシステムと胎児と母体の回路中のグルコース消費量と乳酸産生を測定します。また、胎盤ホルモンヒトchoriongonadotropin（hCGの）および酵素結合免疫吸着検定法（ELISA）によって均質化された組織サンプル及び灌流液中のレプチンの産生を評価する。 </li></ol>

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Toxicol. 30, 138-146 (2010).</li><li>Myllynen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+interlaboratory+comparison+of+the+ex+vivo+dual+human+placental+perfusion+system.">Preliminary interlaboratory comparison of the ex vivo dual human placental perfusion system.</a> Reprod Toxicol. 30, 94-102 (2010).</li><li>Malek, A., Sager, R., Schneider, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal-fetal+transport+of+immunoglobulin+G+and+its+subclasses+during+the+third+trimester+of+human+pregnancy.">Maternal-fetal transport of immunoglobulin G and its subclasses during the third trimester of human pregnancy.</a> Am. J. Reprod. Immunol. 32, 8-14 (1994).</li><li>Prouillac, C., Lecoeur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+placenta+in+fetal+exposure+to+xenobiotics:+importance+of+membrane+transporters+and+human+models+for+transfer+studies.">The role of the placenta in fetal exposure to xenobiotics: importance of membrane transporters and human models for transfer studies.</a> Drug Metab. Dispos. 38, 1623-1635 (2010).</li><li>Poulsen, M. S., Rytting, E., Mose, T., Knudsen, 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=Modeling+placental+transport:+correlation+of+in+vitro+BeWo+cell+permeability+and+ex+vivo+human+placental+perfusion.">Modeling placental transport: correlation of in vitro BeWo cell permeability and ex vivo human placental perfusion.</a> Toxicol. In Vitro. 23, 1380-1386 (2009).</li><li>Mathiesen, L., Rytting, E., Mose, T., Knudsen, 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=Transport+of+benzo[alpha]pyrene+in+the+dually+perfused+human+placenta+perfusion+model:+effect+of+albumin+in+the+perfusion+medium.+Basic+Clin.">Transport of benzo[alpha]pyrene in the dually perfused human placenta perfusion model: effect of albumin in the perfusion medium. Basic Clin.</a> Pharmacol. Toxicol. 105, 181-187 (2009).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

デュアル循環灌流下に答えなければならない質問に応じて可能な他のいくつかの実験的な構成がありますが、ここに示しました。特にオープン胎盤灌流は、一般的に定常状態濃度3に薬物クリアランスを評価するために使用される。再循環血流のセットアップはまた、内因性又は外因性物質の能動輸送を確認するために適用することができる。このアプローチのために生体異物の同じ?...

胎盤は、酸素、二酸化炭素、栄養素および廃棄物と母と互いに分離成長する胎児の二血液回路を維持することができ、同時に交換を担当する複雑な器官である。さらに、母体の免疫系による子供の拒絶反応を防止し、妊娠を維持するホルモンを分泌する。セルラバリアは4,5横細胞膜なしで真シンシチウムを融合して形成栄養膜細胞によって形成される。全体の胎盤は、一胎児絨毛ツリーが含まれており、胎盤の一つの機能単位を表すいくつかの子葉、で構成されています。 胎盤のバリア機能の研究は、1960年代にサリドマイド誘発さ奇形の発見で激化しました。明白な理由のために妊娠中の女性との転座の調査を行うことができない。したがって、様々な代替モデルは6,7開発されている</s&gt;まで。最も有望な、おそらくほとんどの臨床関連モデルはPanigelや同僚2,3によって開発されたex vivoでヒト胎盤灌流モデルです。 多くの女性は、妊娠8時の薬物や環境汚染など、さまざまな異物にさらされている。既に妊娠中に定期的に投与されたいくつかの薬については、 インビボ試験で臍帯血中のそれと母体血中濃度と比較することによって行うことができる。しかし、一般的に胎児およびこれらの物質の催奇形性における薬物動態と力学に関する限られた情報があります。 例えば、ヘロインのようなアヘンを簡単胎盤関門を通過し、子宮内発育制限、早産や自然流産9,10につながることができます。だから、妊娠中行方不明禁欲の場合メタドンによる補充療法が推奨される。 前のインビボでヒト胎盤灌流モデルは、胎児循環へのメタドンの転送が配達12後算出臍帯血対母体血中濃度比とよく相関ごくわずか11であることが明らかになった。 ナノテクノロジーは、特に医学の成長分野である。だから、自然に細かい発生（直径&lt;2.5μm）で、森林火災、火山噴火の煙でと砂漠の塵に超微粒子（直径&lt;0.1μm）で、工業ナノ材料への暴露（少なくとも一次元&lt;0.1μmの13の下）増加している。これは、工業ナノ材料の毒性可能性について疑問を提起した。全く人間の危険がまだ証明されなかったものの、工業ナノ粒子は毒性結果14につながる有害な生物学的反応を引き起こす可能性があることを示す主な実験的研究があります。最近では、いくつかの研究では、への出生前暴露を示した大気汚染は、新生児と子供15,16が高い呼吸必要性と気道炎症にリンクされています。また、小型のナノ粒子は、特に胎児や妊婦のいずれかを治療するための薬物担体として使用されるかもしれない。そのため、異なる生体異物やナノと胎盤関門を通過する能力を広範囲の研究が必要であることが明らかになります。工業ナノ材料への胎盤透過性に関する現在の研究の実際の概要はメネゼスらに要約され、2011年17 Buerki-Thurnherr らは 2012年7。 ex vivoでデュアル循環ヒト胎盤灌流モデルでは、様々な内因性および外因性化合物3,11,12,18,19の胎盤輸送との責任のメカニズムのような胎盤の他の機能の広い範囲を研究するための制御された信頼性の高いシステムを提供しています子癇前症のような病理学的状態の開発&lt;&gt; 20-22（商標）。このプロトコルでは、蓄積効果や生体異物やナノ粒子の広範なセットの転率の研究を可能に設定し、扱い、そして方法に主に焦点を当てています。

<table border="1">
	<tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>NCTC-135 medium</td>
 <td>ICN Biomedicals, Inc.</td>
 <td>10-911-22C</td>
 <td>could be replaced by Medium 199 from Sigma (M3769)</td>
 </tr>
 <tr>
 <td>Sodium chloride (NaCl)</td>
 <td>Sigma-Aldrich, Fluka</td>
 <td>71381</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium chloride (KCl)</td>
 <td>Hospital pharmacy</td>
 <td></td>
 <td>also possible: Sigma (P9541)</td>
 </tr>
 <tr>
 <td>Monosodium phosphate (NaH2PO4 &middot; H2O)</td>
 <td>Merck</td>
 <td>106346</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium sulfate (MgSO4 &middot; H2O)</td>
 <td>Sigma-Aldrich, Fluka</td>
 <td>63139</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium chloride (CaCl, anhydrous)</td>
 <td>Merck</td>
 <td>102388</td>
 <td></td>
 </tr>
 <tr>
 <td>D(+) Glucose (anhydrous)</td>
 <td>Sigma-Aldrich, Fluka</td>
 <td>49138</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium bicarbonate (NaHCO3)</td>
 <td>Merck</td>
 <td>106329</td>
 <td></td>
 </tr>
 <tr>
 <td>Dextran from Leuconostoc spp.</td>
 <td>Sigma-Aldrich</td>
 <td>31389</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine serum albumin (BSA)</td>
 <td>Applichem</td>
 <td>A1391</td>
 <td></td>
 </tr>
 <tr>
 <td>Amoxicilline (Clamoxyl)</td>
 <td>GlaxoSmithKline AG</td>
 <td>2021101A</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium heparin</td>
 <td>B. Braun Medical AG</td>
 <td>3511014</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium hydoxide (NaOH) pellets</td>
 <td>Merck</td>
 <td>106498</td>
 <td>CAUTION: corrosive</td>
 </tr>
 <tr>
 <td>Ortho-phosphoric acid 85% (H3PO4)</td>
 <td>Merck</td>
 <td>100573</td>
 <td>CAUTION: corrosive</td>
 </tr>
 <tr>
 <td>Maternal gas mixture: 95% synthetic air, 5% CO2</td>
 <td>PanGas AG</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal gas mixture: 95% N2, 5% CO2</td>
 <td>PanGas AG</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Antipyrine (N-methyl-14C)</td>
 <td>American Radiolabeled Chemicals, Inc.</td>
 <td>ARC 0108-50 &mu;Ci</td>
 <td>CAUTION: radioactive material (specific activity: 55mCi/mmol)</td>
 </tr>
 <tr>
 <td>Scintillation cocktail (IrgaSafe Plus)</td>
 <td>Zinsser Analytic GmbH</td>
 <td>1003100</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene particles 80 nm</td>
 <td>Polyscience, Inc.</td>
 <td>17150</td>
 <td></td>
 </tr>
 <tr>
 <td>Polystyrene particles 500 nm</td>
 <td>Polyscience, Inc.</td>
 <td>17152</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Water bath</td>
 <td>VWR</td>
 <td>462-7001</td>
 <td></td>
 </tr>
 <tr>
 <td>Thermostat</td>
 <td>IKA-Werke GmbH &amp; Co. KG</td>
 <td>3164000</td>
 <td></td>
 </tr>
 <tr>
 <td>Peristaltic pumps</td>
 <td>Ismatec</td>
 <td>ISM 833</td>
 <td></td>
 </tr>
 <tr>
 <td>Bubble traps (glass)</td>
 <td>UNI-GLAS Laborbedarf</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Flow heater</td>
 <td>UNI-GLAS Laborbedarf</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pressure sensor + Software for analyses</td>
 <td>MSR Electronics GmbH</td>
 <td>145B5</td>
 <td></td>
 </tr>
 <tr>
 <td>Notebook</td>
 <td>Hewlett Packard</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Miniature gas exchange oxygenator</td>
 <td>Living Systems Instrumentation</td>
 <td>LSI-OXR</td>
 <td></td>
 </tr>
 <tr>
 <td>Tygon Tube (ID: 1.6 mm; OD: 4.8 mm)</td>
 <td>Ismatec</td>
 <td>MF0028</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubes for pumps (PharMed BPT; ID: 1.52 mm)</td>
 <td>Ismatec</td>
 <td>SC0744</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 0.8 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.592.81</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 1.2 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.592.90</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 1.5 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.592.94</td>
 <td></td>
 </tr>
 <tr>
 <td>Blunt cannulae (&Oslash; 1.8 mm)</td>
 <td>Polymed Medical Center</td>
 <td>03.952.82</td>
 <td></td>
 </tr>
 <tr>
 <td>Parafilm</td>
 <td>VWR</td>
 <td>291-1212</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber with tissue holder (plexiglass)</td>
 <td>Internal technical department</td>
 <td></td>
 <td>Similar equipment is available from Hemotek Limited, UK</td>
 </tr>
 <tr>
 <td>Surgical suture material (PremiCron)</td>
 <td>B. Braun Medical AG</td>
 <td>C0026005</td>
 <td></td>
 </tr>
 <tr>
 <td>Winged Needle Infusion Set (21G Butterfly)</td>
 <td>Hospira, Inc.</td>
 <td>ASN 2102</td>
 <td></td>
 </tr>
 <tr>
 <td>Multidirectional stopcock (Discofix C-3)</td>
 <td>B. Braun Medical AG</td>
 <td>16494C</td>
 <td></td>
 </tr>
 <tr>
 <td>Surgical scissors</td>
 <td>B. Braun Medical AG</td>
 <td>BC304R</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting scissors</td>
 <td>B. Braun Medical AG</td>
 <td>BC162R</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder</td>
 <td>B. Braun Medical AG</td>
 <td>BM200R</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting forceps</td>
 <td>B. Braun Medical AG </td>
 <td>BD215R</td>
 <td></td>
 </tr>
 <tr>
 <td>Automated blood gas system </td>
 <td>Radiometer Medical ApS</td>
 <td>ABL800 FLEX</td>
 <td></td>
 </tr>
 <tr>
 <td>Multi-mode microplate reader</td>
 <td>BioTek</td>
 <td>Synergy HT</td>
 <td></td>
 </tr>
 <tr>
 <td>Liquid scintillation analyzer</td>
 <td>GMI, Inc.</td>
 <td>Packard Tri-Carb 2200</td>
 <td></td>
 </tr>
 <tr>
 <td>Scintillation tubes 5.5 ml</td>
 <td>Zinsser Analytic GmbH</td>
 <td>3020001</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue Homogenizer</td>
 <td>OMNI, Inc.</td>
 <td>TH-220</td>
 <td></td>
 </tr>
 <tr>
 <td>pH meter + electrode</td>
 <td>VWR</td>
 <td>662-2779 </td>
 <td></td>
 </tr>
	 </tbody>
 </table>

determination of the transport rate of xenobiotics and nanomaterials across the placenta using the ex vivo human placental perfusion model

十年前にヒト胎盤は母親と胎児の間に不可解な障壁であると考えられた。しかし、サリドマイド誘発性の先天性欠損症や多くのその後の研究の発見は、その後、反対のことを証明した。ニコチンのような今日、いくつかの有害な異物、ヘロイン、メタドンまたは薬物と同様の環境汚染物質は、この障壁を克服するために記載された。ナノテクノロジーの使用が増えていると、胎盤は、誤って露出を介して、または意図的に潜在的なナノ医療アプリケーションの場合における新規ナノ粒子と接触する可能性があります。胎盤は、ほとんどの種特異的な哺乳類の臓器1であるため、動物実験からのデータは、ヒトに外挿することはできません。したがって、Panigel らによって開発されたex vivoでのデュアル循環ヒト胎盤灌流、1967 2と連続シュナイダーらによって修正された、1972年 3において、優れたモデルTとして機能することができoが生体異物または粒子の移動を検討する。 ここでは、再現性のある結果を取得するために、ヒト胎盤灌流プロトコルおよびさらなる発展を再循環させるex vivoでの二重焦点を当てる。 胎盤は帝王切開を受け合併症長期妊娠から母親のインフォームドコンセント後に得られた。無傷の子葉の胎児と母体の血管が少なくとも5時間カニューレを挿入し、灌流した。モデル粒子として、直径80〜500nmのサイズを有する蛍光標識ポリスチレン粒子は、母体回路に加えた。 80 nmの粒子は胎盤関門を通過し、500 nmの粒子は胎盤組織または母性回路に保持している間、胎児に胎盤を介して転送された物質のための完璧な例を提供することができました。 ex vivoでヒト胎盤灌流モデルは、約信頼できる情報を提供するいくつかのモデルの一つである予測と臨床関連データを提供しています重要な組織関門における生体異物の輸送挙動。

図4Aは、胎児のコンパートメントに移管されていない大きなポリスチレン粒子（500nm以下）に比べて胎盤を通過して輸送された小さなポリスチレン粒子（80 nm）での灌流プロファイルを示しています。各データポイントは、少なくとも3つの独立した実験の所定の時点までの平均粒子濃度を表す。ポリスチレンの場合は胎盤転送はサイズ依存19であるナノ粒子。 500 nmのポリ?...

Watch this Scientific Journal Video about Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model at JoVE.com

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

ザ<em&gt; ex vivoで</em&gt;デュアル再循環ヒト胎盤灌流モデルはヒト胎盤を横切る異物およびナノ粒子の移動を調査するために使用することができる。このビデオプロトコルでは、胎盤の血流を正常に実行するために必要な機器や技術について説明します。

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 ...

determination-transport-rate-xenobiotics-nanomaterials-across

Research

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Bioengineering

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

17.2K ビュー.  University Hospital Zurich. ザ ex vivoでデュアル再循環ヒト胎盤灌流モデルはヒト胎盤を横切る異物およびナノ粒子の移動を調査するために使用することができる。このビデオプロトコルでは、胎盤の血流を正常に実行するために必要な機器や技術について説明します。

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

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

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

正常と異常ヒト造血のex vivoでの擬態

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

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

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

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

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

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

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

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.

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 ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

調整可能なナノ細孔を介してナノ粒子の転ベロシティを経由してゼータ電位の決意：例として、DNA修飾された粒子を用いました

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

実験および有限要素議定書は、関節軟骨全体で中性および荷電溶質の輸送を調査します

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

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 ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

3 つの補完的な手法を使用して胎盤をターゲットとした薬剤投与の安全性の総合評価

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

ヒト好中球群れの化学化学および分子解析のための生体微粒子マイクロアレイ

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

磁性ナノ材料の生体機能化

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...

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

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