这项工作是由瑞士国家基金会（NRP 64个程序，不授予4064-131232）财政支持。

1。准备灌注系统<ol><li>设置的水浴中，灌注腔室，两列进行氧合，两个蠕动泵，两个气泡陷阱，两个流加热器和一个压力传感器（ 图1）组成的灌注系统。这些组件连接起来的管路部分组成的硅树脂和聚氯乙烯的材料，根据图2中的方案。最后，还有两个分别代表胎儿和母体的电路，电路。 </li><li>打开水浴上，流动加热器和加热灌注室。温度应在37℃下</li><li>热身灌注介质（NCTC-135组织培养基稀释1:2厄尔的缓冲液（6.8克/ L盐水，0.4 g / L的氯化钾，0.14 g / L的磷酸二氢钠，硫酸镁0.2克/升，0.2克/ L的氯化钙，2 g / L的葡​​萄糖）葡萄糖（1克/升），右旋糖酐40（10克/升），牛血清白蛋白（10克补充/ L）的钠，肝素（2500 IU / L），阿莫西林（250毫克/升）和碳酸氢钠（2.2克/升），pH 7.4）中，在水浴。 </li><li>连续再次冲洗动脉系统的胎儿和母体的电路）200毫升蒸馏水，二）50毫升1％的氢氧化钠，三）1％磷酸和d）200毫升蒸馏水中（流速：15 - 20毫升/分钟）。 </li><li>胎儿的连接套管（直径1.2毫米，钝针应该连接到修改后的翅针输液器）到胎儿动脉导管。 </li><li>灌注液冲洗动脉胎儿和母体的电路系统，直到所有的管子包含介质（流速：15〜20毫升/分钟）。在这一步填补了泡沫陷阱除去所有气泡下游的陷阱。然后停止泵。这是非常重要的入球小动脉管始终无气泡，否则插管后，特别是细胎儿血管破裂。 </li><li>打开的气体流。产妇电路被氧化无线第5％二氧化碳和95％与5％二氧化碳和95％氮气的的合成空气和胎儿电路。 </li><li>开始录音的压力传感器。 </li></ol> 2。插管胎盘<ol><li>获得完整初级剖宫产无并发症的足月妊娠后胎盘。给予书面同意（我们的研究中得到的情况下）由母亲分娩前的研究，必须由当地伦理委员会的批准（在我们的研究的情况下）。第一视觉控制应该由助产士，以确保一个健康的和完整的胎盘。 </li><li>插管胎盘是关键的一步！在灌注组织中的每一个细小的中断可导致孕产妇和胎儿的血液循环之间的泄漏。胎盘具有以下方式获得分娩后30分钟内。 </li><li>选择一个完整的无可见中断对产妇胎盘子叶边缘区。在绒毛膜板块，同时配合相关的上游通过手术缝合材料后插管侧（朝向脐带）脐动脉和静脉的分支。总是使两个结。 </li><li>首先胎儿动脉导管插入。胎儿胎盘动脉总是比静脉更小，更薄。 </li><li>使胎儿动脉周围缝合，但不把它立即。保持容器用一个镊子，仔细切断船只，并把小动脉套管（直径1.2毫米）。然后配合缝合（两节）。 </li><li>胎儿静脉中相同的方式继续进行，但使用一个更大的套管（直径1.5-1.8毫米;钝针应附加到的变形的翅针输液器）。 </li><li>打开胎儿泵（2毫升/分钟）。如果没有可见的泄漏和血液来源于胎儿静脉插管，慢慢增加至4毫升/分钟的流量。观察胎儿动脉中的压力，它不应该超过70毫米汞柱。如果液体泄漏在胎儿或配偶的RNAL套管修复他们与另一缝合。 </li><li>将胎盘组织人与胎侧拉的胎盘膜和组织在尖峰。在最后的灌注子叶应该在中间的组织保持器的孔中。 </li><li>稳定的部分，其中只有持有胎盘膜用硅胶膜（直径1毫米），或者两个封口膜件。 </li><li>组装完整的组织支架，拧紧螺丝，并切割悬垂组织。请注意，静脉和动脉插管不捏，而是组织持有人躺在小通道。 </li><li>将纸巾架倒挂，把它放进灌注室，并加盖。现在，产妇方应在顶部。经常检查，如果胎儿的电路仍然保存完好，介质流出来的胎儿静脉管。 </li><li>打开的的产妇泵（12毫升/分钟）。介绍了三个钝套管（直径0.8毫米）日e结束产妇的动脉管入间隙穿透蜕膜板。返回灌流产妇电路一管静脉漏，这也与产妇泵灌注室的上部的最低位置。 </li><li>连接胎儿的静脉插管，胎儿静脉管。 </li></ol> 3。执行前和实验阶段灌注<ol><li>为了使组织恢复缺血期分娩后，冲洗掉血液中的间隙，一个开放的相前的20分钟是必要的。这意味着产妇和胎儿的静脉不会导致动脉水库灌注介质。收集胎儿和母体静脉流出并将其丢弃在一个瓶子经过前期相。 </li><li>灌注的完整性，以评估执行另一个预定阶段20分钟，但在一个封闭的回路。使用两个独立的水库灌注液胎儿和母体的电路，导致胎儿的静脉流出，早在胎儿水库和母亲静脉血流出早在母体水库关闭电路。 </li><li>对于主灌注实验准备两个烧瓶中，用120ml灌注培养基（一个为母体和胎儿水库）。添加放射性标记的14 C-安替比林（4 NCI /毫升;作为阳性对照;注意：放射性物质）和荧光标记的外源性或纳米颗粒要分析产妇水库。以及混合产妇灌流。 </li><li>纯灌注介质交换两个准备瓶（胎儿和产妇水库）开始实验。关闭电路，导致胎儿的静脉流出早在胎儿水库和母亲静脉血流出早在母体水库。 </li><li>继续灌注6小时，并定期采取样本。始终悬浮介质中的胎儿和产妇水库前撤回。 </li><li>灌注过程中的胎儿的动脉（应不超过70毫米汞柱），在两个电路中的pH值（应该是在生理范围内,7.2-7 .4）和两个水塘的体积（，胎儿体积损失不应超过4毫升/小时）中的压力控制。如果有必要，可以使用盐酸或氢氧化钠调节pH值。 </li><li>如果在胎儿储层的体积损失超过4毫升/小时，在组织中会出现泄漏，停止灌注。 6小时无泄漏灌注的成功率是15-20％左右。 </li><li> 6小时后停止灌注。关闭泵，水浴，流量加热器和气体流量。 </li><li>取出胎盘组织人，削减灌注的子叶（亮比unperfused组织）和称重。 </li><li>抽取样本unperfused（部分的胎盘，在开始切割过程中已经采取预相）和灌注组织（约1克），并将其储存在-20°C直至均匀化，或在液氮中用于以后的分析。修复另一个组织样本在4％福尔马林组织病理学评估。样品应包括所有层的胎盘。 </li><li>清洁管灌注后依次漂洗动脉系统的胎儿和母体电路）200毫升蒸馏水，B），50毫升1％的氢氧化钠，三）50毫升1％的磷酸和d）200毫升的蒸馏水再（流量：15〜20毫升/分钟）。 </li></ol>胎盘灌注实验的整个工作过程的图3中描述。 4。分析样品<ol><li>以800×g的10分钟离心灌流液的样品在分析前以除去残余的红细胞。取上清液进行进一步的分析。可将样本在4℃下放置过夜如果在的瘦素和hCG生产的分析，可将样本存储于-20℃。 </li><li>透性来评估的胎盘液体闪烁分析14 C-安替比林。混合300微升的胎儿和母体样品用3ml闪烁鸡尾酒和测量5分钟，用β-计数器。 </li><li>为了评估转移的荧光纳米颗粒或还异生在485 nm激发波长，528发射波长，用酶标仪（表示波长进行分析，我们使用的纳米粒子的黄色的绿色标签）读取荧光。 </li><li>为了确定胎盘组织灌注过程中测量的葡萄糖消耗和乳酸生产中的胎儿和产妇的电路与自动化血气系统的可行性。此外，评估生产人类choriongonadotropin胎盘激素（hCG）的组织匀浆样品的灌流液，用酶联免疫吸附试验（ELISA）和瘦素。 </li></ol>

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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>

作者宣称，他们有没有竞争经济利益。

这里显示下方的双循环灌流，还有其他一些可能的实验配置取决于必须回答的问题。常用特别开放的胎盘灌注，稳态浓度3评估药物清除。的循环灌流设置也可以应用于，确认主动运输的内源性或外源性物质。对于这种方法的生物外源性的相同浓度的已被添加到母亲和胎儿循环。假定不存在逆浓度梯度的主动转运，可以观察到的两个电路中的任一个被检物质的积累24。值得注意的是...

胎盘是一个复杂的器官，它是负责交换，氧气，二氧化碳，营养物和废物和在同一时间能够保持对母亲和胎儿生长彼此分开的两个血电路。此外，它可以防止由母体免疫系统排斥的孩子，并分泌激素，以维持妊娠。由4,5无侧向细胞膜融合可以形成一个真正的合胞体滋养层细胞形成的细胞的屏障。整个胎盘组织在几个子叶，其中包含一个胎儿绒毛树，代表一个功能单元的胎盘。 胎盘屏障功能的研究，发现在1960年的沙利度胺致畸形加剧。出于显而易见的原因，易位研究孕妇，不能进行。因此，各种替代车型已经开发了6,7 </s高达&gt;。最有前途的和可能是最临床相关模型是体外人类胎盘灌注模型开发Panigel和同事2,3。 许多妇女面临不同的外来物质，如药物或环境污染物在怀孕期间8。对于一些已经在怀孕期间定期给药的药物， 在体内研究中，可以通过的产妇与脐带血液中的血药浓度相比，。然而，一般只有有限的信息，这些物质在胎儿致畸的药动学和动力学。 例如阿片类药物，如海洛因容易穿过胎盘屏障，可导致胎儿宫内发育迟缓，早产或流产9,10。因此，在丢失的情况下，节制在怀孕期间美沙酮的替代治疗的建议。 前体内的人类胎盘灌注模型透露，转移美沙酮进入胎儿循环是微不足道的11，以及相关计算脐带血产妇血药浓度比分娩后12。 纳米技术是一项不断发展的领域，尤其是药品。所以，自然发生的罚款 ​​（直径小于2.5微米）和超细微粒（直径小于0.1微米）的森林火灾，火山喷发的烟雾和沙尘，暴露于纳米材料工程（至少有一维小于0.1微米13下方）增加。有关工程纳米材料的毒理学潜力提出了疑问。虽然没有人类的危害还可以证明，也有主要的实验研究表明，工程纳米颗粒可引起不良的生物反应导致毒理学结果14。最近，一些研究表明，产前暴露于空气污染与新生儿和儿童15,16需要更高的呼吸道和呼吸道炎症。此外，小颗粒可能被用来作为药物载体，专门治疗胎儿或母亲。因此，它变得明显，需要广泛的研究，不同的外源性化学物质或纳米材料和他们有能力穿过胎盘屏障。在梅内塞斯等胎盘渗透到工程纳米材料的研究对当前实际的概述总结2011年17和Buerki Thurnherr 等。2012 7。 人体胎盘灌注模型的体外双循环提供了一个控制和可靠的系统研究胎盘胎盘范围广泛的其他功能，如各种内源性和外源性化合物3,11,12,18,19和运输负责的机制开发先兆子痫的病理状态，如&lt;SUP&gt; 20-22。在这个协议中，我们主要集中在一套，处理和方法，使研究的积累，影响和移位率了一套广泛的外来物质或纳米颗粒。

<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

几十年前的人胎盘被认为是一个难以逾越的障碍的母亲和未出生的孩子之间。然而，事后发现沙利度胺致出生缺陷和后来的许多研究证明相反。今天，一些有害的外来物质，如尼古丁，海洛因，美沙酮或描述克服这一障碍的药物，以及环境污染物。随着越来越多的利用纳米技术，胎盘可能接触到新颖的纳米粒子通过曝光是意外或故意的情况下，潜在的纳米药物应用。从动物实验数据，不能外推到人类，因为胎盘是最具体物种的哺乳动物器官1。因此，于1967年2，不断修改施耐德等。体外双循环人体胎盘灌注，开发等Panigel。于1972年3，可以作为一个很好的T型车Ø研究外源性化学物质或微粒的转移。 在这里，我们专注于体外双循环人体胎盘灌注协议及其进一步发展，以获得可重复的结果。 获得知情同意后，无并发症的足月妊娠，剖腹产分娩的产妇的胎盘。一个完整的子叶胎儿和母体的血管插管灌注至少五个小时。作为一个模型颗粒大小为80，直径为500 nm的荧光标记聚苯乙烯颗粒中加入的产妇电路。 80纳米粒子能够穿过胎盘屏障，并提供一个完美的例子是通过胎盘到胎儿，而500纳米粒子被保留在胎盘组织或产妇电路的一种物质。 体外人类胎盘灌注模型提供可靠信息的少数机型之一运输外源性化学物质的行为，在一个重要的组织屏障，提供预测和临床相关数据。

图4A显示小的聚苯乙烯颗粒（80纳米）相比更大的聚苯乙烯颗粒（500纳米），不会转移到胎儿车厢被运往通过胎盘灌注曲线。每个数据点代表给定的时间点中的至少3个独立实验的平均颗粒浓度。聚苯乙烯纳米粒子胎盘传输大小依赖19。胎盘灌注3小时后，已经有20-30％的最初加入80纳米的聚苯乙烯颗粒是从母体转移到胎儿的电路，而500纳米的聚苯乙烯颗粒没有出现在胎儿的电路?...

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

该<em&gt;体外</em&gt;双循环人胎盘灌注模型可用于研究的整个人的胎盘转移的外源物和纳米颗粒。在这个视频协议中，我们描述了一个胎盘灌注成功执行所需的设备和技术。

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

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

JoVE Journal

Bioengineering

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

17.2K 次观看.  University Hospital Zurich. 该体外双循环人胎盘灌注模型可用于研究的整个人的胎盘转移的外源物和纳米颗粒。在这个视频协议中，我们描述了一个胎盘灌注成功执行所需的设备和技术。

视频: 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.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

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

科研

生物工程

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.

体外拟态正常与异常的人造血

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.

人类心脏静脉系统使用对比计算机断层扫描灌注固定的标本解剖重建

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.

的使用<em&gt;前体内</em&gt;钱德勒循环装置，以评估改性聚合物血导管的生物相容性

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.

通过通过纳米孔可调纳米易位流速Zeta电位测定：利用DNA修饰颗粒为例

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.

实验和有限元议定书调查中性和带电溶质的跨关节软骨运输

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.

内镜下黏膜下注射材料性能评价的一种新的体内模型

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.

用三种互补方法综合评价胎盘靶向药物的有效性和安全性

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.

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

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.

磁性纳米材料的生物功能化

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