通过胎盘的外来物质和纳米材料的运输率测定<em>体外</em>人胎盘灌注型号
Summary
该<em>体外</em>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。
Abstract
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。
Introduction
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>。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<SUP> 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。
Protocol
1。准备灌注系统设置的水浴中，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。 打开水浴上，流动加热器和加热灌注室。温度应在37℃下热身灌注介…
Representative Results
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?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of the Reagent | Company | Catalogue Number | Comments (optional) |
| NCTC-135 medium | ICN Biomedicals, Inc. | 10-911-22C | could be replaced by Medium 199 from Sigma (M3769) |
| Sodium chloride (NaCl) | Sigma-Aldrich, Fluka | 71381 | |
| Potassium chloride (KCl) | Hospital pharmacy | also possible: Sigma (P9541) | |
| Monosodium phosphate (NaH2PO4 · H2O) | Merck | 106346 | |
| Magnesium sulfate (MgSO4 · H2O) | Sigma-Aldrich, Fluka | 63139 | |
| Calcium chloride (CaCl, anhydrous) | Merck | 102388 | |
| D(+) Glucose (anhydrous) | Sigma-Aldrich, Fluka | 49138 | |
| Sodium bicarbonate (NaHCO3) | Merck | 106329 | |
| Dextran from Leuconostoc spp. | Sigma-Aldrich | 31389 | |
| Bovine serum albumin (BSA) | Applichem | A1391 | |
| Amoxicilline (Clamoxyl) | GlaxoSmithKline AG | 2021101A | |
| Sodium heparin | B. Braun Medical AG | 3511014 | |
| Sodium hydoxide (NaOH) pellets | Merck | 106498 | CAUTION: corrosive |
| Ortho-phosphoric acid 85% (H3PO4) | Merck | 100573 | CAUTION: corrosive |
| Maternal gas mixture: 95% synthetic air, 5% CO2 | PanGas AG | ||
| Fetal gas mixture: 95% N2, 5% CO2 | PanGas AG | ||
| Antipyrine (N-methyl-14C) | American Radiolabeled Chemicals, Inc. | ARC 0108-50 μCi | CAUTION: radioactive material (specific activity: 55mCi/mmol) |
| Scintillation cocktail (IrgaSafe Plus) | Zinsser Analytic GmbH | 1003100 | |
| Polystyrene particles 80 nm | Polyscience, Inc. | 17150 | |
| Polystyrene particles 500 nm | Polyscience, Inc. | 17152 | |
| EQUIPMENT | |||
| Water bath | VWR | 462-7001 | |
| Thermostat | IKA-Werke GmbH & Co. KG | 3164000 | |
| Peristaltic pumps | Ismatec | ISM 833 | |
| Bubble traps (glass) | UNI-GLAS Laborbedarf | ||
| Flow heater | UNI-GLAS Laborbedarf | ||
| Pressure sensor + Software for analyses | MSR Electronics GmbH | 145B5 | |
| Notebook | Hewlett Packard | ||
| Miniature gas exchange oxygenator | Living Systems Instrumentation | LSI-OXR | |
| Tygon Tube (ID: 1.6 mm; OD: 4.8 mm) | Ismatec | MF0028 | |
| Tubes for pumps (PharMed BPT; ID: 1.52 mm) | Ismatec | SC0744 | |
| Blunt cannulae (Ø 0.8 mm) | Polymed Medical Center | 03.592.81 | |
| Blunt cannulae (Ø 1.2 mm) | Polymed Medical Center | 03.592.90 | |
| Blunt cannulae (Ø 1.5 mm) | Polymed Medical Center | 03.592.94 | |
| Blunt cannulae (Ø 1.8 mm) | Polymed Medical Center | 03.952.82 | |
| Parafilm | VWR | 291-1212 | |
| Perfusion chamber with tissue holder (plexiglass) | Internal technical department | Similar equipment is available from Hemotek Limited, UK | |
| Surgical suture material (PremiCron) | B. Braun Medical AG | C0026005 | |
| Winged Needle Infusion Set (21G Butterfly) | Hospira, Inc. | ASN 2102 | |
| Multidirectional stopcock (Discofix C-3) | B. Braun Medical AG | 16494C | |
| Surgical scissors | B. Braun Medical AG | BC304R | |
| Dissecting scissors | B. Braun Medical AG | BC162R | |
| Needle holder | B. Braun Medical AG | BM200R | |
| Dissecting forceps | B. Braun Medical AG | BD215R | |
| Automated blood gas system | Radiometer Medical ApS | ABL800 FLEX | |
| Multi-mode microplate reader | BioTek | Synergy HT | |
| Liquid scintillation analyzer | GMI, Inc. | Packard Tri-Carb 2200 | |
| Scintillation tubes 5.5 ml | Zinsser Analytic GmbH | 3020001 | |
| Tissue Homogenizer | OMNI, Inc. | TH-220 | |
| pH meter + electrode | VWR | 662-2779 |
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Cite This Article
Grafmüller, S., Manser, P., Krug, H. F., Wick, P., von Mandach, U. Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model. J. Vis. Exp. (76), e50401, doi:10.3791/50401 (2013).