我们感谢T.亨特，M.罗森布鲁斯，和他们的意见和有益的讨论林实验室。我们承认从G.微调，在佐治亚理工学院电子和纳米技术研究所的支持。由NIH资助的系列K08-HL093360 REAC奖，加州大学旧金山分校，美国国立卫生研究院奈米发展中心奖PN2EY018244，从亚特兰大儿童保健内皮细胞生物学中心的资金，为这项工作提供了资金支持。

1。内皮微器件的制备<ol><li>外面罩供应商提交的微流体装置的电脑辅助设计（CAD）绘图，创建一个光罩。使用面膜的钠钙玻璃上镀铬层组成。在这种情况下，微通道宽度为30微米。 </li><li> 15分钟的清洁与食人鱼裸硅晶片（10:1比例的硫酸和过氧化氢）和氢氟酸浸泡30秒。与去离子（DI）水冲洗约10秒。 </li><li>使用旋转涂布机，旋转Microchem SU-8 2025到晶圆光阻到30微米的高度。对于SU-8 2025，3000转的转速建议。 SU-8的其他粘度可将达到这个高度。 SU-8使用的完整说明可在<a href="http://www.microchem.com" target="_blank">www.microchem.com</a> </li><li> SU-8在95℃的烤盘上放置晶圆5分钟，以驱赶过量的溶剂。 注：烤箱将烤盘干的比晶圆不同，不建议。 </li><li>放置在晶圆所需的要素形状的面具，暴露在紫外光（160兆焦耳/厘米2 在365 nm）在光刻（卡尔SUSS，MA-6）。这种交叉连接的光致抗蚀剂。 </li><li>在95℃的烤盘上放置晶圆背面为额外的5分钟，以进一步加快SU-8聚合。 </li><li>沉浸在SU-8开发的晶片，主要成分为4分钟PGMEA（丙二醇甲醚醋酸酯）除去非交联的SU-8。 </li><li>新开发的晶圆100％异丙醇（IPA）冲洗10秒。使用加压氮，然后进行干燥处理的晶圆或使溶剂蒸发干净通风柜晶圆几分钟。 </li><li>磁带到培养皿干晶圆图案的SU-8的边缘预发泄运动。 </li><li> 1 mL的Sigmacote中申请使用吸管的晶圆，覆盖培养皿的顶部，和漩涡晶圆，以确保完整的晶圆涂层，清除覆盖，让晶圆干燥几分钟，直到所有的溶剂蒸发。 </li></ol> 2。的PDMS（聚二甲基硅氧烷）的制备<ol><li>混合的PDMS聚合物和固化剂，并以10:1的比例（W / W）使用真空干燥器除去气泡。德加的PDMS所需要的时间长短不一可用真空系统的力量，但通常从几分钟到一小时不等。对于一个6英寸的Petri菜没有以前倒的，一个60毫升的聚合物总量的PDMS建议。 </li><li>晶圆的混合物倒入，约5毫米厚。也倒入一个平底的盘创建一个PDMS薄板，约1毫米厚。治愈60°烘箱中过夜。 </li><li>使用刀或手术刀，切出的C左右ured PDMS的设备，并从晶圆中删除它。此外，削减的PDMS薄板，略高于较大的设备。 </li><li>创建在PDMS使用1.0毫米的打孔设备的进口和出口孔。这可以使用一个针副，哈里斯统一的核心，或类似的设备。 </li><li>清洁设备表面和表用透明胶带。 </li><li>使用等离子体吸尘器，暴露表面的PDMS设备和PDMS氧等离子体表为30秒。氧等离子体暴露该债券时，身体接触带来的PDMS表面活性物种。 </li><li>小管连接到一个小的长度（几厘米）的大型油管，而这又是与钝针注射器，充满了50微克/毫升从PBS人体血浆纤维连接蛋白。管子和针头的大小摩擦适合油管之间创建。插入小管中的PDMS微装置的入口和pply压力注射器，以填补渠道完全与纤维连接解决方​​案，并创建一个小的出气口在100μL下降，确保渠道保持潮湿。在37°C孵育40-60分钟设备。 </li><li>用PBS填充钝点针，连接一个新的注射器。施加压力注射器，用PBS冲洗设备。 </li></ol> 3。播种与内皮细胞的微流体装置<ol><li> 8％右旋糖酐，准备内皮生长介质中的人脐静脉内皮细胞（内皮细胞）1,000,000细胞/毫升。已成功地应用于50万至200万细胞/ mL范围。除了细胞加载介质葡聚糖增加流体的粘度，从而降低血管内皮细胞的速度，当他们进入纤维连接蛋白涂层的微系统。这反过来，增加细胞将坚持和文化成功地在微装置的可能性。 </li><li> Çonnect步骤2.7新的注射器和油管。 （约1米长），但具有较长的油管。 </li><li>要优化这个系统的性能，它在整个灌注系统，以防止任何泄漏或气泡在这一点上是至关重要的，任何解决方案或气泡在媒体上存在的泄漏会改变水流和防止血管内皮细胞的成功播种。因此，紧身的油管是必不可少的，以消除任何连接之间的泄漏。必须消除注射器和/或油管中的气泡，细胞之前推出的微器件和整个灌注系统应与底漆前的油管连接到微避免引入气泡的电池解决方案。 </li><li>使用注射泵，细胞悬液注入的PDMS设备2小时37 1.23μL/ min的体积流量°C和5％CO 2。取得最佳的效果时，注射泵在<html>作为PDMS设备相同的高度。为进气口，较长的油管，可孵化器内的盘绕，确保细胞和媒体的充分接触与设备前热身。在我们的系统中，1.23μL/ min的体积流量接近中游〜1毫米/秒的速度，最小的微，相应的壁面剪切应力的〜1达因/厘米2使用全血在这些渠道。 </li><li> 1.23μL/ min的比例为2-8天​​使用同一注射器泵和长管，灌注新鲜培养基。一个成功的设备将有越来越多在​​24-48小时内对设备内部的内皮细胞单层。以前的实验表明，适当融合单层整个设备14 VE-cadherin的表达在细胞与细胞路口。 </li><li>血液或细胞悬液注入系统的实验。 </li></ol> 4。代表结果&gt;使用_content“这个协议，标准平版印刷微细加工技术用于创建模具生产所需的微流体通道，生理模仿微血管（ 图1A）sizescale。内皮细胞，然后使用优化灌流技术，种子和confluently文化的整个微系统（ 图1B）细胞接种在24-48小时内表面。由于微系统是透明的，整个微型设备可以放置在明/荧光显微镜成像和数据收集阶段。 然后我们的系统可以应用于研究涉及改变生物物理性质，化学性质，如镰状细胞病，镰刀红细胞和异常白细胞和内皮细胞的粘附增加刚性促进微血管阻塞，血液系统疾病。临床上批准的药物，羟基脲改善症状，但其direct微血管流量的影响是未知的。我们的检测需要考虑到这两种细胞的硬度和附着力，并表明，羟基脲显着改善镰状细胞病（ 图2）流动。 镰状细胞病是只有一个为微血管芯片的应用例子，这个系统非常适合研究在对方的微血管和内皮细胞与血细胞相互作用过程中，任何血液。其他临床相关应用包括炎症性疾病，败血症/肺损伤，血栓microangiopathies，疟疾，癌症转移，而更基本的应用，包括白细胞生物学及造血干细胞生物学，等等。 <img src="/files/ftp_upload/3958/3958fig1.jpg" alt="图1"/> 图1 A）最初的PDMS微内皮前的设备。在这里，微器件注入机智Ĥ食品着色，说明sizescale和整体系统的设计。乙）明场显微镜显示，微流体系统内皮细胞接种后48小时内完全使用这里描述的协议。 <img src="/files/ftp_upload/3958/3958fig2.jpg" alt="图2"/> 图2 A）微血管与微一个芯片的毛细血管后静脉（30微米），最镰状细胞微血管阻塞性事件现场的大小相若。镰状细胞贫血患者接受羟基脲和不接受羟基脲的患者全血流经两个不同的微血管上的一个芯片设备。镰状细胞内的内皮化微羟基流动相对容易接收患者的全血）。 c）在相同的血流动力学条件下，镰状细胞贫血患者不接受羟基脲的全血，然而，表现呆滞与microch流量更annel阻塞。底部的通道完全堵塞，无流和其他内皮化微流速有显着比羟基条件较低。在所有三个图像的比例尺为30μm。

<ol>
	<li>Mezzano, D., Quiroga, T., Pereira, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Level+of+Laboratory+Testing+Required+for+Diagnosis+or+Exclusion+of+a+Platelet+Function+Disorder+Using+Platelet+Aggregation+and+Secretion+Assays.">The Level of Laboratory Testing Required for Diagnosis or Exclusion of a Platelet Function Disorder Using Platelet Aggregation and Secretion Assays.</a> Semin. Thromb. Hemost. 35, 242-254 (2009).</li><li>Young, E. W. K., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+microfluidic+cell+culture+in+controlled+microenvironments.">Fundamentals of microfluidic cell culture in controlled microenvironments.</a> Chemical Society Reviews. 39, 1036-1048 (2010).</li><li>Young, E. W. K., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macro-+microscale+fluid+flow+systems+for+endothelial+cell+biology.">Macro- microscale fluid flow systems for endothelial cell biology.</a> Lab on a Chip. 10, 143-160 (2010).</li><li>Higgins, J. M., Eddington, D. T., Bhatia, S. N., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+cell+vasoocclusion+and+rescue+in+a+microfluidic+device.">Sickle cell vasoocclusion and rescue in a microfluidic device.</a> Proceedings of the National Academy of Sciences. 104, 20496-20500 (2007).</li><li>Rosano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physiologically+realistic+in+vitro+model+of+microvascular+networks.">A physiologically realistic in vitro model of microvascular networks.</a> Biomedical Microdevices. 11, 1051-1057 (2009).</li><li>Meer, A. D. v. a. n. d. e. r., Poot, A. A., Duits, M. H. G., Feijen, J., Vermes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Technology+in+Vascular+Research.">Microfluidic Technology in Vascular Research.</a> Journal of Biomedicine and Biotechnology. 2009, (2009).</li><li>Karunarathne, W., Ku, C. -. J., Spence, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dual+nature+of+extracellular+ATP+as+a+concentration-dependent+platelet+P2X1+agonist+and+antagonist.">The dual nature of extracellular ATP as a concentration-dependent platelet P2X1 agonist and antagonist.</a> Integrative Biology. 1, 655-663 (2009).</li><li>Kotz, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+microfluidics+for+neutrophil+genomics+and+proteomics.">Clinical microfluidics for neutrophil genomics and proteomics.</a> Nat. Med. 16, 1042-1047 (2010).</li><li>Rosenbluth, M. J., Lam, W. A., Fletcher, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+cell+mechanics+in+hematologic+diseases+with+microfluidic+biophysical+flow+cytometry.">Analyzing cell mechanics in hematologic diseases with microfluidic biophysical flow cytometry.</a> Lab on a Chip. 8, 1062-1070 (2008).</li><li>Shelby, J. P., White, J., Ganesan, K., Rathod, P. K., Chiu, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+model+for+single-cell+capillary+obstruction+by+Plasmodium+falciparum-infected+erythrocytes.">A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes.</a> Proceedings of the National Academy of Sciences. 100, 14618-14622 (1073).</li><li>Borenstein, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+endothelialized+microvascular+networks+with+circular+cross-sections+in+a+tissue+culture+substrate.">Functional endothelialized microvascular networks with circular cross-sections in a tissue culture substrate.</a> Biomedical Microdevices. 12, 71-79 (2010).</li><li>Nesbitt, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shear+gradient-dependent+platelet+aggregation+mechanism+drives+thrombus+formation.">A shear gradient-dependent platelet aggregation mechanism drives thrombus formation.</a> Nat. Med. 15, 665-673 (2009).</li><li>Prabhakarpandian, B., Shen, M. -. C., Pant, K., Kiani, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+modeling+cell-cell+and+particle-cell+interactions+in+the+microvasculature.">Microfluidic devices for modeling cell-cell and particle-cell interactions in the microvasculature.</a> Microvascular Research. 82, 210-220 (2011).</li><li>Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+modeling+of+the+microvascular+occlusion+and+thrombosis+that+occur+in+hematologic+diseases+using+microfluidic+technology.">In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology.</a> The Journal of Clinical Investigation. , (2011).</li><li>Green, D. A., Murphy, W. G., Uttley, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemolytic+uraemic+syndrome:+prognostic+factors.">Haemolytic uraemic syndrome: prognostic factors.</a> Clinical &#38; Laboratory Haematology. 22, 11-14 (2000).</li></ol>

没有利益冲突的声明。

最适合我们的内皮化微装置系统结合， 在体内实验中使用时，其还原方法可能有助于阐明在人类和动物模型中观察到的血液进程的物理机制。此外，我们的系统也不是没有限制的。例如，我们的微通道截面是方形。虽然可以制作技术上圆形微10,11，我们选择使用更简单，更标准的制造过程，让其他研究人员，随时这个系统应用到自己的工作。此外，培养内皮细胞自然“轮出”有效?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>钝点针</td><td> OK国际</td><td> 920050 - TE </td><td>精密的TE针20×1/2计“，粉红色</td></tr><tr><td>葡聚糖</td><td> Sigma-Aldrich公司</td><td> 31392 </td><td></td></tr><tr><td>纤维连接蛋白</td><td> Sigma-Aldrich公司</td><td> F0895 </td><td></td></tr><tr><td>打孔器（引脚老虎钳） </td><td>技术创新</td><td></td><td></td></tr><tr><td>人类脐带内皮细胞（内皮细胞） </td><td>龙沙</td><td> CC-2519 </td><td></td></tr><tr><td>等离子清洗机</td><td>血浆</td><td> PDC-326 </td><td></td></tr><tr><td>聚二甲基硅氧烷（PDMS） </td><td> Fisher Scientific则</td><td> NC9285739 </td><td> sylgard 184硅橡胶KIT </td></tr><tr><td> Sigmacote </td><td> Sigma-Aldrich公司</td><td> SL2的</td><td></td></tr><tr><td>苏-8 2025 </td><td> microchem </td><td> Y111069 </td><td></td></tr><tr><td> SU-8开发</td><td> microchem </td><td> Y020100 </td><td></td></tr><tr><td>注射泵</td><td>哈佛仪器</td><td> 70-3008 </td><td> PHD-超</td></tr><tr><td>管（大） </td><td>科尔 - 帕默仪器公司</td><td> 06418-02 </td><td> tygonreg微孔管，0.020“内径x 0.060”外径</td></tr><tr><td>油管（小） </td><td>科尔 - 帕默仪器公司</td><td> 06417-11 </td><td>聚四氟乙烯微孔管，0.012“内径x 0.030”外径</td></tr></tbody></table>

endothelialized microfluidics for studying microvascular interactions in hematologic diseases

在微细加工技术的进步使生产价格低廉，可重复的微流体系统进行生物和生化实验，在微，和nanoscales 1,2。此外，微流体也被专门用于定量分析血液和微血管的过程，因为他们有能力轻松地控制流体的动态环境和生物条件3-6。因此，研究人员最近利用微系统研究血细胞变形，血细胞聚集，微血管血流量，血细胞-内皮细胞相互作用6-13。然而，这些微系统并未包括培养的内皮细胞或较大比sizescale微血管的病理过程有关。与内皮细胞的微流体平台，准确地概括蜂窝，物理，和hemodyn微循环的的AMIC环境需要进一步的我们涉及微血管的血液系统疾病的生物物理基础病理生理学的理解。 在这里，我们报告的方法， 在体外微血管模型创建的“内皮化”，用一个简单的，单一的掩膜微细加工工艺标准的内皮细胞培养技术，研究发生在血液病的病理生物物理微血管相互作用结合。这种“微血管上的单芯片”的研究者提供了一个强大的检测，严格控制生物以及生物物理条件和操作使用标准的注射泵和明/荧光显微镜。如微循环血流动力学条件下，内皮细胞型，血细胞类型（S）和浓度（S），药物/抑菌浓度等参数都可以很容易地控制。正因为如此，我们的微提供方法定量调查疾病微血管流量由于改变细胞粘附，聚集和变形，无法与现有的检测能力受损的过程。

Watch this Scientific Journal Video about Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases at JoVE.com

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

方法文化在整个内部的微血管大小通道（&lt;30微米）的微流体装置的三维表面的内皮细胞单层描述。这<em&gt;在体外</em&gt;微血管模型，使血细胞，内皮细胞，可溶性因子在血液系统疾病之间的生物物理相互作用的研究。

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

endothelialized-microfluidics-for-studying-microvascular-interactions

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15.8K 次观看.  Emory University School of Medicine. 方法文化在整个内部的微血管大小通道（

视频: Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

研究蛋白质折叠的微流体混合器

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

科研

生物工程

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.

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 ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

发展多深的圆形横截面微内皮化的一个芯片的程序

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

细胞的浸入式印刷到改性表面采用连续流动Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

微电极阵列接口微流体研究神经元通信和轴向信号传播

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

研究脑癌和神经退行性疾病的人类神经器官

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...