笔者想苏拉的阿布Mallouh和弗雷德里克·法希姆用于测试的协议，理查德·普雷沃LaVision公司的技术支持，在初始运行，她的帮助，感谢NSERC（自然科学与工程理事会，加拿大）的资金，凯瑟琳Pagiatikis和Guy Cloutier联系蒙特利尔大学的贷款Dalsa公司高速摄像机。

1。微晶片制造第一步是创建或购买你的微通道。微芯片材料有很多的选择。 选择最常见的材料是聚（二甲基硅氧烷）（PDMS）。出版物有很多PDMS加工通过软光刻16,17,18的方向。 PDMS通道一旦被制造的，也有一些表面处理，以扭转其天然疏水性。含氧等离子体处理是一种常见的选择。 周，埃利斯，Voelcker（2009）给表面处理，以及它们如何影响PDMS评论。这些结果是水，但是，血液有不同的性质。研究已经做了什么，表面处理是指血液皮茨等人 （2012A）。 基于这里给出的结果，它们的微芯片的表面和载玻片键合到均暴露于含氧等离子体45秒，然后牢固地压合在一起造成永久粘合。 2。血液制剂<ol><li>收集血液样本认可的设施。例如猪血可以使用乙二胺四乙酸（EDTA）作为抗凝剂。添加1克乙二胺四乙酸，4毫升水，然后添加全血的1升的溶液，轻轻混合10倍。 </li></ol>当拿起血液样本，让它们慢慢冷却到室温。血液样本应该是新鲜的，理想使用的天收集血液的流变学特性，以节省。 冷藏样品可在室温下一次，然而没有抗凝全血冷冻后，将无法使用。红细胞上的抗凝血剂，可以保存在冰箱中长达42天，和全血可冷藏保存35天，但污染将有可能在一个非米兼具医疗设备21。 此外，要小心用牛或猪的样品采集方法，避免污染骨髓。 <ol start="2"><li>离心血液样品3次每次10分钟在3,000 rpm。第一次离心后，除去血浆和白膜，丢弃。它通常是最容易拆卸第一等离子体，然后丢弃或保留以供将来使用，然后以除去血沉棕黄层。 </li></ol>引入的移液管慢慢地，以便不使混回血液的血沉棕黄层。去除白膜后，加入约20毫升的磷酸盐缓冲液（PBS），轻轻混匀，recentrifuge。重复最后一步。第三次离心后，取出PBS和剩余的棕黄色大衣，丢弃。 它也可以使用本机的等离子代替PBS如果你想保持血液聚集性能。确保不能混合任何白膜或白色到血浆中的血细胞。 <ol start="3"><li>清洗红血细胞（红细胞），并悬浮在PBS或等离子在随意的浓度（血细胞比容）。检查离心比容（美国）FisherSci CritSpin，。 </li></ol>在10〜20％的血细胞比容的血应该是更容易的可视化比40或50％（生理血细胞比容）。血液微循环，一般是半宏循环的血细胞比容，所以H = 20是足够的15。 <ol start="4"><li>以所需浓度的血液样本，加入荧光示踪粒子。一般的规则是有10个颗粒，在相关窗口，它可以提前计算。 </li></ol>例如，30微升的颗粒被添加到视频中所示的设置的1毫升的血溶液。或者，可以被荧光标记的红细胞本身。 <ol start="5"><li>此外，进行校准溶液与水和流感颗粒orescing。这将是牛顿的解决方案，需要对系统进行校准。 </li></ol>例如，当使用10倍的目标与通道上的规模为100μm，合适的浓度为300μl的颗粒用15毫升的蒸馏水混合。另一种选择是使用用蒸馏水稀释甘油的粘度为3CP，这是更接近血液的宏观粘度。 3。 μPIV测量<ol><li>激光安全:检查温度和湿度。穿戴合适的激光安全眼镜。关闭激光窗帘或打开门不被暴露保护，以防止有人在实验室的激光标志。 </li><li>戴维斯软件程序在视频显示。欲了解更多详细信息，请参阅特定系统的用户手册。 </li><li>在获取数据之前，需要进行校准相机规模使用千分尺。 </li><li>为了降低系统的合规性，用最短的油管d为最小的刚性注射器可能的，诸如15或50微升汉密尔顿气密注射器。血液或进气口，以减少泄漏到系统中，密封管连接到注射器和芯片。这是可以做到用胶水，PDMS或凡士林（小心不要污染的血液）。 </li></ol>为了填补股价与示踪粒子与水的微量注射器使用回流的方法，因为微量注射器的体积通常是远小于卷以填充。 通过我们的回流方法，包括填充微量注射器和信道的信道使用的二次塑料注射器经由输出。对于这一点，首先除去微量注射器柱塞，然后推入的液体，采用经典的塑料注射器附着到出口的通道，让液体流出来的微量注射器，直到所有的微泡的微型注射器，管或芯片，终于把柱塞回，拔下塑料注射器。的存在可以验证ŗ没有微泡，用显微镜。 <ol start="5"><li>平注射器泵实现横向管。程序注射器泵所需的流量。 </li></ol>要意识到可能短暂影响，像"瓶颈效应"，刚性的系统或系统的合规性，修改实际流量。一个系统的特征时间可估计为一个函数的系统的材料和尺寸。 （第2章，第77-81页，Tabeling，2005年） <ol start="6"><li>显微镜舞台上放置微芯片，并启动泵。使用水与颗粒校准系统到中间的速度分布图和校准DT（脉冲图像之间的时间）。 </li></ol>粒子良好的相关性11 5和10之间移动的帧之间的像素。校准时，小心衡量，在中间的通道。在中间聚焦平面具有最高的速度8。 如果使用一个圆形通道，小心的阴影效果。 图2所示的图像样本，使用脉冲的摄像头，而最佳的图像是第一个脉冲和底部的图像是第二脉冲。可以看出，图中的最亮的点（荧光颗粒）是在焦。 <ol start="7"><li>可选:数据在不同的高度的三维速度分布可以重建成一个单一的图像通过添加不同媒介。视频提出了一种的例程被开发的过程自动化。 </li><li>当患者的血液中的数据时，填充注射器与所需量的血液中的水的方法相同的方法。编程注射泵以所需流速，并采取所需的数据。获得的原始图像的一个例子，提出了如图2所示 。小心红细胞沉降。加注射器每次运行或每其他的运行会降低红细胞的沉降。 </li><li>获取图像后，进行相互相关的图像对取得的图像之间的速度矢量场。重要的是要具有良好的图像，以具有良好的相关性。关联之前，可以做前处理，以消除背景噪音。 </li></ol>在相邻的图像，它可以是多种多样的大小和形状的影响相关的窗口之间进行相互相关。交叉相关性后，可以做图像后处理，删除错误向量或离群。 不同的可用选项及其精度的详细描述，可以发现在皮茨等人 （2012B），最好的处理血液被认为是"图像重叠"与窗口长度延长的方法，与流。这些操作可以像MATLAB程序中手动完成，或在您的系统软件包。该<html>软件并不重要，更重要的是背后的数学操作，每个操作可以影响最终速度的档案的准确性。 由此产生的向量在空间可被平均在整个信道取得瞬时速度分布，然后时刻再次平均，得到的平均的，有代表性的速度分布图。 kloosterman 等人（2011）得到大景深的μPIV测量中的错误的解释。这里介绍的数据处理错误的讨论中可以找到皮茨等人（2012B）的脉冲测量和Chayer 等人 （2012）的高速测量。 速度分布的实例列于图3和图4，图3给出一个单一的速度剖面的中心线10微升/小时流量的RBC在H = 10，在140μm宽的信道。 帐篷"这种单一的配置文件来实现的平均值在整个信道的相关的载体，得到的速度分布，然后取平均值得到有代表性的测量时间，在这种情况下，100对整个视场的时间的平均值。 水标定流量与编程为30微升/小时的流速在100平方微米的信道的三维重建轮廓的一个例子，在图4中找到。在图4中 ，以1微米的间隔测量的档案。 <ol start="10"><li>可选:在所描绘的高速图像设置可以在相同条件下通过切换摄像机（和数据采集系统）。用于可视化的无细胞层中的信道，并用于定量聚合，这可能是有用的。数据分析是略有不同的（Chayer， 等人，2012）。 </li></ol>白光图像与无红细胞聚集预选在图5中，与指向H = 20。最佳的图像显示H = 20除去的红细胞聚集的能力，在PBS中，在1微升/小时。底部图像是原生0.5微升/小时血浆H = 20。 ，聚集在底部的图像是可见的。 <ol start="11"><li>关闭系统时，以安全的方式测量已完成。使用适当的激光安全程序，直到完全关闭激光电源。 </li></ol>

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什么都没有透露。

使用μPIV规模的微循环血流量测量可以洞察到了大量的有关生物医药，机械和化工过程。部分的关键因素考虑到红细胞本身，红细胞聚集或运动的荧光微粒的聚集和变形性，和在通道中的RBC的沉降密度。所有这些都可以被占，则一般遵循上述指引。有一个基本的清单，要记住，使用这个系统的良好的测量。首先，确定有多少合规系统或建议系统。为了尽量减少遵守使用油管可能在最短的和尽可能?...

人体中含有许多血管直径小于50微米，这是主要的血液和组织之间的交流网站。这些血管的血流量的研究表示一个相当大的挑战，由于规模测量的血液的流体性质。这些测量值，包括在小动脉和小静脉的压力梯度，在墙上的剪切速度分布，是与生理反应的关键因素。现在有前所未有的机遇，以解决这些测量挑战，由于新的实验技术，在微观尺度上，研究了微循环和解决这一多尺度问题。 微观粒子的图像测速（μPIV）是一个基于粒子的流量可视化技术，用于评估血液流动的速度分布在微通道通过交叉相关。 μPIV，首先圣地亚哥等人开发的，已用于与血液流变学自杉井等人的研究，在2001年使用的手法是在100μm的圆形的玻璃管1,2测量血流量。不同的方法μPIV存在。可以使用高速摄像机相关联的运动的红血细胞（红细胞），脉冲的图片可被用来示踪粒子的运动相关联。这些选项可以配上直立或倒置显微镜，根据不同的应用。在这两种情况下，其结果是一个二维速度分布图。另一种方法是使用激光共聚焦显微镜实现2D和3D的配置文件。这种方法已被应用到血液3,4,5。 微观尺度PIV有多种并发症较宏观PIV。位宏PIV中的数据可以被限制为一个单一的平面的光通过片材，但在微观尺度上卷照明是必要的。卷照明成像微血流量是一个更大的问题，如红细胞本身相比要大的channels，使用的红细胞作为示踪粒子导致的深度的相关性（DOC），它可以显着降低的准确性的互相关结果6,7,8。继Wereley 等人（1998年）为40微米高的通道与RBC作为示踪剂的DOC是8.8微米，1微米示踪粒子DOC是6.7微米。改变通道高度和放大倍率时，这种差异变得更加明显。此外，红细胞是不透明的，并在流动的红细胞的密度增加会导致成像的困难。荧光示踪粒子，在第一圣地牙哥等人（1998）所使用的，已被作为一种工具，以减少聚焦粒子的影响，倡导使用尽可能最小的颗粒。使用直径为1微米的荧光的微粒用激光耦合的一种方法，可能会降低微血流显像10的深度的焦点问题。有几个当前的状态μPIVTEC评论的hnology，其中每一个突出的重要性μPIV血流研究11,12。几个重要的考虑，必须考虑到当使用血液μPIV。在微观层面上，改善微循环规模，血液可以不被认为是均匀的流体，因为是悬挂灵活的红血细胞（红细胞），大型白细胞，血小板及其他蛋白质悬浮在一个牛顿流体（血浆） 。 的速度分布测量信号可以用来测量微血流量的某些特性。的重要因素在microhemorheology是血液的流速的速度剖面的形状，容器的壁处的剪切应力。此信息具有临床意义，，改善微循环是网站在体内的养分交换，这种交换是剪切速率依赖。有几个目前的审查研究的微循环状态的研究，以及13，14,15。 这里介绍的是μPIV血流量测量中的聚二甲基硅氧烷（PDMS）微通道的协议。按照本协议第1条中的来源渠道，在内部制造PDMS。获得认可的屠宰场猪血液样本和清理后，该协议的第2条。使用LaVision MITAS的μPIV系统，该协议第3条中所描述的所有数据。设置包括一个Nd:YAG激光（新浪潮研究，美国）和CCD照相机（图像激烈，Lavision）由一个可编程触发单元，在荧光显微镜加上3轴载物台移动中，和一台计算机控制的，除了到一个高速照相机（1M150 Dalsa公司，荷兰）溶液中加入红细胞本身的可视化。这两款相机连接到2端口光纤盒（德国蔡司定制）。在典型的在体内的血流量测量，直立的显微镜是用来跟踪的红细胞mselves，而在典型的体外应用倒置显微镜是用来跟踪示踪粒子。在这个独特的双重设置，允许两个示踪剂使用倒置显微镜成像光学框。通过高精密注射器泵的微芯片（Nexus3000，Chemyx公司，美国）被引入到血液。该系统示于图1中 ，图中的顶部部分表示为140μm，40μm的矩形通道的PDMS制作的图表，和底部代表整个系统，包括摄像机，激光，注射泵和显微镜。 电流μPIV集起坐，通常与专有软件，包括TSI，丹特克动力，LaVision。标准的交叉相关算法可以通过众多的软件选项，包括MATLAB的实现。该软件是不是关键，了解的对话方块对应以数学将成为使用ŗ好得多。在这个协议中戴维斯，LaVision的专有软件或MATLAB利用。此协议是不特定的软件，但菜单选项可能会在不同的地方，不同的软件包。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>poly(dimethylsiloxane) (PDMS), i.e. Sylgard-184</td>
			<td>Dow-Corning</td>
			<td>3097358-1004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>E9884-100G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>poshpate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>P5368-10PAK</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fluorescing micro particles</td>
			<td>Microgenics/FisherSci</td>
			<td>R900</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>glycerol (OPTIONAL)</td>
			<td>Sigma Aldrich</td>
			<td>G6279-500 ml</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>microcentrifuge, i.e. CritSpin</td>
			<td>FisherSci</td>
			<td>22-269-291</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>syringe, i.e. 50 &mu;l Gastight</td>
			<td>Hamilton</td>
			<td>80965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>camera, i.e. Imager Intense, high speed</td>
			<td>LaVision, Dalsa</td>
			<td>Imager Intense</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>microscope, i.e. MITAS</td>
			<td>LaVision</td>
			<td>MITAS</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nd:YAG laser</td>
			<td>New Wave Research</td>
			<td>Solo-II</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>syringe pump, i.e. Nexus3000</td>
			<td>Chemyx, Inc.</td>
			<td>Nexus-3000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>flexible tubing, i.e. Tygon</td>
			<td>FisherSci</td>
			<td>14-169-1A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>data processing software, i.e. DaVis</td>
			<td>LaVision</td>
			<td>DaVis</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>centrifuge, i.e. Thermo Scientific CL2</td>
			<td>Thermo Scientific</td>
			<td>004260F</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

micro-particle image velocimetry for velocity profile measurements of micro blood flows

微观粒子图像测速（μPIV）是用于可视化成对的图片，接种在血液流动的微颗粒。图像互相关，给出一个准确的流速剖面。一个协议为μPIV测量血流量在微。规模的微循环，血液不能被认为是一种均匀的流体，因为它是一个灵活的粒子悬浮液悬浮在血浆中，牛顿流体。从这些测量中，可以推导出剪切速率下，最大速度，速度分布图的形状，和流速。如震源深度，粒子浓度，系统符合的几个关键参数，随着各种血细胞比容和流量条件下的实施例和代表性的结果，以确保准确的，有用的数据。

在所有的数字中，流是左到右的原始图像，并在计算速度剖面向上。与血液血细胞比容H = 10以10微升/小时的流动获得的原始数据，例如， 如图2所示。上游的数据可能会被交叉没有任何的数据处理，以实现速度分布密切相关。皮茨， 等人，（2012B）的前处理和数据处理方法的影响进行了讨论。 = 10和10微升/小时的流速得到的速度分布图，从与图2相似在hematorcrit H的数...

Watch this Scientific Journal Video about Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows at JoVE.com

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

微观粒子图像测速（μPIV）是用来配对接种于血流量，给出一个准确的流速剖面互相关微颗粒的图像可视化。剪切速率下，最大速度，速度分布图的形状，和流速，每个都具有临床应用，可以是来自于这些测量。

微观粒子图像测速技术，微血液流动的速度剖面测量

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

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17.2K 次观看.  University of Ottawa. 微观粒子图像测速（μPIV）是用来配对接种于血流量，给出一个准确的流速剖面互相关微颗粒的图像可视化。剪切速率下，最大速度，速度分布图的形状，和流速，每个都具有临床应用，可以是来自于这些测量。

视频: Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus (SCUVA)

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as oceanography, ecology, biology, and fluid mechanics. Field measurements introduce practical challenges such as environmental conditions, animal availability, and the need for field-compatible measurement techniques. To avoid these challenges, scientists typically use controlled laboratory environments to study animal-fluid interactions. However, it is reasonable to question whether one can extrapolate natural behavior (i.e., that which occurs in the field) from laboratory measurements. Therefore, in situ quantitative flow measurements are needed to accurately describe animal swimming in their natural environment.
We designed a self-contained, portable device that operates independent of any connection to the surface, and can provide quantitative measurements of the flow field surrounding an animal. This apparatus, a self-contained underwater velocimetry apparatus (SCUVA), can be operated by a single scuba diver in depths up to 40 m. Due to the added complexity inherent of field conditions, additional considerations and preparation are required when compared to laboratory measurements. These considerations include, but are not limited to, operator motion, predicting position of swimming targets, available natural suspended particulate, and orientation of SCUVA relative to the flow of interest. The following protocol is intended to address these common field challenges and to maximize measurement success.

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus SCUVA

This protocol provides instructions on how to use a self-contained underwater velocimetry apparatus (SCUVA), which is designed for quantification of in situ animal-generated flows. In addition, this protocol addresses challenges posed by field conditions, and includes operator motion, predicting position of animals, and orientation of SCUVA.

定量测量原位流动，使用一个自包含的水下测速仪（SCUVA）

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as ...

科研

生物工程

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

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

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

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

改进方法对血脑屏障的人体细胞为主，接触模型的制备

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

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

从手术组织小学的人结肠癌细胞的分离和培养直接它们柔软的弹性基底牵引术

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

混合μCT-FMT成像和图像分析

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

微流控缓冲液更换为无干扰微/纳米颗粒细胞工程

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

骨再生生物材料的生物相容性曲线

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Meso-Scale Particle Image Velocimetry Studies of Neurovascular Flows In Vitro

Particle image velocimetry (PIV) is used in a wide variety of fields, due to the opportunity it provides for precisely visualizing and quantifying flows across a large spatiotemporal range. However, its implementation typically requires the use of expensive and specialized instrumentation, which limits its broader utility. Moreover, within the field of bioengineering, in vitro flow visualization studies are also often further limited by the high cost of commercially sourced tissue phantoms that recapitulate desired anatomical structures, particularly for those that span the mesoscale regime (i.e., submillimeter to millimeter length scales). Herein, we present a simplified experimental protocol developed to address these limitations, the key elements of which include 1) a relatively low-cost method for fabricating mesoscale tissue phantoms using 3-D printing and silicone casting, and 2) an open-source image analysis and processing framework that reduces the demand upon the instrumentation for measuring mesoscale flows (i.e., velocities up to tens of millimeters/second). Collectively, this lowers the barrier to entry for nonexperts, by leveraging resources already at the disposal of many bioengineering researchers. We demonstratethe applicability of this protocol within the context of neurovascular flow characterization; however, it is expected to be relevant to a broader range of mesoscale applications in bioengineering and beyond.