这项工作是由加拿大自然科学和工程研究理事会的支持。与麦吉尔纳米工具MICROFAB设施麦吉尔大学和电子部卡尔顿大学的支持下进行微细加工。

血液从健康人与渥太华大学（H11-13-06）的伦理委员会的批准收集。 1.制造微通道微通道是基于标准光刻方法18制造。 <ol><li>设计使用计算机辅助设计（CAD）软件的微通道的几何形状和打印在透明光掩模的配置。这些掩码是至关重要的，因为它们在制造过程中决定了光路。在这种情况下，微通道尺寸是120微米厚，60微米的深度和7毫米的长度。 </li><li>在硅晶片上在洁净室，旋涂的环氧基的负光致抗蚀剂以2000rpm进行30秒，以获得所需微通道的深度（60微米）。 </li><li>暴露出晶片和在UV灯下的光掩模，在650毫焦/厘米2为70秒。确保暴露的唯一透明区域光掩膜的UV光。的透明区域的曝光使得导致的硬化链的聚合光致抗蚀剂。沉浸在硅片成显影剂以除去过量的光致抗蚀剂。 </li><li>一旦模具被制造，混合的硅系弹性体和固化剂以10：1的比例以创建聚二甲基硅氧烷（PDMS）的溶液中。放入真空室脱气了的PDMS溶液。倒入模具中并加热它在60℃90分钟，以创建微通道。然后使用氧等离子体结合90秒粘合的各声道到一个载玻片。 注意：为了消除由混合过程所产生的气泡进行的PDMS脱气过程。 </li><li>确保微型槽尺寸允许测试成功。在这个协议中，微通道具有120×60微米的矩形横截面。相比较而言，平均红细胞的直径为8微米，厚度为2微米。该refore，微通道宽度足以遵守适当的聚集和准确地判断速度剖面。 </li></ol> 2.血液制剂<ol><li>采集血液健康志愿者的捐助者，伦理委员会的批准后。治疗存在于收集管的血液与乙二胺四乙酸（EDTA）（7.2毫克乙二胺四乙酸的4ml血液）。 注：使用EDTA来避免血液凝固。 </li><li>以分离的血液成分离心血样三次10分钟，1400×g离心（3000转）。作为离心分离的结果，观察三个不同的层。 注：三个层是红细胞层（位于该管的底部），血沉棕黄层（由位于RBC层的顶部白血细胞和血小板）和血浆层（位于所有其他顶成分）。 <ol><li>第一次离心后，收集使用血液吸管血浆。避免任何Contact与棕黄色的外套。 </li><li>收集并在容器中处理的棕黄层的与漂白稀释在10％以上。 </li><li>添加3mL的PBS pH为7.4的每个与剩余的红细胞层的管子和保证离心机的适当的平衡。轻轻再次混合管和离心机在1400×g离心（3000 rpm）离心10分钟。 注：确保PBS溶液不含有任何镁或钙，因为它们可能会改变的细胞粘附。 </li><li>在第二次离心后在管处置的PBS和如果存在的话除去剩余的血沉棕黄层。重复步骤2.2.3和2.2.4第三离心产生干净的红细胞。 </li></ol></li><li>混合需要在1ml管洁净红细胞与血浆（0.95，0.9和0.85毫升）的量（0.05，0.1和0.15毫升），以获得红细胞悬液与相应的（5％，10％和15％ ）红细胞压积。检查血球计数用的离心。 </li></ol> 3.流体准备<ol><li>添加60微升荧光示踪粒子的溶液（1％固体，D 粒径 = 0。86微米，λ 腹肌 = 542纳米 和λ 发射 = 612纳米）到1ml的红细胞悬浮液管。 注：荧光示踪粒子（内部染色聚苯乙烯粒子）被用来测​​量所述微通道内的流动的速度。这些颗粒发荧光时暴露于激光束（λ= 542纳米）的相应波长。 </li><li>准备一个pH 7.4的PBS溶液，并在1ml的PBS溶液中添加60微升同样的荧光示踪粒子溶液。 </li></ol> 4.骨料的三围尺寸<ol><li>插入在微通道中的流体（RBC-混悬剂和PBS），使用25微升和100微升两个玻璃注射器。这将有助于减少该系统的顺应性。通过管道和接头装配入孔连接到微注射器。补使用流体容器和注射器之间存在的压力差的玻璃注射器。确保无气泡存在于系统中。作为一种替代方法，使用一个压力控制系统中的微通道以驱动两种流体。 </li><li>放置在微通道上倒置显微镜阶段连接到一个高速照相机和μPIV系统。程序注射器泵到所需的流速（ 例如，Q = 2微升/小时血液编发Q = 8微升/小时的PBS中的流速）。 注意：只有一个注射泵被使用。使用两种不同的玻璃注射器，有两个不同内径，有可能会发生变化，以进入Y型微通道的每个分支的流速。 </li><li>插入两种流体在双Y型微通道各自从不同的入口分支，并在不同的流速，如图<strong&gt;的图1，为了测试不同的剪切速率，改变泵的流速。这保持了两个分支19之间（在这种情况下4）同样的适当的比例（ 图2和3）。等待流量获取测量前达到稳定状态：目视检查高速摄像机图像的畅通。 注：该比率是在血液层内获得一个线性速度分布是至关重要的。 </li><li>使用可视化的高速摄像机的红细胞。将相机连接到计算机控制，记录和保存流体流动的图像。一旦两个流体流动达到稳定状态，开始录制。 <ol><li>获得多个图像获得更好的处理结果。确定最优照相机曝光时间为聚集体的清晰的图像。 注：相机曝光时间这种情况下为0.5毫秒。每个帧之间的时间间隔的选择是基于相机帧速率和流量在叔他的微通道。在此过程中处理的各帧之间的时间为60毫秒。因此能够每秒记录18帧的相机是足够的。 注：避免血液沉降在管和注射器，这可能导致错误的测量结果。 </li></ol></li><li>利用图像处理技术（在这种情况下，一个MATLAB程序），以改善图像质量，检测聚集在基于像素强度（ 图4中示出）如下每个图像： <ol><li>提高使用直方图均衡处理方法，以获得针对每个帧一个更好的图像质量的图像对比。 </li><li>灰度图像转换成二进制图像。为了这个目的，设置一个阈值，范围从0到1，即决定每个像素的转换。任何像素低于阈值的图像中会被检测为一个黑象素，而其值大于阈值的像素将被检测为白色像素。 </li><li>填写孔（如果存在的和必要的）对应于所述间隙内的一个骨料获得更好的结果。 </li><li>通过确定，使得相邻小区相关联的聚集体的二进制图象相邻白色像素检测所述细胞。 </li><li>标记检测的不同的聚集体，并转换二进制图象成红 - 绿 - 蓝（RGB）图像以便更好地观察。结合的原始帧与每个相应的处理的图像，以验证的图像处理技术的效率，并确保所有的细胞被考虑， 如图5。执行步骤4.5.1，4.5.2，4.5.3 ，4.5.4和4.5.5的所有捕获的帧。 </li><li>计算在基于检测到的像素的数目的帧中检测到的每个聚集体的区域。基于所使用的镜头倍率，计算出转换系数，使用校正手 ​​提袋，并将结果转换为μm2以下。平均每个检测到的聚集体尺寸帧，然后平均化结果的所有帧以获得对红细胞悬液各记录的平均聚集体尺寸。 </li><li>计算一个无线闭塞中心的区域来确定每个所检测的集合中红细胞的代表估计数目。利用这些结果，计算出的红细胞的百分比每个聚合中的分布为聚集体尺寸（通过细胞在每个聚合所估计的数表示）的函数， 如图6。 注意：为了测量红细胞聚集体，可以使用（而不是MATLAB）的ImageJ软件对每一帧执行相同的任务。 </li></ol></li></ol> 5.流体流速和剪切速率测量确定流体速度，然后利用一个μPIV系统的剪切速率。 <ol><li>一旦使用高速摄像机获得的数据，切换到用于μPIV系统的双脉冲相机。使用图像交流的成像软件quisition流和图像处理速度场的决心。 </li><li>采取所有的预防措施必要，这取决于类激光器，开启激光之前。然后打开系统，相机和激光上。 </li><li>校准基于所使用的透镜倍率的相机。为了这个目的，使用10微米精度的微尺度在显微镜下，并设置1个像素的尺寸的图像中。 </li><li>启动激光和可视化在两种流体的粒子。通过专注于最快颗粒在流动发现微通道的中间平面（ 即，最快的颗粒应该是最亮的）。 </li><li>置中的dt（两个连续帧之间的时间间隔），以确保该颗粒的适当位移。最快颗粒应移动这两个图像之间约5至10个像素。 </li><li>开始录制流程。设置为获得100对图像，5毫秒间隔的软件。执行所有的DIF步骤5.4，5.5和5.6同的红细胞悬浮液和对不同的剪切速率。 注：更多的方法的详细情况在皮特和芬内克20的研究。 </li><li>处理使用互相关方法与成像软件获取的记录。 注意：这包括离散图像对成预定大小的小窗口（基于流体流动和由用户选择的时间间隔）称为相关窗和以下在每个窗口中的粒子的位移。 <ol><li>首先，确定是否获取的图像需要预处理（包括背景减法，“基地剪报”21或图像重叠22,23）更好的数据处理。 </li><li>然后选择相关窗口的大小和形状，以及对图像重叠的百分比。由皮茨等人进行了详细研究。24，以确定最佳参数和图像预处理技术用于血液流入不同的通道配置。表达的结果作为从100图像对和速度的均方根（RMS）误差计算的平均速度场。 </li></ol></li><li>提取的速度分布在通道出口，从处理后的数据， 如图7。为了更好地信息，在空间平均的速度场。 注意：构成所述速度分布的条对应于选择的相对于通道宽度的相关窗口尺寸。在速度RMS误差也示于图7中 ，表示在实验速度估计的误差。 </li><li>关闭激光一旦所有的测量和处理被执行，。关闭系统的所有组件：软件，照相机，移动台，计算机和激光。 </li><li>为了计算剪切速率，第一检测和估算血液厚度在基于高速摄像机拍摄所述微通道。为此，所有平均在特定的记录帧，以获得所述视频的一个背景图像。划定血液层（ 如图8的红细胞悬浮在10微升/小时时悬浮在5％，10％和15％血细胞比容流动）。获得用于相应血液层厚度的速度值和由血液层厚度除以速度值计算的剪切速率。 </li></ol>

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L., Mehri, R., Mavriplis, C., Fenech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-particle+image+velocimetry+measurement+of+blood+flow:+validation+and+analysis+of+data+pre-processing+and+processing+methods.">Micro-particle image velocimetry measurement of blood flow: validation and analysis of data pre-processing and processing methods.</a> Meas. Sci. Technol. 23 (10), 105302 (2012).</li><li>Bhamare, M. G., Patil, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+blood+cell+analysis+by+using+digital+image+processing:+a+preliminary+study.">Automatic blood cell analysis by using digital image processing: a preliminary study.</a> Int. J. Eng. Res. Tech. 2 (9), 3135-3141 (2013).</li><li>Maitra, M., Gupta, R. K., Mukherjee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+counting+of+red+blood+cells+in+blood+cell+images+using+hough+transform.">Detection and counting of red blood cells in blood cell images using hough transform.</a> Int. J. Comput. Appl. 53 (16), 18-22 (2012).</li><li>Jambhekar, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cells+classification+using+image+processing.">Red blood cells classification using image processing.</a> Sci. Res. Repot. 1 (3), 151-154 (2011).</li><li>Otsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+threshold+selection+method+from+gray-level+histograms.">A threshold selection method from gray-level histograms.</a> IEEE Trans. Syst. Man. Cybern. 9 (1), 62-66 (1979).</li></ol>

作者什么都没有透露。

使用本方法，有可能以定性分析和定量红细胞聚集不同流动条件和血细胞比容下。对于成功的测试和总检测，关键的是要确定在所述微通道入口在两种流体之间的适当速度比。这个比例是非常重要的，以获得最佳的血层厚度，其中的速度分布是准线性19。 成功测试的另一个关键因素是一个不错的成像质量。实际上，由于该方法是基于图像处理，这是非常重要的是要有?...

An erratum was issue for <a href="http://www.jove.com/video/52719">Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation</a>. The introduction section was updated.

An erratum was issued for <a href="http://www.jove.com/video/52719">Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation</a>. The introduction section was updated.

Erratum: Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

红血细胞（红细胞）在决定血液的流变行为了至关重要的作用。它们几乎单独地负责的血液在体外和体内的特定性质。在生理条件下，红细胞占40％至45％的血液量。在微循环，红细胞仅占到血量的20％，由于更小的血管直径和等离子体掠过效果1。这种现象在微等离子体还原的被称为Fåhræus效果。在低剪切速率，红细胞能够桥接在一起，形成一维或三个所谓“红细胞钱串”或聚集维结构，从而有助于血液的非牛顿行为。然而，红细胞聚集的机制尚未完全了解。两种理论存在红细胞的聚集模型：细胞理论的桥接由于交联的大分子2和力attrac由于渗透压梯度3所引起的分子的耗尽化的理论。 通常，对于人血，聚集体在非常低的剪切速率4从1到10秒-1形成。高于此范围，红细胞倾向于分解和在容器内分开流动。 了解的聚集形成的条件是一个非常重要的领域血液流变学在确定血液的流变行为的条款。这些聚集经常看到在大循环水平（&gt; 300直径微米）5。以该刻度，血液被认为是作为牛顿流体和均匀的混合物。然而，这些聚集体很少见到在毛细管水平（4-10微米直径），通常的病理状况如糖尿病6和肥胖的指示。等病理状态下可能会改变红细胞聚集包括炎症或感染的条件，心血管疾病如高血压或动脉粥样硬化，遗传性疾病和慢性疾病7。因此，了解红细胞聚集机制和分析这些实体（通过定义这些聚集和流动条件的大小之间的关系）可能导致血液的微观流变行为的理解，因此涉及到临床应用。 红细胞聚集体可以由几个因素来改变，例如血细胞比容（血液中红细胞的体积），剪切速率，血管直径，红细胞的膜刚度和悬浮介质的组合物8-10。因此，控制的条件是必需的，以便有效地分析红细胞聚集体。有几种方法可以提供静态汇聚测量（聚集索引）分析聚集形成，提供约血行为的相关信息。这些方法包括，除其他外，血沉法如图11所示，光传输方法12中，光反射法13和低剪切粘度方法14。 很少有研究试图研究红细胞聚集，并确定聚集在控制流动条件15-17度。然而，这些研究间接调查红细胞聚集体尺寸通过确定基于微观血图像提供关于聚集程度以及本地粘度信息测定的剪切系统的占用空间的比率。 因此，我们提出了一个新的程序直接量化红细胞聚集的微循环，动态的，在可控制的恒定剪切速率。红细胞悬液被夹带，在双Y型微通道（ 如图1），与磷酸盐缓冲盐水（PBS）中的溶液，因此产生在血液层的剪切流动。在这血层恒定乳木果ř速率可以得到。的红细胞悬浮液在不同血细胞比容（H）的水平（5％，10％和15％），并在不同的剪切速率（2-11秒-1）进行测试。的血流速度和剪切速率使用的微粒子图像测速（μPIV）系统流被使用高速照相机显现而确定的。所得到的结果然后被处理以基于图像强度，以检测RBC和确定聚集体尺寸一个MATLAB代码。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>SU8-50 epoxy based negative Photo-resist</td>
			<td>MicroChem Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU8-50 developer</td>
			<td>MicroChem Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(dimethylsiloxane) (PDMS) Sylgard-184</td>
			<td>Dow-Corning</td>
			<td>3097358-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-50 series Plasma system&#160;</td>
			<td>Plasma Etch</td>
			<td>PE-50 series</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood collection tubes with K2 EDTA (ethylenediaminetetraacetic acid)</td>
			<td>FisherSci</td>
			<td>B367861</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, i.e. Thermo Scientific CL2</td>
			<td>Thermo Scientific</td>
			<td>004260F</td>
			<td></td>
		</tr>
		<tr>
			<td>Poshpate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Tracer fluorescent particles solution (15 mL)</td>
			<td>FisherSci</td>
			<td>R800</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggregometer</td>
			<td>RheoMeditech</td>
			<td>Rheo Scan AnD300</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringes (50 &#181;L)</td>
			<td>Hamilton</td>
			<td>80965</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing (Tygon)</td>
			<td>FisherSci</td>
			<td>AAA00001</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed camera (Basler)</td>
			<td>Graftek Imaging Inc.</td>
			<td>basler acA2000-340km</td>
			<td>A camera capable of recording 18 frames per seconds could be used.&#160;</td>
		</tr>
		<tr>
			<td>Double pulsed camera&#160;</td>
			<td>LaVision</td>
			<td>Imager Intense</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope MITAS</td>
			<td>LaVision</td>
			<td>MITAS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nd:YAG laser</td>
			<td>New Wave Research</td>
			<td>Solo-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump (Nexus3000 and PicoPlus)</td>
			<td>Chemyx Inc. and Harvard Apparatus</td>
			<td>Nexus3000 and PicoPlus</td>
			<td></td>
		</tr>
		<tr>
			<td>DaVis software</td>
			<td>LaVision</td>
			<td>Davis</td>
			<td></td>
		</tr>
	</tbody>
</table>

controlled microfluidic environment for dynamic investigation of red blood cell aggregation

血液，作为一个非牛顿生物流体，表示了大量的研究，在血液流变学领域的焦点。血液成分包括红血细胞，白血细胞和血小板是悬浮在血浆中。由于红细胞​​的丰度（40％至45％的血液量），它们的行为决定尤其在微循环的血液的流变行为。在非常低的剪切速率下，红细胞被视为组装和形式的实体称为聚集体，这会导致血液的非牛顿行为。重要的是要了解的聚集体形成的条件领悟血液流变学微循环是很重要的。这里描述的协议详细的实验程序来确定定量的红细胞聚集体在微循环在恒定剪切速率，基于图像处理。为了这个目的，红细胞悬浮液测试，并在120×60微米的聚二甲基硅氧烷（PDMS）微通道进行分析。该红细胞悬浮液耳鼻喉科下雨，以获得血液层内的线速度信息，从而实现了广泛的恒定剪切速率下使用的第二流体。剪切速率是使用微粒子图像测速（μPIV）系统测定，而红细胞聚集体使用高速照相机可视化。捕获的红细胞聚集的视频使用图像处理技术，以确定基于所述图像强度的聚集体尺寸进行分析。

在双Y型微通道中的双流体流程的例子示于图2人类红细胞悬浮在5％，10％和15％的血细胞比容和流速为10微升/小时。 图3示出了聚集体尺寸时的差在通道中的流动是从10微升/小时降低到5微升/小时的10％的血细胞比容。这使这些聚集体的尺寸的定性概念不同的血细胞比容和剪切速率时， 图5所示的四种人红细胞聚集体的位移，对三个连续帧，从而提供所需的相机?...

Watch this Scientific Journal Video about Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation at JoVE.com

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

该协议描述细节的实验步骤，根据一个控制的和恒定的剪切速率量化红血细胞（RBC）的聚集体的基础上，图像处理技术。这个协议的目标是涉及红细胞聚集体尺寸，以在受控的微流体环境中的相应的剪切速率。

微流控控制环境的红细胞聚集动态调查

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

controlled-microfluidic-environment-for-dynamic-investigation-red

Research

JoVE Journal

Bioengineering

11.7K 次观看.  University of Ottawa. 该协议描述细节的实验步骤，根据一个控制的和恒定的剪切速率量化红血细胞（RBC）的聚集体的基础上，图像处理技术。这个协议的目标是涉及红细胞聚集体尺寸，以在受控的微流体环境中的相应的剪切速率。 

视频: Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

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 ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

珠聚集测定假定的细胞粘附分子的表征

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...