Name: controlled microfluidic environment for dynamic investigation of red blood cell aggregation
Uploaded: 2015-06-04T14:00:00
Description: Watch this Scientific Journal Video about Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation at JoVE.com

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><...

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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 ...

Research

JoVE Journal

Bioengineering

Microfabricated Platforms for Mechanically Dynamic Cell Culture

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, ...

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

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

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

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

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

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

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

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

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 ...