Name: a microphysiological system to study leukocyte-endothelial cell interaction during inflammation
Uploaded: 2021-12-09T14:00:00
Description: Watch this Scientific Journal Video about A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation at JoVE.com

这项工作得到了美国国立卫生研究院的支持，拨款号：GM114359和GM134701（M.F.K.和L.E.K.），1F31AI164870-01（J.C.L.）和国防威胁减少局，拨款号：HDTRA11910012（M.F.K.和L.E.K.）。

在获得肝素化人血，用于从健康的成年供体（男性和女性，年龄在21至60岁之间）中分离中性粒细胞，经天普大学机构审查委员会批准（美国宾夕法尼亚州费城）批准的知情同意。
1. 用人纤连蛋白启动和涂覆设备
注意：bMFA有两个入口端口和两个连接到血管室的出口端口。它还具有一个连接到组织室的端口（图2）。
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bMFA再现体内微血管网络的形貌和流动条件，可用于 在 生理现实条件下研究白细胞 - 内皮细胞相互作用和 体外 内皮功能。在小鼠或人类的微血管系统中，微血管网络的几何形状是自相似和分形的，雷诺数<<1，表明血管几何形状不会显着影响流动模式。因此，bFMA可用于研究包括人类在内的不同物种的白细胞 - 内皮相互作用，尽管小鼠cremaster肌肉的微脉管系统被映射以制造bMFA。
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炎症是宿主对感染和损伤的反应，内皮在炎症反应中起重要作用1，2，3。炎症失调是许多疾病病理学的根本原因，例如败血症，心血管疾病，哮喘，炎症性肠病，癌症和COVID-19。白细胞 - 内皮细胞相互作用在这些炎症性疾病中起核心作用。在炎症过程中，从病原体释放的PAMPS（病原体相关分子模式）或从受损组织中释放的DAMPS（损伤相关分子模式）激活免疫细胞释放细胞因子/趋化因子和其他促炎介质，导致内皮激活，导致血管内皮屏障功能改变和通透性增加3，4.炎症期间内皮细胞的活化增加导致白细胞 - 内皮细胞相互作用增强，导致活化的白细胞过度迁移穿过血管内皮进入关键器官1，5，6，7。
白细胞的募集是由由生物活性脂质，细胞因子，趋化因子和补体组分8，9组成的化学多样性化学吸引剂引起的。白细胞募集是一个多步骤的过程，包括五个独立的步骤：1）白细胞切缘和捕获/附着，2）滚动，3）牢固的阻止，4）扩散和爬行以及5）外渗/迁移（图1）。该过程的每一步都需要白细胞和内皮细胞之间的串扰来协调这种动态现象1，9。最终，停滞的白细胞通过由并发的化学吸引剂依赖性信号，粘附事件和血流动力学剪切力1，9，10，11，12控制的多步骤过程外渗到内皮的发炎组织。
鉴于剪切应力在调节内皮细胞功能中的核心作用以及白细胞 - 内皮细胞相互作用的重要性13，在过去的几十年中已经开发了几种 体外 模型来研究白细胞迁移级联的各个方面在更可控的环境中14。研究白细胞- 内皮细胞相互作用的传统流体装置可分为两大类14：a）用于研究白细胞滚动，粘附和粘附分子表达的装置，例如平行板流室和b）用于研究在静态条件下的白细胞迁移的装置，例如透孔室。平行板流室等系统已被用于研究粘附分子及其配体在剪切力15下粘附级联中的作用。然而，一个显著的缺点是这些简单化、理想化的装置（例如，直通道）不能再现 体内 微脉管系统的规模和几何形状（例如，连续的血管分岔、血管形态）和由此产生的流动条件（例如，分岔处的汇聚或发散流动）。因此，这些设备只能模拟粘附，而不能模拟迁移。透孔室只能研究静态条件下的迁移，而不考虑 体内 几何特征和流动条件。因此，这些传统模型不模仿活组织的微环境或在单个测定6中解析粘附和迁移级联反应。
为了解决这一限制，我们开发并广泛验证了一种新的3D仿生微流体测定（bMFA）（图2），该测定在芯片16，17，18上实际再现体内微血管网络。该器件的微加工协议先前已于17发布，此处仅简要介绍。使用改进的地理信息系统（GIS）方法对小鼠cremaster肌肉的微血管系统进行数字化19。然后，使用基于数字化微血管网络14，17，20，21，22的软光刻工艺在聚二甲基硅氧烷（PDMS）上生成合成的微血管网络。简而言之，将数字化的网络图像打印在Mylar胶片上，然后将其用作掩模，在硅晶圆上图案SU-8正光刻胶，以创建用于制造的母带。微加工柱（直径10μm，高3μm）用于创建3μm高，100μm宽的孔，白细胞迁移的最佳尺寸23，24，25，连接血管通道和组织室。PDMS是根据制造商的说明准备的，并浇注在开发的母版上。此外，PDMS脱气并允许在烘箱（65°C）中固化过夜，以在PDMS中产生互补的微通道。随后，固化的PDMS从SU-8主站剥离，然后是入口/出口的冲孔端口。然后，PMDS等离子体粘合到载玻片上。微流体装置的表面包括天然玻璃和PDMS。为了促进细胞附着，扩散和增殖，需要细胞外基质（ECM）包衣。bMFA包括一个微血管网络和一个通过3μm高和100μm宽的孔连接的组织室（图2）。该微流体系统在具有相互连接的血管和分叉的完整微血管网络的生理相关3D环境中再现整个白细胞粘附/迁移级联反应，包括循环，边缘化，滚动，粘附和白细胞在单个系统14，16，17，21，26中进入血管外组织室。
需要注意的是，即使bMFA入口处的流速是固定的，网络中的流动条件在不同位置也不同，无法通过简单的数学公式计算。开发了一个基于计算流体动力学（CFD）的模型来计算网络中不同位置的不同流动参数（例如，剪切应力，剪切速率，速度）。该CFD模型用于模拟bMFA中的染料灌注模式和流动参数。与实验结果的交叉验证表明，计算模型可以很好地预测整个网络的流动阻力（图3）17。然后使用该CFD模型来估计bMFA每个容器中的速度和剪切速率曲线（图4），从而可以分析剪切流和几何形状对白细胞滚动，粘附和迁移的影响16。白细胞优先粘附在分岔附近和体内低剪切区域，并且使用嗜中性粒细胞在bMFA中成功证明了白细胞粘附的空间模式（图5）16。本文描述了制备bMFA以使用人肺微血管内皮细胞（HLMVEC）和人嗜中性粒细胞在炎症条件下研究白细胞 - 内皮细胞相互作用的方案。微生理系统，如bMFA，可用于研究内皮细胞与不同类型的细胞如嗜中性粒细胞，单核细胞，淋巴细胞和肿瘤细胞18，27，28，29，30的相互作用。bMFA可以接种来自不同器官（例如，肺与脑）和不同物种（例如，人与鼠内皮细胞）的原代内皮细胞，以及内皮细胞系21，27，31，32。bMFA可用于研究多种细胞反应，细胞 - 细胞相互作用，屏障功能，药物递送和药物毒性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Biomimetic microfluidic assay (bMFA)</td>
			<td>SynVivo</td>
			<td>SMN1-C001</td>
			<td>Exclusive at SynVivo</td>
		</tr>
		<tr>
			<td>Blunt needle</td>
			<td>Jensen Global</td>
			<td>JG24-0.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C70-500</td>
			<td></td>
		</tr>
		<tr>
			<td>CFDA, SE</td>
			<td>ThermoFisher</td>
			<td>C1157</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, 250,000, Powder</td>
			<td>Spectrum Chemical Mfg. Corp</td>
			<td>DE-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Premium</td>
			<td>GE Health Care</td>
			<td>17-5442-02</td>
			<td>Leukocyte isolation media</td>
		</tr>
		<tr>
			<td>fMLP</td>
			<td>Sigma-Aldrich</td>
			<td>F3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Fisher Scientific</td>
			<td>AAJ1692630</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin</td>
			<td>Fisher Scientific</td>
			<td>33-016-015</td>
			<td>use vendor recommended ECM for different cell lines</td>
		</tr>
		<tr>
			<td>Microvascular Endothelial Cell Growth Medium-2 BulletKit</td>
			<td>Lonza</td>
			<td>cc-3202</td>
			<td>Human lung microvascular endothelial cell culture medium (HLMVEC).</td>
		</tr>
		<tr>
			<td>Human lung microvascular endothelial cells</td>
			<td>Lonza</td>
			<td>cc-2527</td>
			<td>use vedor remommended trypsin-EDTA and TNS</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti2</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>NIS-elements, 5.20.01</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>MT21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PhD Ultra Syringe Pump</td>
			<td>Harvard Apparatus</td>
			<td>70-3007</td>
			<td>Syringe Pump</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>02-003-763</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-alpha</td>
			<td>R&#38;D Systems</td>
			<td>210-TA</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide clamp</td>
			<td>SynVivo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S640-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Synvivo Pneumatic Primer</td>
			<td>SynVivo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, Trypsin Neutralization Solution(TNS)</td>
			<td>Lonza</td>
			<td>cc-5034</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon tubing</td>
			<td>Fisher Scientific</td>
			<td>50-206-8921</td>
			<td>Tubing</td>
		</tr>
	</tbody>
</table>

a microphysiological system to study leukocyte-endothelial cell interaction during inflammation

白细胞 - 内皮细胞相互作用在脓毒症等炎症性疾病中起重要作用。在炎症期间，活化的白细胞通过血管内皮过度迁移到关键器官可导致器官衰竭。已经使用几种实验和计算技术开发和验证了生理相关的仿生微流体测定（bMFA），该技术可以重现整个白细胞滚动/粘附/迁移级联反应，以研究白细胞 - 内皮细胞相互作用。使用地理信息系统（GIS）方法对从啮齿动物 体内 图像获得的微血管网络进行数字化，并在显微镜载玻片上用聚二甲基硅氧烷（PDMS）进行微加工。为了研究剪切速率和血管拓扑结构对白细胞 - 内皮细胞相互作用的影响，开发了计算流体动力学（CFD）模型以生成整个网络的剪切速率和速度的相应图。bMFA能够量化白细胞 - 内皮细胞相互作用，包括滚动速度，响应于不同剪切速率的粘附白细胞数量，迁移白细胞数量，内皮细胞通透性，粘附分子表达和其他重要变量。此外，通过使用人相关样品，如人内皮细胞和白细胞，bMFA为快速筛选潜在疗法提供了一种工具，以提高其临床转化性。

在bMFA中剪切流下培养48小时后，内皮细胞覆盖bMFA中血管通道的表面并沿流动方向排列（图6）。共聚焦显微镜表明血管通道的所有表面都被内皮细胞覆盖，在bMFA18中形成完整的3D管腔。
使用该协议，可以获得中性粒细胞粘附图，表明bMFA中嗜中性粒细胞与内皮细胞的粘附显着（图8）。将该粘附图中嗜中性粒细胞的?...

Watch this Scientific Journal Video about A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation at JoVE.com

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

在该协议中，仿生微流体测定法可用于研究炎症性疾病中的白细胞 - 内皮细胞相互作用，该测定可以再现生理相关的微血管环境并重现整个白细胞粘附/迁移级联反应。

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

白细胞 - 内皮细胞相互作用在脓毒症等炎症性疾病中起重要作用。炎症失调常导致血管内皮屏障功能改变和白细胞过多，从而导致器官损伤。仿生微流体测定，我们称之为bMFA，再现体内微血管网络的形貌和流动条件，并允许实时评估中性粒细胞滚动，牢固粘附，扩散和迁移到组织室。仿生微流体测定的一个优点是能够使用原代人类细胞和临床相关患者样本来增加临床转化并快速筛选潜在的治疗方法。演示这些程序的将是来自我实验室的研究生研究助理杨庆良先生。开始使用细镊子将长度约为一英寸的管子插入端口内，但一个入口端口除外。使用钳口夹，将两个出口端口和组织室夹在一起。用PBS将纤连蛋白储备溶液稀释至每毫升100微克。将连接到24号钝针的一毫升注射器与稀释的纤连蛋白溶液一起装入，并将注射器连接到四英寸长的管道上。将管子插入开放的入口端口，然后推动柱塞，直到人纤连蛋白从另一个入口端口释放并夹紧它。对其余端口重复该过程，直到所有通道，组织室和管道都充满人纤连蛋白溶液。取下针头，但保持四英寸长的管子插入并未夹紧。要进行脱气，请将未夹紧的管道连接到气动底漆，该底漆连接到压缩氮气罐，压力为每平方英寸5磅，持续15分钟。在显微镜下，检查通道或组织室内是否没有气泡，如果存在气泡，请重新连接设备。从气动底漆中取出设备，并在37摄氏度下孵育一小时。使用预热的人肺微血管内皮细胞培养基，冲洗所有通道和组织室。按照文本手稿中所述收获内皮细胞后，G.In 可编程注射泵以150次离心将细胞沉淀五分钟，安装一毫升注射器并将管子连接到钝针上。将大约20微升的细胞悬浮液吸入管中，而不让它进入注射器桶中。从设备的出油口卸下夹具。将管道连接到入口端，而不会将任何气泡引入通道。以每分钟四到八微升的流速停止泵，并在显微镜下观察。当通道充满电池时，停止泵。夹紧出口并切割入口管。将设备在5%二氧化碳和37摄氏度的培养箱中放置四小时。孵育四小时后，准备另一个注射器并用新鲜的细胞培养基填充。将注射器安装在注射器泵中，并将其连接到入口端口。从出油口卸下夹具。以每分钟四到八微升的速度将新鲜培养基通过设备约五分钟，以除去漂浮或未连接的细胞。通过用新鲜细胞培养基填充注射器来准备注射器。将注射器安装在注射泵中，并将其连接到一个入口端口，同时保持出口打开。将设备置于37摄氏度和5%二氧化碳的培养箱中。编程注射泵用于在流动下培养内皮细胞，如文本手稿中所述。使用显微镜，在流动下培养48小时后检查bMFA。共聚焦显微镜表明血管通道的所有表面都被内皮细胞覆盖，在bMFA中形成完整的3D管腔。在制备三种不同的bMFA装置后，分别用细胞培养基或TNF-α或TNF-α联合PKC delta抑制剂加载三个一毫升注射器，其中TNF-α和炎性细胞因子用于刺激内皮细胞和嗜中性粒细胞。PKC δ抑制剂是一种新型抗炎抑制剂。将三个加载的注射器连接到三个 bMFA 设备。使用缓冲液TNF-α或TNF-α添加抑制剂，以每分钟0.1微升的速度治疗人肺微血管内皮细胞四小时。如文稿所述分离人嗜中性粒细胞后，将中性粒细胞重悬于999微升HEPES缓冲液中，并向悬浮液中加入1微升10毫摩尔CFDA SE染料储备溶液，得到10微摩尔CFDA SE工作溶液，并在室温下孵育10分钟。用HEPES缓冲液洗涤细胞两次，方法是将溶液以315倍G离心5分钟，计数细胞后，在细胞培养基中以每毫升200万嗜中性粒细胞重悬于细胞培养基或加入PKCδ抑制剂的TNF-α或TNF-α中，并在室温下孵育15分钟。用在细胞培养基中制备的一微摩尔化学引诱剂fMLP填充注射器。打开 bMFA 的一个入口和一个出口。从组织室中取出端口管，然后插入fMLP管。将约20微升fMLP注射到组织室中以用于所有bMFA，留下用细胞培养基处理的fMLP。切割管子并夹紧它。用大约200微升的中性粒细胞悬浮液填充注射器，并将注射器安装在注射器泵上。将设备放在倒置显微镜载物台上后，将流速设置为每分钟一微升，然后启动泵。等到一小滴中性粒细胞悬浮液从管中出来，然后将管子插入入口端口。荧光标记的嗜中性粒细胞流入血管通道，并与内皮细胞和生理相关的流动条件相互作用。从开始实验10分钟后，打开图像分析软件，将物镜切换到10倍，并使用载物台操纵杆将设备置于显微镜下的中心。要获取附着贴图，请转到采集，单击扫描大图像。将弹出一个新窗口。选择 10 倍物镜并设置场选项，例如 5 x 3。单击扫描，单击文件，然后保存附着贴图。要获得用于迁移分析的迁移图，请使用载物台操纵杆将设备居中在显微镜下。依次单击视图、采集控制、ND 采集。将弹出一个新窗口。设置“路径”以保存文件并键入文件名。选中大图像功能，设置扫描区域，例如，5×3。勾选时间功能，将间隔设置为5分钟，持续时间设置为60分钟。拍摄组织室的延时图像，在接下来的一小时内每五分钟拍摄一张图像。CFD仿真显示了血管通道中的层流模式，但流动模式受到干扰的分岔区域除外。在进行仿生微流体测定后，相差图像显示血管通道的表面被内皮细胞覆盖，并在培养48小时后沿剪切流方向对齐。使用共聚焦显微镜进行荧光成像，表明在bMFA A中获得中性粒细胞粘附图中内皮细胞形成完整的三维腔，这表明bMFA中嗜中性粒细胞与内皮细胞有显着的粘附。bMFA中的中性粒细胞迁移图显示，在TNF-α激活时，组织中室内会发生相当大的中性粒细胞迁移，而在没有TNF-α激活的情况下没有观察到这种迁移。将中性粒细胞的空间分布与剪切速率相关联的粘附图表明，中性粒细胞粘附优先发生在剪切速率低的血管和分岔附近区域。此外，TNF-α治疗显着增加了粘附，其被PKCδ抑制剂抑制。分析延时图像表明，内皮细胞的TNF活化增加了响应fMLP的中性粒细胞迁移，而与TNF-α处理的细胞相比，用PKC delta抑制剂治疗减少了迁移。因此，bMFA可用于测试治疗炎症性疾病的新型疗法的疗效。bMFA还可用于通过测量渗透性和经内皮电阻等变量来研究内皮完整性，所谓的TEER以及炎症过程中的粘附分子表达。仿生微流体测定可以模拟不同器官的微环境，不限于单细胞类型或物种，还可以模拟对器官功能至关重要的细胞/细胞通讯和模拟不同疾病。

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

构建一个低预算的激光干切断系统研究轴突再生 C。线虫</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

在胎儿肺细胞的机械传导的实验系统研究

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

细胞物质相互作用研究微细的纳米多孔金模式

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

评估白细胞内皮的相互作用下水流条件的<em&gt;离体</em&gt;自动注入微流室法

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Live Cell Imaging during Mechanical Stretch

在机械拉伸活细胞成像

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A Microfluidic System with Surface Patterning for Investigating Cavitation BubblesÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œCell Interaction and the Resultant Bioeffects at the Single-cell Level

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...