来自美国国立卫生研究院支持（授予GM092804，DE019938）和烧伤的Shriners医院。

1，微流控设备制造<ol><li>使用标准光刻技术，制作了1000级洁净室的母模晶片。图案的第3μm的薄环氧系负光刻胶层，根据来自制造商的说明来定义迁移通道。图案的第二50微米厚的层来定义小区负载和趋化因子的腔室。 </li><li>使用图案化的晶片投聚二甲基硅氧烷（PDMS）的设备。大力混合的PDMS（20克）与引发剂（2克），使用塑料叉子在大型塑料托盘称重5分钟。 </li><li>小心倒出的PDMS模具上。 </li><li>通过将模具与浇注PDMS在真空干燥器中进行1小时脱气PDMS。 </li><li>烘烤并在烘箱中设定为65℃固化至少3小时的PDMS微流体装置</li><li>冲出来用冲床具有1.5毫米的尖端直径中央WBLCs。 </li><li>使用punc则为切出整个环形装置她的为5.0毫米的尖端直径。 </li><li>使用胶带甜甜圈设备去除水中的微粒。 </li><li>冲洗12孔板，用去离子水，然后用干燥的氮气。放置板在60℃的烘箱中持续5分钟。 </li><li>氧等离子体处理的12孔板的两倍;再次单独为35秒，然后用另一个35秒的甜甜圈设备。 </li><li>小心将设备中使用镊子板的孔中。 </li><li>烘烤板与上热板设定为80℃下进行10分钟的连结装置。 </li></ol> 2，微流控检测制剂<ol><li>氧等离子体处理后，立即素的微流体装置时，该设备是亲水性的毛细管作用可促进在器件中的小通道的起动。 </li><li>将5微升的fMLP [原液10微米]与5微升纤维连接蛋白[原液1毫克/毫升]和490微升的HBSS创建趋化解决方案。 </li><li>慢慢的移液管＆＃160;趋化溶液成WBLC，使用凝胶加载提示（ 图1A）。移液管的额外20微升围绕该装置的外部的趋化。 </li><li>放置板在干燥器中15分钟。通过将真空施加到该装置，将溶液滴入侧通道的装置和移位的空气扩散通过所述PDMS的。 </li><li>从干燥器中取出板和显微镜下确认设备通道的润湿性。看着泡变小趋化解决方案进入室内。没有泡沫应该是经过真空处理装置内的存在。 </li><li>洗WBLC和装置外部彻底除去过量的趋化溶液。这个步骤产生趋化从每个焦点趋化室（FCC的）到设备中心渐变。 <ol><li>补一个1毫升的注射器用PBS，加30 G钝针尖注射器。轻轻地插入在注射器的针尖到圆环孔的中心。轻轻推100μl的PBS中成孔以使PBS的形式在设备的顶部的液滴。 </li><li>倾斜板和移液管1ml的PBS周围装置，以使液体收集在井的底部。吸出液体，然后重复过程总的3倍使用（而不是使用缓冲介质，如果分离的嗜中性粒细胞会在介质上加载）新鲜的缓冲溶液9。 </li></ol></li><li>每个媒体以及填补，直到设备的顶部下液体被淹没。让设备静置15分钟，使梯度达到稳定。实验结果和有限元模拟的理论数据表明，在这些设备中的梯度稳定长达24小时，小分子（ 例如 ，的fMLP）和最多为大分子量（ 例如 IL8）一个数天。 </li><li>用凝胶加载提示，慢慢吸取2微升血液（或孤立的中性粒细胞）到每个全血洛阿鼎室（WBLC）（ 图1A）。 </li></ol> 3，样品制备人类嗜中性粒细胞从毛细血管血<ol><li>收集毛细血管血液从健康志愿者，谁是没有免疫抑制剂的手指刺。所有病人的样本，并以书面的知情同意书获得，并通过批准的麻省总医院和施赖纳斯机构审查委员会的程序。 <ol><li>对于扎手指采血，洗手用水和肥皂和干燥的皮肤。加入1 ml HBSS + 0.2％HSA和10微升的Hoechst染色（32.4微米）到肝素的血液收集管（1.65 USP/50微升血液）准备anti-coagulant/stain原液。使用SurgiLance安全柳叶刀（2.2毫米深，22 G）刺破手指，擦干血第一滴，收集50微升的血液在Eppendorf管中含有股票抗凝血剂和赫斯特荧光染料溶液（10微升），轻轻混合。 </li></ol></li&gt; <li>孵育血液和Hoechst的染色10分钟，以允许核荧光染色。 1小时内采血运行示例。 </li></ol>人类嗜中性粒细胞从静脉血<ol start="3"><li>画出加入10ml外周血，静脉血入含有33美国药典肝素管。 </li><li>加入50微升的静脉血媒体和赫斯特染色如前所述。孵育血液和Hoechst的染色10分钟，以允许核荧光染色。 1小时内采血运行示例。 </li></ol>中性粒细胞分离从全血 - 阳性对照<ol start="5"><li>分离中性粒细胞，使用无菌技术，从使用羟乙基淀粉10毫升静脉全血样品中分离嗜中性粒细胞（6％重量/体积）密度梯度后跟一个阴性选择磁珠分离试剂盒按照制造商的协议。 </li><li>嗜中性粒细胞重悬的最后等分试样在concentr通报BULLETIN 4000个/μl在1X HBSS + 0.2％的人血清白蛋白。细胞保持在37°C，直到准备好运行实验。 </li></ol> 4，显微镜和图像分析为中性粒细胞趋化测量<ol><li>设置biochamber温度至37℃，湿度为80％，和CO 2为5％。 </li><li>开始使用时间推移成像在显微镜（10X或更高的放大倍率）立即成像。 </li></ol> 5，统计分析<ol><li>每个样本中手动追踪至少50中性粒细胞。 </li><li>计数结束时进入FCC的细胞（ 图2）。 </li><li>计算嗜中性粒细胞的速度在使用ImageJ（NIH）（ 图2）WBLC和FCC之间的信道。 </li><li>通过计数细胞，通过分叉的数量，并计算细胞的转向FCC和细胞退出次的数量之间的比率量化嗜中性粒细胞的方向性E设备。细胞是不定向不遵循趋化梯度，因此迁移朝向FCC和出口流道（ 图2）相等的数字。 </li></ol>

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有没有利益冲突披露。

在这项工作中，我们开发了一个微流体平台，从血液滴（2微升）测量嗜中性粒细胞的趋化性。红细胞从积极的中性粒细胞迁移的芯片上的机械过滤规避了繁琐的细胞分离方法如密度梯度10，正选择11或阴性选择12，这很容易通过激活嗜中性粒细胞引入伪像。红细胞的机械过滤区分我们的技术与其他微流控芯片，用于探测中性粒细胞迁移。例如，从本实验室13和以往?...

<html>嗜中性粒细胞贩卖起着决定的许多炎症性疾病，包括动脉粥样硬化1，细菌感染或败血症2的进度和分辨率的关键作用，以及烧伤3。对于健康和疾病状况做出的巨大贡献，中性粒细胞计数的标准血液分析通常被认为在临床和科研实验室的一部分。然而，尽管是一种最普遍的测试，中性粒细胞计数在感染和败血症的诊断价值已频频质疑4。例如，在烧伤病人的中性粒细胞的一项研究发现，嗜中性粒细胞计数和中性粒细胞迁移的功能不相关;标志着单独的中性粒细胞计数是不是免疫状态3的准确指标。虽然越来越多的难以衡量，中性粒细胞功能的能力提出了更有价值的各种条件。 T“&gt;重要的是，许多嗜中性粒细胞缺陷是短暂的，不会被永久的遗传缺陷，已经在很大程度上忽略了诊所，直到最近的区分触发。在烧伤的情况下，中性粒细胞迁移能的过程中被监控病人的治疗，炎症状态或感染3的指标。目前在实验室（Boyden小室，邓恩室，微量法）使用传统迁移实验不能转换成临床上，因为它们需要大量的血液和繁琐费时嗜中性粒细胞分离技术（ 表1）。这些测定也不能用来监测在小的实验室动物，如小鼠的嗜中性粒细胞趋化性的瞬时变化，由于血液所需的嗜中性粒细胞隔离的体积只允许有一个样品甚至常常需要汇集血液从多个动物为一个单一的测定，例如，涉及的研究米在多个时间点ultiple条件和治疗方法可以使用​​目前的趋化实验可能需要上千小鼠。这限制了基础生物学研究是可以做到的，了解的损伤，感染的情况下免疫功能的复杂动态或烧伤经常研究在小鼠模型5。 以满足对嗜中性粒细胞功能测定法是快速，健壮，同时要求最小的血液量，我们已经开发了直接从一个小液滴的全血测定中性粒细胞趋化性的微流体装置。它是已知的许多因素中的全血，血清，包括6和血小板7，影响嗜中性粒细胞的功能。因此，它是有益的，该全血的微流体测定最大限度地减少样品处理测量的变化趋当与体外测定8维持体内微环境中的中性粒细胞。这种方法降低时间从采血到使用传统技术时中性粒细胞迁移实验，以短短几分钟内（ 表1）。全血的微流体平台产生用于实验的长度的稳定的线性趋化梯度，没有移动部件，并且不需要外部压力源（ 例如注射器泵）。在全血中的微流体设备的设计中的关键特征是红细胞（RBC）的过滤梳机械地过滤进入设备的迁移通道的RBCs的掺入。这个过滤梳的右转弯防止需要排阻过滤，这将可能由红细胞堵塞，因此到达在WB的积极迁移阻断中性粒细胞趋化梯度。全血的微流体装置的一个12或24孔板的掺入促进人或鼠中性粒细胞趋化性的Si多种介质的筛分multaneously。

<table border="0" cellpadding="0" cellspacing="0" style="width:771px;" width="771">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:179px;">Catalog Number</td>
			<td style="width:191px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Device Fabrication</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">SU8&#160;</td>
			<td style="width:191px;">Microchem</td>
			<td style="width:179px;">Y131273</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:191px;">Ellsworth Adhesives</td>
			<td style="width:179px;">Sylgard 184 1.1 lb. Kit</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Standard glass slides</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">125495</td>
			<td style="width:191px;">1 X 3 inches</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Glass-bottom plate</td>
			<td style="width:191px;">MatTek</td>
			<td style="width:179px;">P12G-1.5-14-F</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Harris Uni-Core, Tip Diameter 5.0mm</td>
			<td style="width:191px;">Ted Pella, Inc.</td>
			<td style="width:179px;">15081</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Harris Uni-Core, Tip Diameter 1.5 mm</td>
			<td style="width:191px;">Ted Pella, Inc.</td>
			<td style="width:179px;">15072</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Microfluidic Assay Preparation and Analysis</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Gel-loading pipet tip</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">02-707-139</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Syringe</td>
			<td style="width:191px;">Fisher Scientfic</td>
			<td style="width:179px;">309602</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Blunt tip needle, 30g &#189; in.</td>
			<td style="width:191px;">Brico Medical Supply</td>
			<td style="width:179px;">BN3005</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Vacutainer, Heparin</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;"></td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">HBSS&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Human serum albumin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">A5843-5G</td>
			<td style="width:191px;">0.2% final concentration in HBSS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Fibronectin</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">F0895-1MG</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">fMLP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:179px;">F3506-10MG</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">SurgiLance safety lancet, 2.2mm depth, 22 gauge</td>
			<td style="width:191px;"></td>
			<td style="width:179px;">SLN240</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Hoescht stain</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:179px;">H3570</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Positive Control&#160;</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">HetaSep</td>
			<td style="width:191px;">STEMCELL Technologies Inc.</td>
			<td style="width:179px;">7906</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">EasySep Human Neutrophil Enrichment Kit</td>
			<td style="width:191px;">STEMCELL Technologies Inc.</td>
			<td style="width:179px;">19257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;"></td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Equipment</td>
			<td style="width:191px;"></td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Plasma Asher</td>
			<td style="width:191px;">March Instruments</td>
			<td style="width:179px;">P-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Lindberg/Blue M Oven</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:179px;">13-258-30C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Stainless Steel Precision Tweezers</td>
			<td style="width:191px;">Techni Tool</td>
			<td style="width:179px;">758TW458</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Bel-Art Scienceware Chemical-Resistant Vacuum Desiccator</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:179px;">08-594-15A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Dataplate Digital Hot Plate</td>
			<td style="width:191px;">Alpha Multiservices</td>
			<td style="width:179px;">PMC 720&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Nikon TiE inverted microscope</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">MEA53100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">CFI Plan Fluor DL 10x na 15.2wd Objective</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">MRH20101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Lumen 200 with 2 Meter Light Guide for Nikon</td>
			<td style="width:191px;">Nikon - Micro Video Instruments Inc.</td>
			<td style="width:179px;">500-L200NI2</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:211px;">DAPI/Hoechst/AMCA Narrow Band 32mm Exciters - 25mm Emitters</td>
			<td style="width:191px;">Chroma - Micro Video Instruments Inc.</td>
			<td style="width:179px;">31013v2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Retiga R 2000 cooled CCD Camera 1600x1200 pixels</td>
			<td style="width:191px;">Qimaging - Micro Video Instruments Inc.</td>
			<td>RET-2000R-F-M-12-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood

中性粒细胞在抵御感染和它们的数量在血液中发挥至关重要的作用，经常测量在诊所。在血液中较高的中性粒细胞计数通常持续感染的指标，而低中性粒细胞计数是一个警告标志的感染风险较高。为了实现其职能，中性粒细胞也必须能够有效地从那里度过大部分生命的血液移动，进入组织，在感染发生。因此，在嗜中性粒细胞的迁移能力的任何缺陷可以提高风险的感染，即使在中性粒细胞是存在于血液中的相应的数字。但是，衡量在诊所中性粒细胞迁移能力是一项艰巨的任务，这是费时，需要大量的血液，和专家知识。为了解决这些限制，我们设计了一个强大的微流体检测中性粒细胞的迁移，这需要未处理血单个液滴，circum气孔的必要性嗜中性粒细胞分离，并且容易量化的简单的显微镜。在该测定中，嗜中性粒细胞直接从血液滴，迁移通过小通道朝趋化因子的来源。为了防止通过同一信道的红血细胞的颗粒流，我们实现了机械过滤器具有直角转弯，其选择性地阻挡红细胞的前进。我们通过从手指刺和静脉血采集的血液滴比较中性粒细胞迁移的验证实验。我们还比较了这些全血（WB）的来源与中性粒细胞纯化样品中性粒细胞迁移，发现三个来源之间保持一致的速度和方向。此微流体平台将使人类中性粒细胞迁移的研究在临床和研究环境，以帮助增进我们的健康和疾病中性粒细胞功能的理解。

全血（WB）中性粒细胞的趋化性试验，通过测量中性粒细胞朝着渐变的fMLP（ 动画S1）的积累验证。结果证实，红细胞通过的过滤梳困而嗜中性粒细胞（蓝色）能够积极地迁移出的全血（ 图3A和电影S1）。由全血的微流体装置所形成的稳定的线性趋化梯度（绿色）用FITC-标记的葡聚糖（ 图3A中的插图）的确定和测量的荧光水平随着时间的推移。在这?...

Watch this Scientific Journal Video about Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood at JoVE.com

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

这个协议的细节设计，从全血具有强大的可重复性一滴测量人体中性粒细胞的趋化作用的试验。这种方法绕过了必要的中性粒细胞的分离和需要测定的准备时间仅几分钟。微流控芯片使中性粒细胞的趋化作用的重复测量随着时间的推移在婴儿或小型哺乳动物，其中样品体积是有限的。

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 ...

microfluidic-platform-for-measuring-neutrophil-chemotaxis-from

Research

JoVE Journal

Bioengineering

11.2K 次观看.  Massachusetts General Hospital. 这个协议的细节设计，从全血具有强大的可重复性一滴测量人体中性粒细胞的趋化作用的试验。这种方法绕过了必要的中性粒细胞的分离和需要测定的准备时间仅几分钟。微流控芯片使中性粒细胞的趋化作用的重复测量随着时间的推移在婴儿或小型哺乳动物，其中样品体积是有限的。 

视频: Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

科研

生物工程

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

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 ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

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 ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

影像学在两层微流控平台拟南芥幼苗的根头发形态

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...