这项工作是由研究奖由美国加州理工学院定量生​​物科学（QB3），在缩小差距奖由罗杰斯家族基金会，加州大学旧金山分校/桑德勒基金会计划为突破生物医学研究，从巴斯夫赠款和美国国家科学基金会通过支持学院早期职业发展（CAREER）计划（DBI-1253293）。

1. SU8主制造<ol><li>设计微流体结构中使用AutoCAD软件两层制作，并与10微米的分辨率由印刷电路板上胶片供应商的设计。器件设计的细节在一个附加的基准11和通道几何形状示于图1中 。这些层应该包括对准标记，以帮助从每个制造层 12并置的特征。 </li><li>放置一个预净化3英寸直径的硅晶片上旋涂机和打开真空将其粘贴到卡盘。申请1 ml的SU8-3035在晶片和旋转的中心，持续20秒在500rpm，然后在30秒以2000rpm，提供50μm的层厚度。 </li><li>取出晶片并烘烤上的135℃热板上30分钟。允许晶片移动到下一步骤之前，冷却至室温。 </li><li>揭露涂晶圆到1层掩膜（ 图2A </strong&gt;的）下一个准直190毫瓦，365纳米LED 90秒。曝光后，放置在一个135℃热板上的晶片1分钟，然后冷却至RT在进行下一步骤之前。 </li><li>放置在晶片上旋涂机和打开真空将其粘贴到卡盘。申请1 ml的SU8-2050在晶片和旋转的中心，持续20秒在500rpm，然后在30秒在1375转，从而产生层，可提供135微米的附加厚度。 </li><li>移动到下一步骤之前，取出晶片并烘烤上的135℃热板上进行30分钟，然后冷却至室温。 </li><li> 第 2次层掩模（图2B）对齐到图案化在1.3的几何形状和涂布的晶片暴露于准直的190毫瓦，365纳米LED 3分钟。曝光后，放置在一个135℃热板上进行1分钟，然后冷却至RT在进行下一步骤之前。 </li><li>通过浸入丙二醇单甲基醚乙酸酯的搅拌浴中30分钟，开发口罩。洗片在异丙醇和烘烤在135℃热板上进行1分钟。将发达师傅在100毫米的培养皿的PDMS成型。 </li></ol> 2. PDMS设备制造<ol><li>通过组合50个克硅酮基料而言，用5克在塑料杯中的固化剂1的PDMS:准备10。混合内容物用装有搅拌棒的旋转工具。脱气30分钟，或直至所有的气泡除去的干燥器内的混合物。 </li><li>倒入PDMS，得到厚度为3毫米以上的主站和放置放回干燥器进行进一步脱气。一旦所有的气泡被除去，烘烤设备在60℃下2小时。 </li><li>从使用上清洁的表面与图案的一面了手术刀和地点的模切设备。切PDMS模具一半用刀片从硕士2人（ 图3a）单独的主1。在含有50微米流体处理几何大师1印片，冲流体入口和出口有0.75毫米活检穿孔。 </li><li>等离子治疗装置在1毫巴O 2等离子体60秒，一个300瓦等离子清洗。润湿未打孔片的PDMS的表面上一滴去离子水，以暂时延缓PDMS-PDMS键合，并作为润滑剂。同时通过立体显微镜，地点硕士1人观看的主2面相对滑动面，直至机械锁，实现当凹入框架和图 3A队友伸出帧。 </li><li>将设备放置在一个60℃的烘箱烤的组装的装置（图3B）两天，在60℃以蒸发水和完全粘结。 </li></ol> 3.制备试剂<ol><li>填充1ml注射器用蒸馏水内相。 </li><li>填写1毫升注射器与HFE 7500氟油与1重量。 ％生物相容性表面活性剂的表面活性剂13的中间阶段。 </li><li>填充10毫升注射器用10重量％polyethylenë乙二醇（PEG）在含有1重量水溶液。 ％吐温20和1重量％十二烷基硫酸钠为连续相。 </li></ol> 4.系统准备<ol><li>放置在微流体芯片上的倒置显微镜加上能够&lt;100微秒的快门速度的数字照相机的阶段。 </li><li>所有装入注射器上的注射泵和连接27G的针头。附加〜PE-2管30厘米长的针头插入有始有终到适当的打孔设备中的漏洞。 </li><li>插入PE-2的10cm长到设备的出口，并放置在一个垃圾收集容器的另一端。 </li><li>素的装置，通过运行速度的高速率的注射泵（2000微升/分钟），直到流体在管段到达装置的入口端口。 </li></ol> 5.乳液代<ol><li>聚焦显微镜对包含50微米×50微米孔和一个区域下游出口通道。 </li><li>设定注射器泵将流体输送到双乳剂发生器在250微升/小时的内相，加入100μl/小时为中间相，和700微升/小时的连续相的流速，并等待10分钟以达到平衡。 </li><li>保持内和中间相的流速，在250微升/小时和100微升/小时，分别。在1050微升/小时设定外相的流率。等待3-5分钟为双乳液一代在这组流状况稳定。 </li><li>采集5秒的视频图像时，透过手动图像分析30赫兹脱机处理。 </li><li>重复5.3和5.4 与表1中给出的流速，内和中间相的流速保持恒定和载波相位的流速是通过调节注射器泵的设置来改变。 </li></ol>

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The authors have nothing to disclose.

这里所描述的双乳液生成几何形状设计模仿的玻璃毛细管装置8的物理过程。在这些，对齐圆筒形玻璃毛细管用于创建被剪切成均匀的双乳液液滴三相同轴射流。我们的3D PDMS装置的功能是依赖于用50微米高的制造具有载波相位通道是320微米的总高度形成小的特征的中心对准。有一个显著潜力以错开的第二层掩模，在步骤1.7图案相对于50μm的高大几何的高功能，如果掩模不能准确地对准。适当的对准可以通过设计对准标记，例如同心圆入口罩被共同定位过程中的照片图案化来辅助。的装置的两个半体的PDMS等离子体键合是可能导致最终器件的显著不对准的第二过程。等离子焊接PDMS至PDMS的一般是瞬间的，所以在步骤2.4，我们描述了装置表面的润湿用DI水以延迟接合并允许处理，使得在图3A中所示的对准帧可被允许以锁定。如果这是试图在没有足够的湿润，在PDMS表面将不可逆转键进入适当的调整之前，和设备必须加以处置和新的PDMS模具制作。 双乳液装置设计成采取的制造技术优点，即该铅均匀疏水表面性质。然而，在该协议中描述的参数之外的操作要求所要求的流体方法的一些了解。内层和中间相的交界处（图1D，点"1"），内相和中间相的低流量的相对高的流创建的两相喷射，与疏水性中间相涂敷的通道壁。如果中间相的比例流量增加，离散水包油微滴的产生将开始发生，消除以形成相干的三相喷射在孔（图1D滴形成的能力，点"3" ）。后的信道的扩展（图1D，点"2"），一个显著量的载体相流的需要来创建中间相和疏水通道壁之间的几何距离。在载波相位流量减少，最终将导致中间相润湿的疏水性设备的壁。显著减少载波相位流动可产生流动条件不足以剪切的内相为长，细丝，从而从根本上改变的双乳液液滴形成的物理过程。 一旦建成，该装置的设计，生产从14双乳液到50微米，一个方便的大小商业FACS分拣使用仪器。如果超出该尺寸范围双乳液期望，孔尺寸需要从这里使用的50微米×50微米大小缩放。因为该设备被设计为产生水/油/水双乳液有均匀的疏水表面性质，油/水/油双乳液不能创建除非有施加到使设备均匀地亲水表面处理。 这项工作表明一个简单的制造PDMS设备能够健壮形成水/油/水双重乳液。虽然以前的研究报告双乳液的使用3D光刻14,15创建的设备的形成，形成了他们的设备的双乳液有直径分别测量微米100S的。这里所报告的设备适合于生产双乳液幅度比这个更小的顺序，提供卷类似于哺乳动物细胞和适合于由FA排序CS。 虽然这些结果也可以用玻璃毛细管微流控技术，制造玻璃的设备来实现是费力的，需要很多动手的每个设备的步骤。对于我们所有的PDMS设备制造主要包括铸造，焊接，烘烤PDMS板，这是简单的，重复的，并且易于扩展到大量进程。 一个光刻制造的器件的效用来生成使用共轴流聚焦已证明双乳液。我们希望简单的制造和这双乳液发生器的设计强大的功能应该导致其适应的科学和工业应用。以前在玻璃毛细管微流控芯片工作所需的专业技能阻止调查人员，使用硅橡胶软光刻，现在常见的实验室技术应该会更舒适。此外，可以产生小的液滴尺寸很适合于perfor米细胞并以小滴的生物测定法，定量和使用FACS分选。对于工业应用，它已经被证明，这些类型的设备可被制造成阵列和并行化10，使双乳剂产生率相比，单个设备，以提高到几个量级。此外，能够形成大的同轴流量聚焦通道小双乳液应使设备抵抗结垢和堵塞，并行旨在在不干预运行较长的时间的设备时，这是至关重要的。

<html>双乳液包括由一中间，不混溶流体层从载体相分离的液滴的，并且是特别令人感兴趣的，因为它们在工业，制药潜在用途，以及生物应用1。在某些情况下，以封装高价值的化合物在双乳剂的核心的能力使材料受到保护和释放以控制的方式。例如，药物可能不适合于外部载体流体 2的溶解度的条件下被封装。此外，中间层的油可以被用作用于药物，化妆品，和营养物质3的封装和递送的胶囊模板。在生物学中，双乳液，可以在高通量筛选有用的，因为它们允许进行子纳升实验数量庞大出来，然后检测和分选使用荧光激活细胞分选（FACS）仪器4,5。 ENT"&gt;双乳液具有所需的性能特性的设计需要的双乳液的尺寸，组合物和均匀性的精确控制。虽然散装乳化过程，如膜乳化，在工业中使用，所得到的乳液是高度多分散的，显示出各种各样的功能特性1。液滴的微流体的字段被自然地适合单分散乳剂的产生与仔细控制组合物6。微流体双乳液生成已经实现了两个主要策略，顺序降制作和玻璃毛细管流聚集。双乳剂可以在使用两步骤降决策过程的平面PDMS的设备来产生。首先，含水包油乳液用的是水包油滴制作具有疏水通道壁的装置的区域。接着创建的，该乳液可以是流入或重新注入与适用于水包油型亲水墙壁下降，使得区域落制作4。然而，PMDS的亲水性表面处理需要额外的制造步骤和通常是暂时的7。最可控和可重复的方法，以形成双乳液是通过共轴流聚焦，一种技术使用玻璃毛细管微流体，由此形成含有三个阶段的同心射流通过一个小孔剪切以产生单分散的液滴8首创。这种技术允许用于生产液滴比通道尺寸小得多的，与双乳液是各相的流速的函数的精确大小和组成。液滴和信道的大小和保护性外鞘流之间的大的差异防止液滴从接触通道壁，使表面处理不必要。然而，这种玻璃设备需要锥形毛细管提示定制加工，以及精心的组装和密封。以前的研究已经用3D软光刻造影生成使用流聚集物理双乳液，但这些设备生产的乳剂，直径&gt;150μm的9,10，要比一般分拣用FACS较大的物体大约一个数量级。一个有吸引力的替代方案将包括强大的功能的玻璃毛细管同轴流量和小液滴产生聚焦与易于制造的硅橡胶软光刻技术。 在本文中，我们描述了使用同轴流动聚焦以产生≤50μm的乳剂和完全使用三维软光刻11构成的双乳液生成器。我们的设备采用了翻盖的方法来制造的设备，其中包括一个小剪切通道（图1）来近似所述乳液形成过程中拉玻璃毛细管喷嘴。更重要的是，这些设备不需要特定的表面处理，并且所有的聚合物结构提供简单和可重复的制造SCalable到大量的重复装置。在这里，我们概述了设计，制造和测试的双乳液生成器。双乳液生成被证明是健壮的，可重复至14微米的微滴直径。功能轻松制作的耦合使得该器件一个有吸引力的选项，新的双乳液的应用开发。

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Photomasks</td>
			<td style="width:135px;">CadArt Servcies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">3&#34; silicon wafers, P type, virgin test grade</td>
			<td style="width:135px;">University Wafers</td>
			<td style="width:119px;">447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 3035</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y311074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 2050</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y111072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:135px;">Krayden</td>
			<td style="width:119px;">4019862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">10 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27 gaugue needles</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">PE 2 polyethylene tubing</td>
			<td style="width:135px;">Scientific Commodities, Inc.</td>
			<td style="width:119px;">B31695-PE/2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Novec 7500</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Biocompatable surfactant</td>
			<td style="width:135px;">Ran Biotechnologies</td>
			<td style="width:119px;">008-FluoroSurfactant</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">35,000 MW PEG</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">1546660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tween 20</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">P1369</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sodium dodecyl sulfate&#160;</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">L3771</td>
			<td></td>
		</tr>
	</tbody>
</table>

double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

双乳剂发生器由使用三维PDMS制造（图1A）产生的共轴流聚焦装置的。几何图形，可允许形成一个三相同轴射流被剪切成一个正方形，50微米×50微米的孔，允许水/油/水双乳液（图1B， 图1C）的形成。的内水相和中间油相都汇集于连接处为10微米的通道尺寸x 50微米（图1D，点"1"）。由于PDMS的疏水性，氟化油拥抱通道壁和内相停留在通道作为流体的中心在连续喷射行进，直到突然膨胀通道扩展达到（图1D，点"2" ）。在这个位置中，内部两相被注入到一个320微米的高大JUNC的中心针对这样可以使相对同心引进水载波相位。这三个阶段被强制进入一个50微米×50微米的孔（图1D，点"3"），从而使载波相位剪的高流速的内两相到细长卷须而分解，从均匀的液滴（ 图1E）。 三维PDMS制造需要的两个独特的硅橡胶模具在翻盖配置成型两种层光刻大师之后的耦合。甲50微米高的层被用于形成所述内和中间流体处理通道，连同上万事达1（图2A）的剪切孔，以及免费凸出和凹进帧上相对的主站。一个附加的135微米高的层被用于创建载体流体和出口通道（图2B）。双乳液发电机组件利用牛逼他凹和等离子体处理（图3B）经过几何对准突出帧（ 图3A）。 双乳液装置在各种流量条件下进行测试以证明不同大小的，单分散的双乳液的形成。对于这些实验中，内和中间相的流速保持恒定和载波相位流速改性液滴生成期间影响剪切力。实验条件是由载波相位流量（Q c）在所述内两相的总和的比率参数流（Q 总和）。产生液滴的以Q C / 3至57 之和q进行的实验图像示于图4中 。观察到含有内两相的细长区域伸入50微米×50微米孔和断裂成液滴被对流下游。一世D.加强载波相位的流量（提高Q C / Q 总和 ）导致所述内相被剪切成能产生更小的液滴逐渐变薄的区域。由该装置在不同的流速产生双乳液显示出5.2％的变化的平均直径系数。液滴直径为q的C / Q 总和选择值的直方图也显示中所产生的微滴（图5）的大小相对均匀性。该装置展示了，以形成双乳液显著较小孔口宽度的能力，并显示出具有增加Q C / Q 总和 （图6）的明确减少的趋势。在最高载波相位流量测试，使用50微米×50微米的小孔形成14微米的双乳液。 <img alt="图1" src="/files/ftp_upload/53516/53516fig1.jpg" /> 图1.几何域金字塔之戒的Ë乳液发电机。在制造的器件的（A）3D模型。 （B）的中心通道表示引入的内（灰色）的垂直横截面，中间（红色），以及载体（蓝色）相。 （C）的横截面示出包含内部两个阶段进入方孔中射流。 （D）的乳液产生在设备顶视图。交界（1）在注入疏水中间相是由亲水性的PDMS，这会导致它的外套通道壁辅助。交界（2）的信道扩展和内两相的射流被剪切成孔（3）由较高的速率流动的连续流体的一个点，其中物理原因液滴形成。 （ 五）复乳一代器件的显微图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。&lt;</a>/ P&gt; <img alt="图2" src="/files/ftp_upload/53516/53516fig2.jpg" /> 图2.平版印刷生产的主人 。 （A）的用于制备50μm的特征的掩模。主站1是用来模制流体入口，所述内/中间相交界，该乳液产生孔口，以及一个凹槽对准。硕士2包含用于对准凸起的脊。 （二）用于制备135微米功能的面膜。主人是包含载液布线通道和出口通道镜像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53516/53516fig3.jpg" /> 图3.大会PDMS设备 。 （ <s仲&gt; A）捏造PDMS板包含免费的凹陷和凸出的框架。 （B）组装，联锁，提供最佳的功能定位框架。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53516/53516fig4.jpg" /> 图生成双乳液4.图像以不同的流速。外相的流速被改变，以改变Q C / Q 总和 ，这是考虑到每个图像的左侧。增加Q C / Q 总和变窄通过节流孔被剪切内液体的喷射，产生越来越小的液滴。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5.直方图双乳液液滴的尺寸在不同的流速 。在给定的流动条件下生产的乳滴的直径平均变异系数为5.2％。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53516/53516fig6.jpg" /> 图6.雾滴直径与归一化的流量参数 。调整连续相的流速可以生产双乳液是30％的孔径-100％的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53516/53516fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </P&gt; <table fo:keep-together.within-page="1"><tbody><tr><td> 数 Q i [微升/小时] </td><td> Q·M·[微升/小时] </td><td> 之和q [微升/小时] </td><td> Q C [ 微升/小时] </td><td> QÇ <fon> / Q 总和 </fon></td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 1050 </td><td> 3 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 2100 </td><td> 6 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 3850 </td><td> 11 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 5950 </td><td> 17 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 8050 </td><td> 23 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 10150 </td><td> 29 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 11900 </td><td> 34 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 17150 </td><td> 49 </td></tr><tr><td> 100 </td><td> 250 </td><td> 350 </td><td> 19950 </td><td> 57 </td></tr></tbody></table> 用于实验表1流速的参数。内相和中间相的流速（Q I，Q 米 ）被保持不变，从而以恒定的组合流速（Q 总和）。载波相位流动速率（QC）是变化的，以产生双乳液具有不同的直径。的比例Q C / Q 总和是主无量纲参数描述的实验条件。

Watch this Scientific Journal Video about Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device at JoVE.com

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

复乳代使用聚二甲基硅氧烷（PDMS）同轴流量聚焦设备

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

double-emulsion-generation-using-polydimethylsiloxane-pdms-co-axial

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

16.0K 次观看.  University of California, San Francisco. Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

视频: Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

科研

生物工程

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

在硅橡胶膜拉伸微图案细胞

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

自动化脂双层膜的形成使用聚二甲基硅氧烷薄膜

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

一种单片机操作装置, 用于生成传统卷烟烟雾和电子烟气溶胶中的液体萃取物

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

使用伦迪病毒载体方法生成转基因大鼠

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...