我们感谢迈克Vahey从弗莱彻实验室在加州大学伯克利分校进行的microjetting参数的建议。这项工作是由赞助国立卫生研究院授予DP2 HL117748-01。

1。无限制造商会<ol><li>设计的无穷室使用电脑辅助设计（CAD）软件（命名为它的形状），然后保存文件，这样它是用激光切割机兼容。在由中心到中心的距离的0.15英寸在透明的丙烯酸用激光切割器切割腔室从1/8- 3/16创建这种形状，直径0.183分开两个圆。无穷大的形状有利于液滴界面双层的形成和稳定。 </li><li>通过丙烯酸系室的边缘钻一个1/16英寸的孔到无穷大形井。重复的相反侧。切离为0.2mm的丙烯酸板用剪刀或激光切割器，以充当一个底部至孔中的小矩形。 </li><li>涂上薄薄但完整层的快干粘合剂，以0.2mm的丙烯酸类的一侧，并且它粘附到所述腔室的底部。持紧密代替0.2毫米丙烯酸对腔室的底部和分配胶在界面以允许胶以形成密封，但避免覆盖可视面积。一定要对齐丙烯酸，使其边缘与室壁的边缘齐平，它完全覆盖了无穷室缺口。这将允许足够的射流穿透和防止井漏。 </li><li>切成两小块天然橡胶的覆盖钻孔。孔周围施加快干粘合剂。将橡胶过孔，然后按上所有区域有一对钳子固定的。重复这两个钻孔。确保所有胶合连接完成密封，以防止任何泄漏。 </li><li>在针使用23 G，1，戳在腔室的两侧中的天然橡胶的孔，以便插入在压电喷墨头的。 </li></ol> 2。实验准备在氯仿在-20℃下冷冻储存股票脂质的解决方案。在这项研究中，无论是1,2 - diphytano基-sn-甘油基-3 -磷酸胆碱（DPhPC）或1,2 -二油酰-sn-甘油基-3 -磷酸胆碱（DOPC）中的使用。 2毫升25毫克/毫升脂质溶液通常制备在本协议中并储存在-20℃下时，将持续两个月<ol><li>重悬脂中的正癸烷中，首先从一个股票的小瓶将其传送到一个小的玻璃瓶中，并在氩气或氮气轻轻擦干。握住瓶子成一角度而干燥，从而暴露更多的表面积与气体，从而使脂质干燥更快。 </li><li>只略微拧在瓶子的盖子，让干燥的溶液中，以在真空下坐于干燥器中1-2小时。然后加入正癸烷再溶解脂质在25毫克/毫升。涡流脂质溶液短暂超声处理它在水浴超声15分钟。存储在正癸烷的重组脂质在-20℃下</li><li>准备一只股票的蔗糖溶液。在1.5毫升微量离心管中，加入900微升300mM的蔗糖和100微升的1％甲基纤维素（MC）。甲基纤维素中加入，以使溶液，这有助于喷射的粘度。可选步骤：添加一个可选的1微升的深色食物染料或荧光珠，借出更多的对比度或荧光成像分别。该解决方案是一个300毫渗量的蔗糖溶液，设计用于蜂窝渗透压和microjetting期间提供的对比。 </li><li>绘制溶液倒入一次性1毫升塑料注射器。握住注射器针尖朝上，反复轻弹轴驱逐向着尖端任何气泡，并推动柱塞弹出被困空中。一定要撤离所有空气，然后再继续注射器，因为它会与适当的压电收缩负责喷射干扰。 </li><li>在注射器的端部安装一个0.22微米的过滤器。以防止空气被截留在过滤器中，垂直握住注射器和推动柱塞，直到液滴的顶端的上方形成。注意：33毫米直径的注射器过滤器单元被发现工作情况最好，但也可用于替代过滤小至3毫米直径的注射器过滤器，以减少死体积。 </li><li>拧开喷墨的阴路厄接头，并牢固地压在适当位置在过滤器的端部。同样，喷射流体，以防止截留的空气。旋入喷墨的顶部。流体应行进到喷墨的前端之后它完全附着。 </li></ol> 3。准备好装备<ol><li>使用v型钳，安装在显微镜载物台的注射器组件。从喷墨连接导线到喷墨控制器。注：自定义阶段是专为这个协议，而舞台设计可以由用户来决定，它有注射器和样品架的XY控制的独立XYZ控制是至关重要的。 </li><li>确定放大率和必要的透镜组合来实现期望的成像。在这里，一个10倍的目标和10X目镜使用。 </li&gt; <li>使用高速摄影机（≥1000帧），以形象化的喷射和囊泡的生成。在此之前成像，进行必要的摄像机标定。利用相机软件中基于图像的自动触发启动图像捕捉。 </li><li>安装无穷室到显微镜载物台。通过录音到位的舞台上固定室。 </li><li>小心地将喷墨针尖刺破用在天然橡胶的孔（参见图1b）。要做到这一点，把喷墨靠近腔和调整由眼定位，然后通过显微镜镜头进行更为精确的调整。步步为营，因为粗动作可能会损坏喷墨一角。 </li><li>一旦喷墨对齐，备份注射器组件远离该腔室，以防止装在井的过程中喷墨的任何损坏。肯定的是，喷墨的运动是单向的，使得它保持与膜中的孔对准。 </li><li>轻轻按下plun注射器组件的GER直到小液滴形式的喷墨喷嘴。这将提供一些初始背压。 </li><li>输入喷射参数。对于双极性梯形波，请使用以下参数：20 kHz脉冲频率，3微秒的上升时间，35微秒的脉冲持续时间，3微秒的下降时间，65 V外加电压（脉冲幅度），并触发每100喷气脉冲（脉冲数） 。然而，所施加的电压（脉冲幅度）和脉冲数（每个触发喷射脉冲）可以变化，以控制泡囊的尺寸。 </li></ol> 4。囊泡生成<ol><li>添加悬浮于正癸烷的无穷室25-30微升脂质溶液，覆盖两孔的全部表面上。 </li><li>加25μl的葡萄糖（同渗容摩为蔗糖溶液）的一个孔中，移液缓慢而平稳的最外边缘。当沉积，减少葡萄糖的形成应​​该的，因为血糖和血脂的解决方案不要混用。添加另一个251; l葡萄糖的相对良好，这将使得再次下降，并在5-10分钟，形成在腔室中部的脂双层膜。 </li><li>插入喷墨穿过天然橡胶，并小心地引导它向着液滴界面双层。接近双层慢慢地，随着引进喷墨将取代量和断裂能的双层。 </li><li>当喷墨内〜200微米，适用于喷射与步骤3.8中所述的设置。从双层的距离可以变化，主要取决于电压和脉冲数，除其他参数。本协议建议缓慢增加设置（​​电压和脉冲数），并观察双层变形。 </li></ol> 5。清洁设备<ol><li>从显微镜载物台取下注射器组装，并处理1毫升的塑料注射器和过滤器。 </li><li>要清洁喷墨，浸渍尖端的solutio吸，以下列解决方案ñ7-10x的各：70％乙醇，2％Neutrad在温水中，70％的乙醇和双蒸2 O溶液如果喷墨不会对吸液管牢固地配合，切枪头，以形成一个变压器。 </li><li>干燥室与组织。将无穷室在250毫升烧杯中，用2％体积/体积Neutrad在温水中，并超声处理5-10分钟。超声处理后，彻底干燥下的压缩空气的水井。任何水分的井可以妥协的脂双层膜的稳定性，因此它也被建议该室被置于烘箱中在60℃下持续15分钟。 </li></ol>

<ol>
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没有利益冲突的声明。

许多技术已被开发为小泡的产生，包括electroformation，乳液和微滴产生14〜16。然而，新的实验技术是必要的，以允许生物系统的不断增长的相似度来生活系统的设计。特别是微流控方法都提供控制水平的增加管膜unilamellarity，尺寸的单分散性和内部内容17,18，使泡囊模型更接近生物学。此外，使用微流体喷射特性和实验表明定向膜蛋白的有效结合，膜的不对称性，以及封装3,13。...

<html>它已成为越来越明显，细胞生物学是涉及整合我们的理解从分子到细胞的多尺度问题。因此，知道分子正是如何独立工作并不足以理解复杂的细胞行为。这部分是由于多组分系统的涌现行为的存在，作为例证的肌动蛋白网络的互动与脂质双层囊泡4，有丝分裂纺锤体组装在非洲爪蟾的重建中提取5，和细菌细胞分裂机械6空间动态。补充解剖生命系统的分子过程的还原论的方法的一个办法是采取重构的细胞行为使用一组生物成分最小的相反的做法。这种方法的一个关键部分涉及生物分子在一个密闭容积可靠的封装，细胞的一个关键特征。 e_content“&gt;几种策略，用于封装的生物分子为研究仿生系统存在，最生物学相关的系统是脂质双层膜，它模仿了细胞的质膜上施加的生化和物理约束。巨单层囊泡（GUVs）由electroformation 7组，最广泛使用的技术GUV代14之一，通常具有差的包封收率，由于其不兼容的高盐缓冲液8。Electroformation也需要大体积样品（&gt; 100微升），这可能是一个问题，用纯化的蛋白加工和低效地结合由于紧密间隔的脂质层之间扩散的难度大的分子。已经开发了几种微流体方法，用于产生脂质囊泡的双乳剂的方法，它通过多层水 - 油 - 水之间的两个接口传递部件（W / O型/ W）时，依赖于一个VO的蒸发latile溶剂驱动脂质双分子层形成9。他人已经使用产生的脂质双层囊泡10或在两个独立的步骤11的连续流微流体装配线。我们已经开发了基于快速应用流体脉冲对液滴的界面双层12生产控制规模，组成和封装GUVs的替代技术。我们的做法，被称为微流控喷射，提供了从现有几个小泡生成技术的综合优势，为创建功能生物分子系统研究各种生物问题的方法。

<table border="0" cellpadding="0" cellspacing="0" style="width:633px;" width="633">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Name</td>
			<td style="width:163px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:160px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:153px;">Piezoelectric Inkjet</td>
			<td style="width:163px;">MicroFab Technologies</td>
			<td style="width:157px;">MJ-AL-01-xxx</td>
			<td style="width:160px;">xxx denotes orifice diameter in microns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Jet Drive III Controller</td>
			<td style="width:163px;">MicroFab Technologies</td>
			<td style="width:157px;">CT-M3-02</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">High-speed camera</td>
			<td style="width:163px;">Vision Research</td>
			<td style="width:157px;">MiroEX2</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:153px;">DPhPC lipid in chloroform</td>
			<td style="width:163px;">Avanti</td>
			<td style="width:157px;">850356C</td>
			<td style="width:160px;">Ordered in small aliquots in vials</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:153px;">33mm PVDF filters, 0.2 &#181;m</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:157px;">SLGV033RS</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">1ml syringes</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:157px;">14823434</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">n-Decane</td>
			<td style="width:163px;">Acros Organics</td>
			<td style="width:157px;">111871000</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Glucose</td>
			<td style="width:163px;">Acros Organics</td>
			<td style="width:157px;">410950010</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Sucrose</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:157px;">S7903-1KG</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">Methylcellulose</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:157px;">NC9084958</td>
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		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:153px;">1/8&#34; Acrylic</td>
			<td style="width:163px;">McMaster Carr</td>
			<td style="width:157px;">8560K239</td>
			<td style="width:160px;">CAD designs for the infinity-shaped chamber are available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:153px;">0.2 mm Acrylic</td>
			<td style="width:163px;">Astra Products</td>
			<td style="width:157px;">Clarex clear 001</td>
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			<td height="21" style="height:21px;width:153px;">Acrylic Cement</td>
			<td style="width:163px;">TAP Plastics</td>
			<td style="width:157px;">10693</td>
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			<td height="21" style="height:21px;width:153px;">Loctite 495 Superglue</td>
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			<td height="63" style="height:63px;width:153px;">Loctite 3494 UV Strengthening Adhesive</td>
			<td style="width:163px;">Strobels Supply</td>
			<td style="width:157px;">30765</td>
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			<td height="21" style="height:21px;width:153px;">Natural rubber</td>
			<td style="width:163px;">McMaster Carr</td>
			<td style="width:157px;">85995K14</td>
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			<td height="42" style="height:42px;width:153px;">Custom stage</td>
			<td style="width:163px;">Home made</td>
			<td style="width:157px;">N/A</td>
			<td style="width:160px;">CAD designs are available upon request</td>
		</tr>
	</tbody>
</table>

lipid bilayer vesicle generation using microfluidic jetting

自下而上的合成生物学提出了一种新的方法来调查和重构生化系统和潜在的，最小的生物。这一新兴领域从事工程师，化学家，​​生物学家和物理学家来设计和组装基本生物成分为复杂的，功能从下往上的系统。这种自下而上的系统可能导致人造细胞的基本生物学查询及创新疗法1,2的发展。巨型单层囊泡（GUVs）可以作为一个模型平台合成生物学，由于其细胞样膜的结构和尺寸。微流喷射，或microjetting，是一种技术，它允许GUVs具有控制尺寸，膜组成，膜蛋白掺入和封装3的生成。该方法的基本原理是利用由压电致动器的喷墨变形暂停升产生多个高频流体脉冲IPID双层成GUV。该过程类似于从肥皂膜吹肥皂泡。通过改变喷出的溶液中，涵盖溶液的组成和/或各组分的组合物包括在双层中，研究人员可以应用这种技术来创建定制的囊泡。本文介绍的程序通过microjetting生成简单的小泡从液滴界面双层。

我们已经组建了一个微流体喷射建立在传统的倒置荧光显微镜用自定义的阶段，从加工零件和人工微米组装（ 图1）。喷墨表征提供了洞察囊泡生成过程。不同的喷墨喷嘴和脂质双分子层之间的距离会影响所施加的力，以使膜的变形。靠近双层聚焦的喷流，并防止所述膜从分散能量相差囊泡产生点。涡流行程随施加到压电致动器，与我们的预期（ 图2）相一致的电压。囊?...

Watch this Scientific Journal Video about Lipid Bilayer Vesicle Generation Using Microfluidic Jetting at JoVE.com

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

对液滴界面脂双层微流体喷射提供了一种可靠的方法来产生囊泡膜多不对称，跨膜蛋白的结合，以及材料的封装控制。该技术可用于研究多种生物系统，其中隔离的生物分子是理想的。

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 ...

lipid-bilayer-vesicle-generation-using-microfluidic-jetting

Research

JoVE Journal

Bioengineering

14.7K 次观看.  University of Michigan. 对液滴界面脂双层微流体喷射提供了一种可靠的方法来产生囊泡膜多不对称，跨膜蛋白的结合，以及材料的封装控制。该技术可用于研究多种生物系统，其中隔离的生物分子是理想的。 

视频: Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

科研

生物工程

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 ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

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 ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

在微加工微流体平台中生成简化的三维芯片皮肤模型

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

细胞模拟支持和悬浮脂质双分子层模型的组装用于分子相互作用研究

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice.

测试通过微流控混合配制的mRNA-脂质纳米颗粒的 体外 和 体内 效率

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

用于组合插头生产的微流体装置的设置

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

作者聚焦：在精准肿瘤学中整合计算和实验方法

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

用于组合塞生产的双层微流控装置

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...