This work was supported by a grant from the National Health Research Institutes (03-A1 BNMP11-014).

注意:我们的微流体装置的制造的光掩模的设计通过使用计算机辅助设计（CAD）软件绘制。然后设计被用于制造使用的是商业服务镀铬光掩膜。 PDMS的设备用软光刻技术制成。11 1.制作母模的通过光刻<ol><li>光刻工艺12之前，使用4英寸硅晶片作为基板，并在120℃下脱水在常规烘箱晶片10分钟。 </li><li>通过在100瓦特使用氧等离子体处理中的等离子体清洁器30秒清洗脱水硅晶片。 </li><li>预热在65℃两个分别电炉和95℃，对下列烘烤过程。 </li><li>涂层5克，以通过旋涂机的清洗硅晶片负光致抗蚀剂（PR）的;旋在1,200rpm下（SU-8 50）30秒，以产生微通道层。 </li><li>放置PR在65℃涂覆的晶片上的预热加热板上12分钟，并在95℃下转移至另一预热加热板上33分钟（100微米厚的图案），以执行软烘烤过程。 </li><li>烘烤后，放置在一个半自动化掩模对准的支架的PR涂覆的硅晶片，并对准一个25400 dpi分辨率透明度光掩模。 </li><li>的剂量为500mJ / cm 2的中暴露的PR涂覆的硅晶片到UV光（365纳米）来创建在硅晶片上的PR图案。 </li><li>从对准器中取出晶片，并放置在烘烤后在95℃下12分钟的加热板上。 </li><li>浸泡在SU-8显影剂（丙二醇单甲醚乙酸酯，PGMEA）溶液晶片以洗掉未交联PR为12分钟，并用氮气轻轻擦干以暴露对准标记。 </li><li>再次，涂层5克，以通过旋涂在晶片的负光致抗蚀剂;旋在700rpm（SU-8 100）30秒和1,200rpm下（SU-8 10），用于为300 30秒＃181;米厚的格局和27微米厚的图案分别使微孔层。 </li><li>放置在电炉上的PR涂覆的晶片在65℃下进行4分钟，并在95℃下8分钟（27微米深捕集阱层）;并在65℃40分钟，并在95℃110分钟（300微米深培养阱层）。 </li><li>冷却后，放置在装有UV光掩模对准器的PR涂覆的硅晶片。 </li><li>剂量250毫焦耳/ 平方厘米（27微米厚的图案）和700兆焦耳/厘米2（300微米厚的图案）暴露PR涂硅片紫外光灯（365纳米）。 </li><li>烘烤在95℃下的晶片5分钟，（27微米厚图案）和30分钟（300微米厚图案），分别。 </li><li>由PGMEA为分别为6分钟（27微米厚图案）和25分钟（300微米厚图案），洗掉未交联PR，然后用氮气干燥。 </li><li>测量高度的图案展示了通过放置在晶片上扫描激光轮廓曲线的在xy阶段用扫描激光表面光度仪晶片。 <ol><li>调整焦平面清楚地显示了由一20X物镜下使用"相机视图"观察模式在晶片上的图案特征。 </li><li>从"摄像机视图"到"激光视图"切换观察模式，以及设置特征的上下位置。 </li><li>设定分别测量模式，面积，质量，和z音调高达透明（上），1线（1,024×1），高准确度和0.5微米，然后按下开始底部开始测量。 </li></ol></li></ol> 2.准备PDMS器械的单细胞分离<ol><li> PDMS铸造之前，silanize主模具与三氯硅烷以创建一个疏水性表面，这使得它更容易从主模具剥离PDMS复制品。 <ol><li>将主模具和含权舟三氯硅烷的200微升在干燥器，和15分钟，施加真空（-85千帕）。 </li><li>停止真空，然后离开主模具在干燥器中silanize在室温下将主模具至少1小时。 </li><li>从干燥器中取出主模具和各母模放置在一个10cm培养皿。 </li></ol></li><li>混合含有碱和以10比的固化剂17.6克PDMS聚合物试剂盒的总量:1，然后倒入PDMS上的培养皿的母模。 </li><li>放置培养皿在干燥器和1小时施加真空（-85千帕），以除去在PDMS中的气泡。 </li><li>从干燥器中取出培养皿并将其放置在常规烘箱中在65℃下3 - 6小时以固化的PDMS。 </li><li>从主模中取出固化的PDMS复制品，并通过与内径-0冲床冲两个孔作为入口和出口处的捕集阱阵列的PDMS复制品所述微通道的两端。75毫米;流体通道（ 图2A）。 </li><li>使用磁带清洁PDMS复制品的表面上，然后将PDMS复制品在短暂氧等离子体处理（100瓦14秒）的等离子体清洁器。 </li><li>除去由氧等离子体机器与PDMS复制品。 </li><li>对齐的顶（包含捕获微孔）和一个底部PDMS（含有培养微孔）复制品用手在立体显微镜下，并使其接触。 </li><li>放置在烘箱对准PDMS复制品在65℃下放置24小时以实现PDMS复制品之间的永久接合，以形成最终器件。 </li><li>浸泡PDMS设备在去离子（DI） - 水填充容器，并将该容器在干燥器真空（-85千帕）下15分钟，以从PDMS设备的微通道除去空气。 </li><li>放置的DI水填充PDMS设备在组织培养罩，并使用UV光（光的波长:254nm），以30英里消毒装置的ñ。 </li><li>在PDMS设备与在1×PBS溶液，5％牛血清白蛋白（BSA）替换DI水，并在37℃下孵育30分钟，以防止细胞粘到PDMS表面。 注意:BSA涂层是至关重要的，以提高从分离的细胞的转移效率捕获孔到培养孔。 </li><li>替换在PDMS设备用无菌1×PBS溶液中的5％BSA溶液。 </li></ol> 3.单细胞悬浮液的制备<ol><li>培养的神经干细胞KT98和人癌A549细胞和MDA-MB-435，我们在常规细胞培养孵育箱（37℃，5％CO 2和95％湿度）的培养皿来制备细胞用于PDMS设备细胞实验。 </li><li>卸下并丢弃废培养基从生长至70细胞培​​养皿（含10％胎牛血清和1％抗生素的供给DMEM基础培养基） - 80％汇合。 </li><li>轻轻洗涤细胞用无菌PBS清洗3次。&lt;/ LI&gt; <li>卸下并丢弃PBS，加入2毫升蛋白，胶原溶解和DNA酶的活动重组酶混合物（请​​参阅材料清单的详细信息）。 </li><li>在室温下孵育培养皿5分钟，然后点击培养皿，以促进细胞脱落。 </li><li>添加4毫升灭菌PBS以分散细胞，然后将细胞悬浮液转移到15毫升锥形管中。 </li><li>离心管中，在300×g离心3分钟，去除上清。 </li><li>轻轻重悬在1ml的灭菌PBS细胞沉淀和计数使用标准台盼蓝排除法13的活细胞数目。 注意:此悬浮步骤是制备以及解离的单细胞悬浮液，以提高单细胞分离效率是至关重要的。 </li></ol> 4.单细胞的分离和克隆培养<ol><li>负载50微升细胞悬浮液中的2.2的浓度 - 2.5×10 <suP&gt; 6个细胞/ ml成经由与手持移液器设备的出口孔与PDMS装置的微通道。 注意:将细胞悬浮液装载吸管需要停止，并在第一停止位置保持，以避免引入气泡进入微通道。 </li><li>通过进口孔加载其他50微升细胞悬液，以均匀地填充了细胞的整个微通道。 </li><li>用密封塞（3毫米长，直径1毫米的尼龙切鱼线），以避免流由从入口和出口孔滴静水压力感应出水孔。 注意:插头是紫外线使用前组织培养罩内灭菌。 </li><li>填补了用培养基1毫升无菌注射器，从中弹出气泡，并将其设置在注射器泵。 </li><li>经由23政钝针和聚四氟乙烯（PTFE）管道连接介质装载注射器与PDMS装置的入口孔。 </li><li>从出线孔，所有拔下插头流2分钟的时间间隔，使细胞沉淀成重力单细胞捕获井。 </li><li>洗去未捕获的细胞用300μl培养基以600微升/分钟通过注射器泵驱动的流速。 </li><li>等待2分钟，以稳定装置，和密封带插头的入口和出口孔，以形成一个封闭的培养系统。 </li><li>手工翻转设备将捕获的单细胞转移到培养微孔。 </li><li>放置PDMS设备在100毫米组织培养皿，并在该装置周围添加灭菌的PBS 10毫升，以避免从所述微通道培养基蒸发。 </li><li>移动至培养皿的常规细胞培养孵育箱（37℃，5％CO 2和95％湿度）为单细胞的克隆培养。 </li></ol> 5.培养基补货<ol><li>培养1天后，具有提高细胞PR新鲜培养基替换PDMS设备培养基oliferation。 </li><li>放置在邻近所述入口孔和出口孔的区域的PDMS设备的顶部培养基的两个液滴以避免引入气泡进入所述微通道在执行下一个步骤。 </li><li>从微通道作为入口的两端附近的PDMS装置的顶部和用于所述微通道的出口冲两个孔。 注意:不要通过PDMS设备的整个两层冲;相反，仅仅通过与PDMS的顶层打孔。 </li><li>通过连接一个23政钝针和PTFE管含有对进口新鲜培养基1毫升的塑料注射器。 </li><li>流120微升新鲜培养基到设备5分钟，更换旧培养基。 </li><li>插入两个塞子以密封所述入口和出口孔，并且该设备返回到细胞培养孵化器。 </li><li>通过除去该密封塞，然后通过重复步骤5.4至5.5刷新培养基中培养的期间每2天。 </li></ol>

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The authors declare that they have no competing financial interests.

基于微孔的设备系统6,14已用于单细胞操纵和分析，例如大型单细胞俘获6和单造血干细胞增殖15。虽然井大小，数量和形状可以调节为特定的应用中，当孔的大小增加时，单细胞分离效率总是受到损害。9,15 为了克服此限制，Park等人报告了具有高的单细胞的捕集率（58.34％）的三角形的微孔的微流体芯片，而微孔尺寸放大以允许细胞扩散?...

目前将单个细胞单独在培养空间通常是通过使用有限稀释或荧光激活细胞分选（FACS）来实现的。对于许多实验室，有限稀释是一个方便的方法，因为它仅需要一个吸管和组织培养板中，这是容易获得的。在这种情况下，细胞悬浮液连续稀释至适当的细胞密度，然后通过使用手动移液管放置到培养孔。然后，这些隔间单细胞被用于细胞分析，如遗传异质性筛选1和集落形成2。然而，该方法是低通量和劳动密集的，不利用用于协助一机械臂，因为有限稀释法的泊松分布性质限制了单细胞事件的37％3的最大概率。 FACS机，集成机械臂可以通过准确PLAC克服泊松分布的限制ING在文化井一单细胞在同一时间4。然而，高机械剪切应力（因此，降低细胞活力）5和机购买和操作成本在许多实验室已经限制了它的使用。 为了克服上述限制，微型装置已经发展到高效的单细胞装入微孔6。然而，微孔不提供足够的空间用于装载的细胞增殖，由于需要使每个的尺寸微孔关闭到单个细胞的最大化单细胞加载的概率。作为培养测定在许多基于细胞的应用（ 例如，克隆形成实验7），较大微孔需要（从90 - 650微米的直径或边长）也被用于允许延长细胞培养物。然而，像有限稀释法，它们也具有低的单细胞负载效率，范围从10 - 30％的8，9 以前，我们已经开发出一种高通量微流体平台，单个细胞中分离出在个体微孔，并在分离的细胞的克隆形成实验证明其应用10的装置，用聚二甲基硅氧烷（PDMS）制成，并包括两组微孔阵列具有不同微孔尺寸，从而可以大大提高在微孔的大小加载单个细胞的效率是比电池显著大。值得注意的是，这种"双阱"的概念允许在不影响单细胞捕获效率，使得它直接地调整该装置的设计，以适应不同的细胞类型和应用程序灵活调整培养面积的大小。这种高效率的方法应该是对于长期的细胞培养实验对于细胞的异质性的研究和单克隆细胞系建立有用的。

<table border="0" cellpadding="0" cellspacing="0" width="1323">
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		<tr height="21">
			<td height="21" style="height:21px;width:401px;">AutoCAD software</td>
			<td style="width:225px;">Autodesk</td>
			<td style="width:235px;">AutoCAD LT 2011</td>
			<td style="width:463px;">Part No. 057C1-74A111-1001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon wafer&#160;</td>
			<td>Eltech corperation</td>
			<td style="width:235px;">SPE0039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conventional oven</td>
			<td>YEONG-SHIN company</td>
			<td style="width:235px;">ovp45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasma cleaner</td>
			<td>Nordson</td>
			<td style="width:235px;">AP-300</td>
			<td>Bench-Top Plasma Treatment System</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 50 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131269</td>
			<td style="width:463px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 100 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spin coater</td>
			<td>Synrex Co., Ltd.</td>
			<td>SC-HMI 2&#34; ~ 6&#34;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>YOTEC company</td>
			<td>YS-300S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Msak aligner</td>
			<td>Deya Optronic CO.</td>
			<td>A1K-5-MDA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>Grand Chemical Companies</td>
			<td>GP5002-000000-72GC</td>
			<td>Propylene glycol monomethyl ether acetate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scanning laser profilometer</td>
			<td>KEYENCE</td>
			<td>VK-X 100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trichlorosilane</td>
			<td>Gelest, Inc</td>
			<td>SIT8174.0</td>
			<td style="width:463px;">TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL.&#160; Hazardous. Corrosive to the respiratory tract., reacts violently with water.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Desiccator</td>
			<td>Bel-Art Products&#160;</td>
			<td>F42020-0000</td>
			<td>SPACE SAVER VACUUM DESICCATOR 190MM WHITE BASE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polydimethylsiloxane (PDMS) kit</td>
			<td>Dow corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Harris Uni-Core puncher</td>
			<td>Ted Pella Inc.</td>
			<td>15072</td>
			<td>with 0.75 mm inner-diameter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Removable tape</td>
			<td>3M Company</td>
			<td>Scotch Removable Tape 811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>Leica E24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin (BSA)</td>
			<td>Bersing Technology</td>
			<td>ALB001.500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM basal medium</td>
			<td>Gibco</td>
			<td style="width:235px;">12800-017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Thermo Hyclone</td>
			<td>SH30071.03HI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotics</td>
			<td>Biowest</td>
			<td>L0014-100</td>
			<td>Glutamine-Penicillin-Streptomycin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant enzyme mixture</td>
			<td>Innovative cell technology</td>
			<td style="width:235px;">AM-105</td>
			<td>Accumax</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DiIC12(3) cell membrane dye</td>
			<td>BD Biosciences</td>
			<td style="width:235px;">354218</td>
			<td>Used as a cell tracker</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td style="width:235px;">703007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic syringe (1 mL)</td>
			<td>BD Biosciences</td>
			<td style="width:235px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">23 gauge blunt needles</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td style="width:235px;">TD21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-tetrafluoroethene (PTFE) tubing</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td style="width:235px;">TFT-23T</td>
			<td>&#160;inner diameter, 0.51 mm; outer diameter, 0.82 mm</td>
		</tr>
	</tbody>
</table>

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.

对于单细胞的分离和培养的微流体平台包括一个微通道（200微米的高度）与两套微孔阵列（ 图2A）。两套微孔阵列被称为捕捉阱（25微米直径27微米的深度），并培养良好（285微米直径300微米深度）分别为单细胞分离和培养，每从顶视图（ 图2B）观察时捕获阱被定位在培养孔的中心。对于设备操作（概略操作流程在图1中示出），所需?...

Watch this Scientific Journal Video about A Microfluidic Platform for High-throughput Single-cell Isolation and Culture at JoVE.com

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

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 ...

a-microfluidic-platform-for-high-throughput-single-cell-isolation

Research

JoVE Journal

Bioengineering

11.2K 次观看.  National Health Research Institutes, Taiwan. 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. 

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

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

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

高吞吐量的单细胞和多细胞微胶囊

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip:一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

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 ...

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 ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

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 ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

微流体平台，用于用动态压缩刺激软骨细胞

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...