Name: microscale vortex-assisted electroporator for sequential molecular delivery
Uploaded: 2014-08-07T20:15:00.000+00:00
Duration: 10 min 51 s
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这项工作是由罗兰初级研究员计划的支持。作者想表达感谢的科学家和工作人员在罗兰研究所是哈佛克里斯·斯托克斯，他的定制，计算机辅助压力控制设置，黛安Schaak博士的帮助下发展她输入的生物样品处理，温菲尔德山开发的电设置，Alavaro桑切斯博士授予访问流式细胞仪，斯科特贝维斯，肯尼斯宾塞和唐·罗杰斯用于加工所需的压力设置机械管道部件。微流控大师是捏造的中心纳米系统（CNS）的哈佛大学。

1，细胞的制备<ol><li>板1×10的转移性乳腺癌细胞系MDA-MB-231中的体积每组织培养的T75烧瓶10毫升在Leibovitz的L-15培养基补充有10％（V / V）胎牛血清和1 5个细胞/毫升％青霉素 - 链霉素。 </li><li>培养MDA-MB-231细胞在潮湿的培养箱中在37℃下用0％CO 2环境中。 </li><li>收获细胞用于实验接种后第2天通过用细胞用0.25％胰蛋白酶-EDTA进行2分钟，并通过加入8毫升生长培养基的灭活胰蛋白酶的酶活性。 </li><li>沉淀细胞离心5分钟，在200×g下，重悬在Dulbecco磷酸盐缓冲盐水（DPBS，1，不含Ca 2 +和Mg 2 +），以5×10 5个细胞/ ml的终浓度。 </li></ol> 2，设备的设计与制造注:本面膜，母模捏造和微封装过程要一个干净的房间内进行的，而聚二甲基硅氧烷（PDMS）微铸造工艺可以定期实验室工作台进行。 <ol><li>设计一个微流体装置， 如图1与AutoCAD。该装置应包括具有多个喷射口，粗过滤器和两个平行的直的矩形惯性聚焦通道的入口（L = 7毫米，W = 40微米，和H = 70微米）。通孔的铝电极个人直通道由5个电室，其中放置800微米外（W C = 400微米）有两个。两个横向相邻的电穿孔室共用孔用于负极。两个直的通道合并成一个出口处的电穿孔区域的下游。 </li><li>微型图案CAD文件转换为使用LinkCAD一个GDSII文件。写微图案上5"×5"光掩模坯使用激光面膜作家。通过下面的制造商的方案开发的掩模。注:面膜的开发过程，包括光致抗蚀剂显影，蚀刻铬和抵制搬迁。另外，微图案印在高分辨率的透明度（&gt; 20000 DPI）可从第三方厂商购买和使用作为光掩模。在光掩模最终微观特征应是透明的负光致抗蚀剂的极性匹配。 </li><li>使用标准软光刻技术23用4英寸的硅晶片上形成负光致抗蚀剂纺涂层以2000rpm到具有70微米的最终厚度制造该设备设计的铸造模具。 </li><li>预烘和负光致抗蚀剂的晶片20分钟，在95℃下。 </li><li>与晶片接触 ​​的掩模，并使用掩模对准器暴露到UV光源（17.4毫焦/厘米2）为80秒。 </li><li>制定4分钟的SU-8开发者暴露的晶片。 </li><li>测量发达的抗蚀剂厚度使用表面轮廓。 </li><li>严格地混合以10:1比例的弹性体与固化剂（有机硅弹性体盒）和上铸型浇注混合物以生成PDMS复制品。脱气浇铸弹性体30分钟，并固化该铸塑模具用脱气的弹性体在烘箱中于70℃下搅拌2小时。 </li><li>分层固化的PDMS副本与模具和冲压孔用销钳入口，出口和电极插入微图案。注:针钳的正常切削刃直径应0.76毫米，足以紧紧地抱着PEEK管（外径32"）和铝电极（0.029 OD"）代替。 </li><li>创建封闭的微流体通道由接合PDMS复制品，以在氧等离子体清洁器在75瓦和500毫乇，分别射频功率和氧分压处理7秒的载玻片上。 </li></ol> 3，流动实验<ol><li>插入铝电极（0.029"OD）和一个出口的PEEK管（32"OD）为通过在所述微通道的孔的指定地点（参见图3B）。 </li><li>连接的电气设备18负责产生高电压的短方波脉冲的铝电极（ 见图3C），这些在接触中的PDMS模具流动的解决方案。注:该设备应包括一个脉冲发生器和一个内部内置高电压放大器18。 </li><li>制备4的50ml离心管中分别含有的DPBS，溶液与细胞和生物分子，并且每个管连接到连接到所述气动流控制系统的各自的小瓶保持器（ 见图3A）。18 </li><li>连接入口PEEK管从药瓶持有者成在微流体装置中的各个进气孔。 </li><li>为了有电动呸设置方波脉冲，V的大小，以100伏LD强E = V / L E，整个电室相当于0.7千伏/厘米。请注意:在这里，L e为两点，其中正电极和负电极是在与流动溶液接触之间的距离（参见图1）。 </li><li>将压力调节到40磅。注:单个手动调节氮源用于均匀加压所有样品瓶和高速歧管采用采用定制的LabVIEW软件，及时启动个性化解决方案的端口18 </li><li>对于阀门控制，打开标阀门亚军的定制LabVIEW软件，单击运行，从经营资格的下拉菜单。 </li><li>通过点击每个相应的阀门图标打开阀门。注:该阀应的图标变成绿色，当它被激活。点击活动图标将停用并关闭阀门。关闭阀门图标应该变成灰色。 </li><li>打开阀门的DPBS水库（即，洗涤液），以素所需的稳定细胞俘获涡流产生1.5分钟的流速。 </li><li>从洗涤溶液中的细胞溶液以捕获细胞在电穿孔室中，持续30秒（ 即 ，小区捕获步骤）切换活动溶液端口。 </li><li>通过该装置同时向细胞俘获步骤流既洗涤和细胞溶液，持续10秒之前，以确保在溶液中切换步骤不间断流动。注:此简短的联合流程的步骤应重复在每个解决方案切换的步骤。 </li><li>把洗涤端口上和为了除去非截留污染细胞冲洗设备，持续20秒。 </li><li>注入含有目的插入设备（3000沓中性和阴离子葡聚糖分子或1.5毫克/毫升70000沓中性葡聚糖分子的0.2毫克/毫升）的第一分子的溶液。 </li><li>及时注射后可申请五短脉冲（ΔT= 30毫秒，2秒间隔）分子溶液并监测实时使用示波器所施加的电脉冲的幅度和持续时间。 </li><li>重复步骤3.10的次数的额外分子的数量来提供。为每个分子递送，孵育细胞为100秒，在分子的溶液，然后用100秒以除去过量的分子的洗涤溶液冲洗该装置。 </li><li>通过降低低于5磅的操作压力释放的细胞在96孔板中用于下游分析。注:大约100μl用100细胞溶液从每个释放收集。 </li><li>运行实验步骤3.9至3.16三次收集到足够的细胞用于流式细胞仪。 </li><li>离心96孔板中含有处理细胞5分钟，在228×g下。 </li><li>删除含有过量荧光分子上清。 </li><li>重悬细胞DPBS流式细胞仪分析。 </li><li>称重传感器在流式细胞仪分子üptake效率​​分析。 </li></ol>

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作者什么都没有透露。

与新的并行电平台，10倍的增强吞吐量和多分子递送效率，除了所有的先前开发的单室型系统提供的优点来实现。18之前提供的优点包括:（i）预净化的靶细胞与活力增强粒度分布均匀，（ⅱ）精确和个别分子的剂量控制，以及（iii）低操作电流。荧光标记的葡聚糖被选择作为感兴趣的分子，因为它们在很宽范围的分子量，缀合的荧光团的类型和电荷的容易获得的。这些大分子都适合这样的研究，因为他们是天生防渗膜对活体细胞和不显示细胞毒性。荧光右旋糖酐，培养时间为24最佳浓度，以及电参数，确定了当前系统之前吨澳电的实验。优化过程中是相当简单和有效的，由于该平台的能力，以监测实时的分子吸收过程。这个过程的可视化，如果少到没有知识的电参数的分子或细胞类型正在测试非常有利的。温育时间对测试的所有分子被设定为100秒通过的细胞俘获在所有10个室呈现实时检测的荧光强度信号，表示完成分子递送成功的。注意，这不是必需的分子递送的最小温育时间。 有一种关于使用电，分子分娩过程中争论由来已久无论是通过扩散4,19,25或电泳单独调节。26鉴定分子传递的主要机制涉及一系列艰苦的单项测试，比较吨他结局取决于分子大小，电荷和电参数。以最好的作者的知识，这些繁琐的优化过程没有报告与传统的电穿孔系统。4,26-28所开发系统的简单参数优化功能允许识别的主要分子传递机制，通过在电效率变化研究取决于分子和电参数。结果表明，阴离子3000沓葡聚糖的效率并不比它的中性对应物（分别为83和75％）的显著不同，这表明所观察到的分子的摄取可能是通过扩散介导的。不像其他电穿孔系统，处理的细胞连续整个电穿孔过程中该系统搅拌。在轨道单元的荧光信号逐渐增加，观察，表明扩散将是多米纳NT交付机制。中性70000沓葡聚糖分子的高效率分子吸收（90％）还表明，即使在高的分子量的输送过程中发生的扩散的结果。最后，成功的连续交付3000大阴离子和中性葡聚糖分子的巩固的分子传递时，尽管分子费用索赔。这个结果还表明，膜再密封之内没有电穿孔的3分钟（每分子递送100秒）完成。 所提出的分子交付平台可以提供连续的分子大小，不论其电荷的广泛范围。适时微调和各个参数的修改可以毫不费力的实时过程监控能力辅助来实现。该系统的尺寸为基础的细胞纯化过程无需费力的样品制备和费时离心分离前后的电步骤。此外，该系统的低工作电流显着减少细胞死亡。

在最近几年，使用电脉冲，以促进胞内递送的细胞外分子已经成为一种有吸引力的操纵哺乳动物细胞的方法。1此过程中，也被称为电穿孔，可逆permeabilizes细胞膜，允许固有膜不可渗透的分子，以获得到细胞的细胞内环境。因为几乎所有的分子可以在任何类型的采用电穿孔的细胞的膜通过临时创建的孔被引入到细胞溶质中，该技术已被报告为更可再现的，普遍适用的，并且比其它的方法包括病毒介导的，化学更高效和光学方法。2-3这一技术已被用来引入荧光分子，4种药物5和核酸6-7，同时保持细胞存活和完整性。考虑到这些好处，电已被批准为一个共同的劳动用于DNA转染atory技术， 体内基因治疗8和细胞疫苗接种研究。它是，但是，仍然难以常规电穿孔系统，可同时实现实用的效率和可行性与大的异质性大小的样品，因为需要成功电穿孔的电场强度密切的细胞的直径相关。此外，这些系统不允许的多个分子的量的精确控制被适当传递到依赖散装随机分子递送过程。9为了解决这些问题，许多组织已经开发微流体电平台，将提供较低的穿孔电压的优点，更好的转染效率，大量减少细胞死亡，并有能力提供多分子。由于微型电系统，其电极间距小脚印10-13这些优点能够成为可能长度为亚毫米，显着减少所需的成功交付电压。此外，这些微型电系统可以实现均匀的电场分布，并迅速消散产生的热量，产生降低细胞死亡率，同时提高配送效率。这些芯片进一步透明材料的运用使得原位观察电过程中的提示参数修改。2,12然而，精确的剂量控制和molecular-和蜂窝相关的参数控制，需要新的研究和治疗应用，6， 14-16仍然没有得到解决。 这项工作提出了一种微流体涡流辅助电穿孔系统，能够提供多种分子依次进入靶细胞的预先选定的相同人口。细胞均匀分布在使用之前报道SI隔离之前，电泽选择性捕获机制。17-18通过具有粒度分布均匀，变化较少的电效率和每个人实现给定的电场强度增强生存能力。19此外，不断搅拌用微型涡被困细胞允许在整个统一发货分子整个细胞质中，与该结果相一致以前报道使用另一个涡旋辅助电平台20为了证明这个系统将适用于范围广泛的生物应用中普遍使用的分子，具有宽范围分子量的大分子递送到转移性乳腺癌细胞。此外，实时过程监控的帮助下，这项工作提供了更多的证据来制止有关的分子传递的机制，长期的辩论中electrporation，主要是电泳介导的抗扩散介导的14 </sup&gt;与其他电穿孔系统，该平台独一无二地提供精确的多分子递送，高分子递送效率，最小的细胞死亡，大小和递送分子的电荷的跨度很广，以及实时可视化的电穿孔的组合优势流程。考虑到这些功能，开发电系统具有实用潜力的多功能工具细胞重编程研究，6,14,21-22给药应用10,19和应用需要进行深入的了解电的分子传递的机制。

<table><tbody><tr><td>MDA-MB-231 cancer cell line</td><td>American Type Culture Collection (ATCC)</td><td>HTB-26</td><td></td></tr><tr><td>Leibovitz&amp;rsquo;s L-15 Medium</td><td>Cellgro, Mediatech, Inc.</td><td>10-045-CV</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco, Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (DPBS)</td><td>Cellgro, Mediatech, Inc.</td><td>21-030</td><td></td></tr><tr><td>Trypsin</td><td>Gibco, Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Flow Cytometer easyCyte HT</td><td>Millipore</td><td>0500-4008</td><td></td></tr><tr><td>Oxygen Plasma Cleaner</td><td>Technics Micro-RIE</td><td></td><td></td></tr><tr><td>Dektak 6M surface profiler</td><td>Veeco</td><td></td><td></td></tr><tr><td>KMPR 1050</td><td>Microchem</td><td></td><td></td></tr><tr><td>SYLGARD 184 SILICONE ELASTOMER KIT</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Compressed Nitrogen gas</td><td>Airgas</td><td>NI 300</td><td></td></tr><tr><td>High Pressure Regulator</td><td>McMaster-Carr</td><td>6162K22</td><td></td></tr><tr><td>Downstream regulator</td><td>McMaster-Carr</td><td>4000K563</td><td></td></tr><tr><td>High-speed 3/2way-8 valve manifold</td><td>Festo</td><td></td><td></td></tr><tr><td>Inline Check Valve</td><td>Idex Health and Science</td><td>CV3320</td><td></td></tr><tr><td>5/32&quot; OD x 3/32&quot; ID Polyurethan tubes</td><td>Pneumadyne</td><td>PU-156F-0</td><td></td></tr><tr><td>1/4&quot; OD X 0.17&quot; ID Polyurethan tubes</td><td>Pneumadyne</td><td>PU-250PB-4</td><td></td></tr><tr><td>1/16&quot; PEEK tubings</td><td>Festo</td><td>P1533</td><td></td></tr><tr><td>1/32&quot; PEEK tubings</td><td>Idex Health and Science</td><td>P1569</td><td></td></tr><tr><td>PEEK tubing unions</td><td>Idex Health and Science</td><td>P881</td><td></td></tr><tr><td>Pulse Generator</td><td>HP</td><td>8110A</td><td></td></tr><tr><td>Aluminum Wire</td><td>Bob Martin Company</td><td>6061 ALUM</td><td></td></tr><tr><td>Oscilloscope</td><td>Agilent</td><td>DSO3062A</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>VWR</td><td>21008-178</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>VWR</td><td>21008-216</td><td></td></tr><tr><td>T75 culture flask</td><td>VWR</td><td>82050-862</td><td></td></tr><tr><td>Dextran, Tetramethylrhodamine, 3,000 MW, Anionic</td><td>Gibco, Life Technologies</td><td>D3307</td><td></td></tr><tr><td>Dextran, Tetramethylrhodamine, 70,000 MW, Neutral&amp;nbsp;</td><td>Gibco, Life Technologies</td><td>D1819</td><td></td></tr><tr><td>Dextran, Texas Red, 3,000 MW, Neutral</td><td>Gibco, Life Technologies</td><td>D3329</td><td></td></tr></tbody></table>

microscale vortex-assisted electroporator for sequential molecular delivery

电越来越受到重视，在过去几年中，因为它是一个非常强大的技术引入物理不可穿透物外源性分子探针进入细胞。这项工作汇报微流体电平台，能够执行多种分子运送到哺乳动物细胞具有精确和分子有关的参数控制。该系统的分离的细胞具有均匀尺寸分布的能力允许在每个给定的电场强度电穿孔效率的变化少;因此，提高样品的可行性。此外，它的过程可视化特征允许观察在实时荧光分子的吸收过程，其允许迅速分子递送参数的调整在原位对提高效率的。来显示报告平台的广大的能力，大分子具有不同尺寸和电荷的（ 例如，右旋糖酐具有3000和70000沓MW）为传送到转移性乳腺癌细胞具有高输送效率（&gt; 70％）对所有测试的分子。该开发平台已经证明了自己的潜力，在研究领域，其中片内电穿孔技术是有益的扩展使用。

所开发的并行微电穿孔交付大分子具有不同尺寸和电荷为生活转移性乳腺癌细胞。成功的分子递送定性通过监测电穿孔的轨道细胞原位的荧光强度来确定，并通过流式细胞仪分析定量测量证实， 图4A显示了90％的经处理的细胞摄取的70000沓中性葡聚糖。对于统计分析，对每个荧光强度阈值的建立，使得未处理的活细胞中，大部分（&gt; 99％）被计数低于阈值（参见图4 的（c））。的效率被定义为单元的数目之比成功占用感兴趣的分子来处理的细胞的总数。 图4B示出了效率基本上不取决于分子重量或电荷（P&gt; 0.1）。所有被测试的葡聚糖分子被递送到与效率大于70％的细胞溶胶。此外， 图4D显示出已成功用56％的使用阴离子和中性葡聚糖具有相同的分子量（MW = 3000道尔顿）的双分子递送效率进行连续的分子传递。比以前报道的系统18中的当前系统能够处理的细胞具有10倍更高的吞吐量和多分子递送效率和（，82％和70％的阴离子和中性葡聚糖分别）这种改进并不影响单分子递送。 <img alt="图1" src="/files/ftp_upload/51702/51702fig1highres.jpg" /> 图一，（一）微流控电系统的原理图，包括入口的细胞（记为C），分子（表示为M1和M2）和冲洗SOLUT离子（记为F）中，两个直的通道，其中惯性聚焦时，10电穿孔室与电极和一个出口（B）溶液交 ​​换，在用1μM的FITC溶液和冲洗溶液（DPBS）入口示范表明活性溶液可以被均匀地注入到腔室的所有阵列的下游。图像的对比度是通过调整查找表（LUT）的增强。比例尺是250微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51702/51702fig1large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/51702/51702fig2highres.jpg" /> 图2的照片用于短脉冲高电压应用的15针电极的方法，包括10个正（+）和5个负电极（ - ），每个正极隔开间隔2mm从负电极，和电子相同极性的ACH电极间距1.35毫米分开。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51702/51702fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/51702/51702fig3highres.jpg" /> 图3示意了实验装置中，由（A）中的流体控制单元，（B）中的微流体电穿孔，和（C）的电气设备。（A）含有具有分子，细胞和清洁的缓冲液的小瓶被分别加压关于使用LabVIEW控制的气动流系统的需求。从加压管形瓶中的溶液通过PEEK管安装有单向阀注入微流体电穿孔仪（B）和（C）的电信号被发送到15针的电极在接触与F在电穿孔步骤中的微流体系统降脂溶液。使用脉冲发生器和电脉冲是由高电压放大器放大到100伏的幅值产生的电脉冲的编程时间。所有应用的电气参数，实时使用示波器监视。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51702/51702fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/51702/51702fig4highres.jpg" /> 图4。代表性的流式细胞仪的数据（A）的MDA-MB-231细胞，成功地拿起了70000大阴离子葡聚糖分子相比，在控制对方的荧光信号（B）的效率为每转葡聚糖分子不表现出显著分子依赖性变化（P&gt; 0.1）（C）流式细胞术对细胞的轮廓，这是不以电穿孔法（对照）治疗的代表性流程。荧光阈值表示分子递送成功从使得来自对照样品的信号被发现低于阈值的数据集（D）流式细胞仪，用于顺序地电穿孔的细胞的数据代表流动。在右侧积仪和荧光条纹图像流绿色，红色和黄色的方块代表从细胞中入土3000大中性葡聚糖只，阴离子葡聚糖只有两者葡聚糖分子，分别为荧光信号。比例尺为100 m。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51702/51702fig4large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery at JoVE.com

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

微流体涡辅助电平台的连续交付的多个分子进入相同的细胞群与精确，独立的剂量控制开发。该系统的大小基于靶细胞纯化步骤前电穿孔辅助以增强分子递送效率和处理的细胞活力。

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

microscale-vortex-assisted-electroporator-for-sequential-molecular

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.5K 次观看.  Harvard University. 微流体涡辅助电平台的连续交付的多个分子进入相同的细胞群与精确，独立的剂量控制开发。该系统的大小基于靶细胞纯化步骤前电穿孔辅助以增强分子递送效率和处理的细胞活力。 

视频: Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.
Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.
Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

以减少液滴表面的相互作用来优化Bioanalytes运输中的数字微流体

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; ...

科研

工程学

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

遗传学

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

基于液滴在微流体装置的被动抽水高速输送系统

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

生物学

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 ...

生物工程

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

一种基于液滴的单链 dna 放大术和微球 pcr 扩增法

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

用于长期无细胞基因表达传导的多层微流体平台

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

免电力，顺序的核酸和蛋白质的分离

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

具有透化检测功能的连续流动微电穿孔系统的制造和操作

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

用于从人尿液中连续分离细胞外囊泡的微流控装置

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

作者聚焦：用于生物反应器集成的不对称场流分离

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

使用分叉 A4F 微流控装置从大体积样品中单步分离细胞外囊泡

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...