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?...

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 border="0" cellpadding="0" cellspacing="0" width="1056">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:475px;">MDA-MB-231 cancer cell line</td>
			<td style="width:297px;">American Type Culture Collection (ATCC)</td>
			<td style="width:119px;">HTB-26</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Leibovitz&#8217;s L-15 Medium</td>
			<td style="width:297px;">Cellgro, Mediatech, Inc.</td>
			<td style="width:119px;">10-045-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">fetal bovine serum (FBS)</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">penicillin-streptomycin</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4333</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:475px;">Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td style="width:297px;">Cellgro, Mediatech, Inc.</td>
			<td style="width:119px;">21-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Trypsin</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Flow Cytometer easyCyte HT</td>
			<td style="width:297px;">Millipore</td>
			<td style="width:119px;">0500-4008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Oxygen Plasma Cleaner</td>
			<td style="width:297px;">Technics Micro-RIE</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dektak 6M surface profiler</td>
			<td style="width:297px;">Veeco</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">KMPR 1050</td>
			<td style="width:297px;">Microchem</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">SYLGARD 184 SILICONE ELASTOMER KIT</td>
			<td style="width:297px;">Dow Corning</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Compressed Nitrogen gas</td>
			<td style="width:297px;">Airgas</td>
			<td style="width:119px;">NI 300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:475px;">High Pressure Regulator</td>
			<td style="width:297px;">McMaster-Carr</td>
			<td>6162K22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Downstream regulator</td>
			<td style="width:297px;">McMaster-Carr</td>
			<td style="width:119px;">4000K563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">high-speed 3/2way-8 valve manifold</td>
			<td style="width:297px;">Festo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Inline Check Valve</td>
			<td style="width:297px;">Idex Health and Science</td>
			<td style="width:119px;">CV3320</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:475px;">5/32&#34; OD x 3/32&#34;ID Polyurethan tubes</td>
			<td style="width:297px;">Pneumadyne</td>
			<td>PU-156F-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">1/4&#34; OD X 0.17&#34; ID Polyurethan tubes</td>
			<td style="width:297px;">Pneumadyne</td>
			<td style="width:119px;">PU-250PB-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">1/16&#34; PEEK tubings</td>
			<td style="width:297px;">Festo</td>
			<td style="width:119px;">P1533</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">1/32&#34; PEEK tubings</td>
			<td style="width:297px;">Idex Health and Science</td>
			<td style="width:119px;">P1569</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">PEEK tubing unions</td>
			<td style="width:297px;">Idex Health and Science</td>
			<td style="width:119px;">P881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Pulse Generator</td>
			<td style="width:297px;">HP</td>
			<td style="width:119px;">8110A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Aluiminum Wire</td>
			<td style="width:297px;">Bob Martin Company</td>
			<td style="width:119px;">6061 ALUM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">oscilloscope</td>
			<td style="width:297px;">Agilent</td>
			<td style="width:119px;">DSO3062A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">50 mL centrifuge tubes</td>
			<td style="width:297px;">VWR</td>
			<td style="width:119px;">21008-178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">15 mL centrifuge tube</td>
			<td style="width:297px;">VWR</td>
			<td style="width:119px;">21008-216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">T75 culture flask</td>
			<td style="width:297px;">VWR</td>
			<td style="width:119px;">82050-862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dextran, Tetramethylrhodamine, 3000 MW, Anionic</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">D3307</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dextran, Tetramethylrhodamine, 70,000 MW, Neutral&#160;</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">D1819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Dextran, Texas Red, 3000 MW, Neutral</td>
			<td style="width:297px;">Gibco, Life Technologies</td>
			<td style="width:119px;">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?...

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

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

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

科研

生物工程

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

皮肤纹身作为一种新的方法DNA疫苗交货

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

阿普利西亚 单一神经元电生理学和分子分析的甘利亚制备

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

多孔硅微粒进行交付的siRNA治疗的

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

由微型条纹分析技术在微流控设备混合不均匀

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

微型涡辅助电穿孔的顺序传递分子

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Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

由微型条纹分析技术在微流控设备混合不均匀

适应静电过程中提供三个独特的环境为一个三层次

聚电解质复合物肝素结合域成骨生长因子交货

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

微型涡辅助电穿孔的顺序传递分子

摘要

探索更多视频

此视频中的章节

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

皮肤纹身作为一种新的方法DNA疫苗交货

自我报告的支架3维细胞培养

阿普利西亚 单一神经元电生理学和分子分析的甘利亚制备

多孔硅微粒进行交付的siRNA治疗的

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

由微型条纹分析技术在微流控设备混合不均匀

适应静电过程中提供三个独特的环境为一个三层次

聚电解质复合物肝素结合域成骨生长因子交货

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

阿普利西亚单一神经元电生理学和分子分析的甘利亚制备