这项工作是由卫生部资助R01 DK091526（JO），美国国家科学基金会0852416（DTE）全国学院和芝加哥糖尿病项目的支持。

1。准备小鼠胰岛<ol><li>解剖C57BL / 6小鼠，并通过胶原酶消化和Ficoll密度梯度分离孤立小岛。 （参见在2,3 JOVE引用的文章）。 </li><li>培养在含有10％FBS，1％青霉素/链霉素，和20mM HEPES在培养皿中的胰岛在RPMI-1640培养基（37℃，5％CO 2）。后分离，培养胰岛实验中使用前24小时。使用1-2天以内的小岛，以确保一致的结果。 </li></ol> 2。使得微流体平台<ol><li>生成的微观结构几何形状的透明的光掩模对每个器件层：1）的入口和出口，2）葡萄糖微流体层与表面灌流室（直径8毫米×3毫米，150微升），3）200微米的膜具有微孔（500微米的直径， 100微米深），以及4）气体的微流体层。 </li><li>为了制造入口和出口层，倒脱气，预混PDMS到为1.5毫米的空白培养皿中并固化在80℃下进行2小时的高度。冲头直径为2毫米的输入/输出端口。 </li><li>到制作葡萄糖微流体层，自旋2 350微米层的SU8-2150，以形成一个单一的700微米的层上形成4中的硅晶片。 <ol><li>通过适当的光掩模暴露紫外光显影后到微通道图案转印到SU8，产生700微米高的主人。 </li><li>倾脱气，预混合的PDMS到这个主到3mm的高度和固化在80℃下搅拌2小时。切这个PDMS塑造一个刀片。 </li><li>冲头直径为2毫米的输入/输出端口以及将直径8毫米的腔室。 </li></ol></li><li>为了制作出200微米的滤膜微孔，旋SU8-2100为100微米。采用同样的紫外线光刻技术，以微孔图案转移到这个100微米高高手。 <ol><li>旋转脱气，预混合的PDMS到这个主，在900 rpm离心30秒并固化在80℃下10分钟。 SPI使用相同的条件下在第一顶呐第二层，导致在含有微孔图案的200微米的硅橡胶膜。 </li><li>通过此膜打孔直径为2毫米的输入/输出端口</li></ol></li><li>为了制造微流体气体层，旋SU8-2100为100微米。采用同样的紫外线光刻技术，以煤气微流体图案转印到这100微米高高手。 <ol><li>倾脱气，预混合的PDMS到这个主到1.5毫米的高度和固化在80℃下搅拌2小时。切割PDMS塑造用刀片。 </li><li>通过此层打孔直径为2毫米的输入/输出端口</li></ol></li><li>粘接的多个层，它们通过清洗用胶带，暴露于电晕放电电弧，然后用手按以下顺序排列的准备。 <ol><li>粘结膜与微孔朝上底部的气体层。 </li><li>债券于微孔膜顶部的葡萄糖微流体。 </li><li>结合进口和出口层Õn个极顶，封装整机装配。 </li><li>通过在顶部加上1千克的重量，并在100℃烘烤3小时加强粘接。 </li></ol></li><li>通过加载的水进入水层中，然后浸没在水中整个设备泄漏测试完成的器件。然后，通过气体层流空气用空注射器。 </li><li>通过将水层，然后冲洗，用PBS缓冲液中，在该装置被储存在4℃直至使用消毒用70％乙醇无泄漏的装置。 </li></ol> 3。微型分配器设置<ol><li>获得2微分配器，5 V和20 V DC电源和数字I / O板卡构建氧气混合和交付安装。 <ol><li>该微分配器“控制线连接到包含驱动单元。 </li><li>驱动程序连接到数字I / O板在对应于Labview的控制端口。 </li><li> 5 V和20 V电源连接到相应的驱动器单向接触TS。 </li><li>数字I / O连接到一台笔记本电脑来执行Labview的代码气体控制（见附录）。 </li></ol></li><li> 1微型分配器连接到氮，而另一个以压缩空气，都以5％的CO 2。设置两种气体为2磅。胡克了掌柜&#39;输出一个丁字路口进入微流体装置前。 </li><li>使用泄漏测试设备，通过循环5-21％的氧气之间的微分配器，同时测量水中的溶解氧与光纤氧传感器（见设备清单及代表性成果）表征氧调制的瞬态响应。 </li></ol> 4。设置在显微镜<ol><li>该装置安装在倒置显微镜加热阶段。 </li><li> 0％和21％的O 2气体连接到设备和校准用的氧传感器。 </li><li>葡萄糖微流体的输出端口连接到馏分收集器。 </li><li>准备克雷布斯Ringer碳酸氢盐缓冲液（KRB）：129 mM氯化钠，5毫米碳酸氢钠3，4.7毫米氯化钾，1.2 mM的KH 2 PO 4，1毫米氯化钙2·2H 2 O，1.2毫米硫酸镁4·7H 2 O，10毫米的HEPES pH值为7.35-7.40 2毫基础葡萄糖和5％FBS中。 </li><li>制备含有2mM和14mM的葡萄糖分别在50ml锥形管中沐浴在37℃水浴中2缓冲溶液。 </li><li>连接蠕动泵绘制缓冲区250微升/分钟进油管。进入微流体装置前流管在37℃烤盘。 </li><li>染色的小岛，在100％的乙醇添加的Fura-2 AM在DMSO和罗丹明〜2毫升的KRB达到5微米最终的染料浓度和2.5微米。 </li><li>拿起胰岛用10微升移液管，在37℃下孵育中的染料30分钟</li><li>通过葡萄糖微通道入口装入约20胰岛到器件中。通过吸缓冲区从插座上回T直拨胰岛进入室他入口。 </li><li>灌注在缓冲器中的胰岛10分钟以洗去多余的染料。 </li></ol> 5。运行同步氧气和葡萄糖刺激<ol><li>递送葡萄糖的刺激脉冲以5分钟的基线由KRB2成立，随后的14 mM的刺激15分钟，然后洗涤15分钟，在KRB2。 </li><li>记录三百八十○分之三百四十零nm激发波长的Fura-2和534发射的罗丹明。 </li><li>每次实验前进行正常的脉冲（14毫米常氧）的测量。 </li><li>对于氧气调制（缺氧/间歇性缺氧），刺激和洗涤步骤和其他步骤没有流动时只适用缓冲流，最大限度地减少对流扰动。 </li><li>收集在1分钟的间隔为酶联免疫吸附胰岛素测定葡萄糖出口废水。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

集成在这个小岛缺氧技术的多模式现在几点注意这里进行故障排除。首先，分离出的胰岛继续降解，在文化瓦解由于来自腺泡细胞消化酶。后胰岛分离标准化的实验来1-2天，因此，在获得一致的结果至关重要。第二，所述水性流缺氧和间歇性缺氧期间被停止，以防止对流的间隙在层流和扩散之间的边界。这似乎限制了胰岛预处理的持续时间。在水通道的气体交换未来整合可以消除这一微小的间隙?...

动态缺氧是很重要的生物学，专为胰岛移植动态缺氧是一种重要的生理，以及在许多生物组织的病理生理参数。变化中的氧，例如，是在血管生成一种强效的发育信号。此外，在缺氧的空间和时间模式调节HIF1-α，像胰腺癌疾病中发挥作用。缺氧也是影响胰岛移植结果的混杂因素。近日，缺氧，或间歇性低氧（IH）的时间上振荡“预处理”小岛1展示了好处。然而，胰岛生理静态和暂态缺氧的影响还没有得到很好的理解或学习，主要是由于缺乏适当的工具来控制胰岛的微环境。 胰岛血管化以及在体内 胰岛是50-400<html> 56;米球形聚集体内分泌细胞，包括β-细胞和α-细胞是负责葡萄糖稳态。当胰岛在血液中，摄取和糖酵解导致ATP的产生，这带来了ATP敏感性钾离子（K ATP）通道，并导致钙离子内流触发胰岛素颗粒的胞吐暴露于刺激的葡萄糖。氧气是很重要的，以驱动此沉重的代谢过程和胰岛素分泌显著由血流量和氧供应的动力除了葡萄糖梯度的影响。胰岛容易在体内内从毛细血管一个单元格的长度执行此葡萄糖-胰岛素反应，因为它们是高度灌注胰腺，每个。然而，密集的网络intraislet毛细血管被胶原酶期间胰岛分离2,3移除。因此，氧气和养分供应被约束到一个100微米的周长由于扩散限制。 在重建胰岛微环境的步骤“&gt;目前的技术已经成功的限制重建胰岛的原生氧和葡萄糖动力学，关键建模的生理和病理生理条件下，很难达到与需要复杂的流量和缺乏连续监测的胰岛功能的标准低氧室中。此外，移植治疗I型糖尿病露出分离胰岛缺氧在肝门系统4相比，生理胰腺（5.6％，40毫米汞柱），其具有低得多的氧分压pO 2（&lt;2％，5〜15毫米汞柱）。移植后的胰岛移植需要两个星期或更长时间来吻合血管。它已被证明，低氧损害胰岛的葡萄糖 - 胰岛素偶联机制。其中刺激分泌耦合系数，钙信号，线粒体潜力和胰岛素动力学可以很容易地监测使用微流体。我们以前的微流体技术证明了这一点再人的实时监控与周围单个胰岛5,6水微环境的精确调节。然而，胰岛的缺氧损伤的定量是由缺乏同时刺激和监测技术的阻碍。因此，结合氧和胰岛监测微流体控制可以改善胰岛缺氧的研究。 微流控可以重新和调节水和氧微环境该标准技术组织和文化缺氧的研究是基于对缺氧室。在一般情况下，低氧室提供单独的氧浓度与平衡时间在〜10-30分钟，用分钟缩放动态缺氧不相容。最近的两项研究中使用自定义的小室用于对全小鼠间歇低氧暴露，对葡萄糖诱导的胰岛素反应7,8相互矛盾的结果。请记住，在整体动物水平，呼出氧气不能直接转录定于胰岛毛细血管氧分压 ，由于呼吸系统的控制。此外，这些研究并没有标准化的氧含量，也没有提供实时措施胰岛的组织水平。 另一方面，氧气微流体可以通过经由气体通道网络控制氧超过这些限制。此外，微流体与实时成像兼容的过程中氧气的调制，一个壮举目前无法与标准缺氧室。许多这些新的微流体方法利用聚二甲基硅氧烷的透气性溶解的氧气浓度成流媒体在靶细胞9-14微通道。这些器件还集成了多个分立的氧浓度，基于荧光氧传感器，甚至化学氧代芯片上。 液体溶剂化 - 基于微流控也很难保持稳定的，连续的梯度，因为我T依赖于对流混合是敏感的流动条件。相比较而言，我们在这里使用的技术专注于降低氧输送的扩散路径。气体溶剂化和剪切流是通过直接扩散穿过接种细胞或胰岛组织膜的氧消除。这消除控制溶剂化需要额外的微流体和防止不必要的剪切应力的小岛，它本身可以引发胰岛素的释放。该平台已经被用来证明活性氧（ROS）的上调在两个高氧和缺氧的极端（2-97％O 2）在细胞培养1,15。因为直接运送氧气和去除剪切流动的，我们的扩散型平台为研究胰岛缺氧的最佳微流体解决方案。 多式联运刺激和监控基于扩散的微流体时改编为研究胰岛英里也带来了额外的好处crophysiology。通过使用膜作为扩散阻挡层，所述液体可从氧的调制进行分离，使之含水葡萄糖刺激的控制独立地选自缺氧的刺激。这将创建一个类似三明治的同时刺激在空间上针点送货到小岛。此外，由于气体经由计算机化microinjectors时间上调制的，我们可以从21-0％数字短暂时间小于60秒的调节氧气浓度。中的氧和葡萄糖的微环境在显微镜的动态控制允许实时多峰协议使用标准的低氧室那将是不可能的或极其麻烦的。使用这种装置，钙信号（的Fura-AM），线粒体电位（罗丹明123），和胰岛素动力学（ELISA）进行了监测，以提供在缺氧条件下的动态葡萄糖 - 胰岛素反应的全貌。

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			<td></td>
			<td>Reagent/Material</td>
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		<tr>
			<td>Spinner</td>
			<td>Laurell</td>
			<td>WS-400</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8</td>
			<td>MicroChem</td>
			<td>SU8-2150/SU8-2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Hotplate</td>
			<td>PMC Dataplate</td>
			<td>722A</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Curing Lamp</td>
			<td>OmniCure</td>
			<td>S1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PMDS</td>
			<td>Dow Chemical</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Corona Wand</td>
			<td>ETP</td>
			<td>BD-20AC</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Chamber</td>
			<td>Bel-Art</td>
			<td>420220000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdispensers</td>
			<td>The Lee Company</td>
			<td>IKTX0322000A</td>
			<td></td>
		</tr>
		<tr>
			<td>5 V and 20 V DC Power</td>
			<td>Radio Shack</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NI USB</td>
			<td>National Instrument</td>
			<td>NI USB-6501</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Omega Engineering, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Gilson</td>
			<td>Minipulse 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen Sensor</td>
			<td>Ocean Optics</td>
			<td>NeoFox</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Collector</td>
			<td>Gilson</td>
			<td>203</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippette</td>
			<td>Fisher Scientific</td>
			<td>Finnpipette II 100&#956;l</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Epifluorescence Microscope</td>
			<td>Leica</td>
			<td>DMI 4000B</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Conical Tubes</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2 Fluorescence Dye</td>
			<td>Molecular Probes, Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine 123 Fluorescence Dye</td>
			<td>Molecular Probes, Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Media</td>
			<td>Sigma-Aldrich</td>
			<td>RPMI-1640</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30 in Silicone Tubings</td>
			<td>Cole-Parmer</td>
			<td></td>
			<td>1/16 in x 1/8 in</td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf Tubes</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Y-connectors</td>
			<td>Cole-Parmer</td>
			<td></td>
			<td>1/16 in and 4 mm</td>
		</tr>
		<tr>
			<td>Syringe Connectors</td>
			<td>Cole-Parmer</td>
			<td></td>
			<td>female Luer plug 1/16 in</td>
		</tr>
		<tr>
			<td>Straight Connectors</td>
			<td>Cole-Parmer</td>
			<td></td>
			<td>1/16 in</td>
		</tr>
		<tr>
			<td>Elbow Connector</td>
			<td>Cole-Parmer</td>
			<td></td>
			<td>1/16 in</td>
		</tr>
	</tbody>
</table>

quantitative and temporal control of oxygen microenvironment at the single islet level

同时氧化葡萄糖刺激分泌耦合系数在一个单一的技术和监测是模拟胰岛缺氧的病理生理状态的关键，尤其是在移植环境。同时标准的缺氧室技术无法调节两个刺激，也不提供葡萄糖刺激分泌耦合系数的实时监控。为了解决这些问题，我们应用了多层微流控技术，通过扩散膜水性和气相调制集成。这将创建在周围的透明聚二甲基硅氧烷（PDMS）设备的microscaled胰岛刺激夹层，可以监控通过荧光显微镜前述的耦合因素。此外，气体输入由一对微分配器的控制，提供了定量，分分钟之间0-21％氧气的调制。这种间歇性缺氧是适用于调查小岛的一个新现象吨预处理。此外，手持多式联运显微镜，我们能够在这些缺氧事件来看看详细的钙和K ATP通道的动态。我们设想微缺氧，特别是这个同步双相位技术，如胰岛细胞研究以及许多离体组织的重要工具。

中央到此胰岛缺氧的方法是调节水相和气相的刺激在相同微流体腔室与微小尺度瞬变的能力。 图1是一个）的有代表性的结果胰岛室中测量的双重刺激和b）快速调制。水调制，通过引入荧光素所显示的腔，实现了在三到四分钟混合平衡。此外，氧可以从5-21％会加强与快速瞬变，使氧气与循环周期短至2分钟。不同循环深度和频率也可以实现为如图2所示。 <p class="jove_cont...

Watch this Scientific Journal Video about Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level at JoVE.com

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

微流控氧赋予的不仅仅是方便和速度超过低氧室的生物实验。通过扩散穿过膜实现的，特别是当，微氧可以在微尺度级同时提供液相和气相的调制。这项技术使动态多参数实验的关键学习islet病理生理学。

氧微环境中的单个胰岛水平的定量和临时控制

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

quantitative-temporal-control-oxygen-microenvironment-at-single-islet

Research

JoVE Journal

Bioengineering

9.1K 次观看.  University of Michigan-Dearborn. 微流控氧赋予的不仅仅是方便和速度超过低氧室的生物实验。通过扩散穿过膜实现的，特别是当，微氧可以在微尺度级同时提供液相和气相的调制。这项技术使动态多参数实验的关键学习islet病理生理学。 

视频: Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

一个单粒子为基础的微流控液力陷阱

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

科研

生物工程

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

在活细胞中的小GTPase活性时空操纵在亚细胞水平上几秒钟的时间刻度

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

工程双层凝胶控制升序分化

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

单个RNA分子的光镊纳米操作

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&amp;#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

用的表面图案化微流体系统用于在单细胞水平调查空泡（S） - 细胞的相互作用及产生的生物效应

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

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 ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.