Name: quantitative and temporal control of oxygen microenvironment at the single islet level
Uploaded: 2013-11-17T14:00:00
Description: Watch this Scientific Journal Video about Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level at JoVE.com

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?...

<ol>
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T., Lee, D., Oberholzer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multi-Parametric+Islet+Perifusion+System+within+a+Microfluidic+Perifusion+Device.">A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device.</a> J. Vis. Exp.. , e1649 (2010).</li><li>Mohammed, J. S., Wang, Y., Harvat, T. A., Oberholzer, J., Eddington, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+device+for+multimodal+characterization+of+pancreatic+islets.">Microfluidic device for multimodal characterization of pancreatic islets.</a> Lab Chip. 9, 97-106 (2009).</li><li>Carreras, A., Kayali, F., Zhang, J., Hirotsu, C., Wang, Y., Gozal, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Effects+Of+Intermittent+Hypoxia+In+Mice:+Steady+Versus+High+Frequency+Applied+Hypoxia+Daily+During+The+Rest+Period.">Metabolic Effects Of Intermittent Hypoxia In Mice: Steady Versus High Frequency Applied Hypoxia Daily During The Rest Period.</a> AJP - Regu Physiol. 303 (7), 700-709 (2012).</li><li>Lee, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+changes+in+glucose+and+insulin+regulation+during+intermittent+hypoxia+and+continuous+hypoxia.">Time-dependent changes in glucose and insulin regulation during intermittent hypoxia and continuous hypoxia.</a> Eur. J. Appl. Physiol. , (2012).</li><li>Kane, B. J., Zinner, M. J., Yarmush, M. L., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver-specific+functional+studies+in+a+microfluidic+array+of+primary+mammalian+hepatocytes.">Liver-specific functional studies in a microfluidic array of primary mammalian hepatocytes.</a> Anal. Chem. 78, 4291-4298 (2006).</li><li>Lam, R. H. W., Kim, M. C., Thorsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+aerobic+and+anaerobic+bacteria+and+mammalian+cells+with+a+microfluidic+differential+oxygenator.">Culturing aerobic and anaerobic bacteria and mammalian cells with a microfluidic differential oxygenator.</a> Anal. Chem. 81, 5918-5924 (2009).</li><li>Polinkovsky, M., Gutierrez, E., Levchenko, A., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+temporal+control+of+the+medium+gas+content+and+acidity+and+on-chip+generation+of+series+of+oxygen+concentrations+for+cell+cultures.">Fine temporal control of the medium gas content and acidity and on-chip generation of series of oxygen concentrations for cell cultures.</a> Lab Chip. 9, 1073-1084 (2009).</li><li>Mehta, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+and+control+of+oxygen+levels+in+microfluidic+poly(dimethylsiloxane)+bioreactors+during+cell+culture.">Quantitative measurement and control of oxygen levels in microfluidic poly(dimethylsiloxane) bioreactors during cell culture.</a> Biomed. Microdev. 9 (2), 123-134 (2007).</li><li>Vollmer, A. P., Probstein, R. F., Gilbert, R., Thorsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+integrated+microfluidic+platform+for+dynamic+oxygen+sensing+and+delivery+in+a+flowing+medium.">Development of an integrated microfluidic platform for dynamic oxygen sensing and delivery in a flowing medium.</a> Lab Chip. 5, 1059-1066 (2005).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+oxygen+gradients+in+microfluidic+devices+for+cell+culture+using+spatially+confined+chemical+reactions.">Generation of oxygen gradients in microfluidic devices for cell culture using spatially confined chemical reactions.</a> Lab Chip. 11, 3626-3633 (2011).</li><li>Lo, J. F., Sinkala, E., Eddington, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+gradients+for+open+well+cellular+cultures+via+microfluidic+substrates.">Oxygen gradients for open well cellular cultures via microfluidic substrates.</a> Lab Chip. 10, 2394-2401 (2010).</li></ol>

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。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		</tr>
		<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 ...

Research

JoVE Journal

Bioengineering

A Microfluidic-based Hydrodynamic Trap for Single Particles

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

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

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

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

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

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

A Microfluidic System with Surface Patterning for Investigating Cavitation BubblesÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œCell Interaction and the Resultant Bioeffects at the Single-cell Level

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

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

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 ...