A.微流体装置的微细加工<ol><li> 4英寸硅片的处理与活性氧离子（Harrick科学30W，5分钟，NY）。 </li><li>负型光刻胶（SU - 8 2015年，Microchem，MA）是在一个硅晶片上的1分钟900转的旋涂。 </li><li>晶圆是软烤95℃，在烤盘6分钟，通过一个面罩含微膜4分钟，以紫外灯（200W）。 </li><li>晶圆是出炉后在95℃，6分钟和开发利用SU - 8光刻胶开发。 </li><li>含微的光致抗蚀剂图形晶片被放置在一个Petri盘。 </li><li>聚二甲基硅氧烷（PDMS）中（Sylgard 184）模具制造混合的有机硅弹性体和固化剂（10:1）。 </li><li> PDMS的混合物倒入四母模，放在真空干燥器，以消除泡沫。 </li><li> PDMS的是固化在70 ° C，1〜2小时。 </li><li>硅橡胶模具，然后从四母模揭下。 </li></ol> B.组装设备<ol><li>从两个不同的四掌握模具获得40微米厚的顶部流体通道和40微米厚的底部microgrooved通道（25，50，75和100μm宽）。 </li><li>通道的入口和流体设备的电源插座是拳打脚踢。 </li><li>流体的渠道和细纹渠道是不可逆的保税的​​活性氧等离子（Harrick科学30W，5分钟，纽约州）。 </li><li>微流体装置内涂外基质（ECM）（即纤维连接蛋白）的培养箱中培养1小时（37℃）。 </li></ol> C.细胞种植和实验装置<ol><li> NIH - 3T3小鼠成纤维细胞培养使用贝科改良Eagle的媒体（DMEM），含10％胎牛血清（FBS），在组织文化烧瓶。 </li><li>细胞胰酶消化分离。 </li><li>游离细胞装入细胞密度3 × 10 6细胞/ ml（图1）细纹渠道。 <img src="/files/ftp_upload/old/270_2008_8_24_3/270_Figure-01.png" alt="图1" width="191" height="300" /> 图1 </li><li>使用注射泵（1μL/分钟）的通道注入2毫升媒体，100毫米的H 2 O 2，检测细胞凋亡（20μLAnnexin V和40μL的碘化丙啶，Invitrogen公司，CA ）。 </li><li>细胞是实时监测，通过倒置显微镜（尼康TE的2000）。 </li></ol>

<ol>
	<li>Chung, B. G., Park, J. W., Hu, J. S., Huang, C., Monuki, E. S., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+microfluidic-vacuum+device+for+interfacing+with+conventional+cell+culture+platform.">A hybrid microfluidic-vacuum device for interfacing with conventional cell culture platform.</a> BMC Biotechnol. 7, (2007).</li><li>Khademhosseini, A., Yeh, J., Eng, G., Karp, J., Kaji, H., Borenstein, J., Farokhzad, O. C., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+docking+inside+microwells+within+reversibly+sealed+microfluidic+channels+for+fabricating+multiphenotype+cell+arrays.">Cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays.</a> Lab Chip. 5, 1380-1386 (2005).</li><li>Whittemore, E. R., Loo, D. T., Watt, J. A., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+analysis+of+hydrogen+peroxide-induced+cell+death+in+primary+neuronal+culture.">A detailed analysis of hydrogen peroxide-induced cell death in primary neuronal culture.</a> Neuroscience. 67, 921-932 (1995).</li></ol>

细胞固定，并在microgrooved基板图案中的微流体装置。暴露在过氧化氢的细胞凋亡过程中实时观察和分析，通过Annexin V和碘化丙啶。因此，这种微流体装置，包含细纹渠道，可用于高通量药物筛选。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PDMS</td>
<td>Reagent</td>
<td>K.R. Anderson Co. Inc. </td>
<td>2065622 </td>
<td>Poly(dimethylsiloxane), Dow Corning Sylgard 184 (8.6 lb)</td>
</tr>
<tr>
<td>DMEM</td>
<td>medium</td>
<td>Invitrogen</td>
<td>11965 </td>
<td>Dulbecco&#x2019;s Modified Eagle&#x2019;s Media </td>
</tr>
<tr>
<td>FBS</td>
<td>serum</td>
<td>Invitrogen</td>
<td>10082-147 </td>
<td>Fetal Bovine Serum
</td>
</tr>
<tr>
<td>Hydrogen peroxide</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td> H1009 </td>
<td>&nbsp;</td>
<tr>
<td>Apoptosis assay</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>V13242 </td>
<td>Annexin A, propidium iodide</td>
</tr>
<tr>
<td>Negative photoresist </td>
<td>&nbsp;</td>
<td>Microchem</td>
<td>SU-8 2015</td>
<td>&nbsp;</td>
<tr>
<td>Si wafer </td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4 inch silicone wafer</td>
</tr>
<tr>
<td>Reactive oxygen plasma </td>
<td>Reagent</td>
<td>Harrick Scientific</td>
<td>&nbsp;</td>
<td>treat wafer 5 min at 30W</td>
</tr>
<tr>
<td>inverted microscope </td>
<td>Tool</td>
<td>Nikon </td>
<td>TE 2000</td>
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					</div>

a microfluidic device with groove patterns for studying cellular behavior

我们描述了一个微流体装置与microgrooved模式，研究细胞行为。这种微流体平台包括一个顶级的流体通道和自下而上microgrooved基板。编造microgrooved渠道，一个顶级的聚二甲基硅氧烷（PDMS）模具包含微流体通道的印象是对齐和粘结到microgrooved基板。使用这种设备，小鼠成纤维细胞被固定在microgrooved基板图案（25，50，75，宽100微米）。为了研究细胞凋亡在微流体装置，介质中含有过氧化氢，膜联蛋白V和碘化丙啶灌注到2小时的流体通道。我们发现，暴露在氧化应激的细胞变得凋亡。通过Annexin V结合在细胞凋亡过程中的质膜外单张磷脂酰证实这些细胞凋亡。 microgrooved模式使用这种微流体装置，细胞凋亡过程中实时观察，并包含一个孵化室（37℃，5％的CO 2）使用倒置显微镜分析。因此，这microgrooved基板中的微流体装置可用于研究细胞行为和进行高通量药物筛选。

Watch this Scientific Journal Video about A Microfluidic Device with Groove Patterns for Studying Cellular Behavior at JoVE.com

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

我们描述了一种用于制造微流体装置，可以使细胞的捕获和文化协议。在这种方法中，如微流体通道内的沟槽图案的微观结构是用来建立低剪切应力区域内的细胞可以停靠。

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

a-microfluidic-device-with-groove-patterns-for-studying-cellular

Research

JoVE Journal

Biology

12.3K 次观看.  Brigham and Women's Hospital. 我们描述了一种用于制造微流体装置，可以使细胞的捕获和文化协议。在这种方法中，如微流体通道内的沟槽图案的微观结构是用来建立低剪切应力区域内的细胞可以停靠。

视频: A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

条块索玛的神经元和轴突的一个微流体设备制造

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

科研

生物学

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

一个渐变产生的微流体装置，细胞生物学

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

准备在微流体装置E18条块皮质大鼠神经元

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Cell Capture Using a Microfluidic Device

细胞捕获使用一个微流体装置

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

使用琼脂糖凝胶为基础的微流体装置的评估神经干细胞的运动

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 &mu;m diameter and 150 &mu;m depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 &mu;m in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

一个多参数的胰岛内的微流控Perifusion装置Perifusion系统

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

窗口上的一个微观世界：简单的微流体系统为研究多孔介质中的微生物运输

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

一个微流体装置量化细菌趋稳定的浓度梯度

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...