A.梯度产生的微流体装置微细<ol><li>硅晶片被视为与活性氧离子（Harrick科学30W，5分钟，NY）。 </li><li>负型光刻胶（SU - 8 50，Microchem，MA）是在一个硅晶片上的1分钟1000转的旋涂。 </li><li>晶圆是软的烤在65 ° C为10分钟，随后在95 ° C为30分钟一个电磁炉。 </li><li>晶圆暴露在紫外线灯（200W），通过一个30微米的最小特征尺寸的透明度面具3分钟。 </li><li>晶圆是出炉后在65 ° C为1分，在95 ° C为10分钟。 </li><li>四掌握模具100微米厚的渠道是开发利用SU - 8光刻胶开发。 </li><li>含微晶圆被放置在一个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>泗母模的PDMS混合物倒入。 </li><li>四母模是放在真空干燥器除去10分钟的气泡。 </li><li> PDMS的是固化在70 ° C，1〜2小时。 </li><li>从四母模的PDMS模具揭下。 </li></ol> B.实验装置<ol><li>细胞的入口，出口，并注入进气口的PDMS微设备使用锋利打孔器打孔。 </li><li>是不可逆转的保税的活性氧等离子（Harrick科学30W，5分钟，纽约州）的设备和载玻片（2 × 3英寸）。 </li><li>聚乙烯管（PE 20，流式细胞Dickinon，MD）被插入到微流体装置注入进气口，随后连接到一个注射器泵。 </li><li>异硫氰酸荧光素（FITC）-葡聚糖（MW = 10kD，10μM，Sigma公司）和缓冲液（PBS，Invitrogen公司，CA）注入到微流体装置内的流体设备，以确认稳定的梯度。 </li><li>培养箱中培养1小时（37℃）的微流体装置内涂外基质（ECM）（即纤维连接蛋白）。 </li><li>的NIH 3T3成纤维细胞，胰酶消化分离。 </li><li>游离细胞被加载到微流体装置（宽800微米），在细胞密度2 × 10 6细胞/ ml 。 </li><li>注入2毫升媒体和50 ng / mL表皮生长因子（EGF）是一个微流体装置，使用注射泵（0.05微升/分钟）生成可溶性梯度。 </li><li>细胞实时监测每5分钟，通过倒置显微镜（尼康TE的2000）。 </li></ol>

<ol>
	<li>Jeon, N. L., Baskaran, H., Dertinger, S. K. W., Whitesides, G. M., Van de Water, 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=Neutrophil+chemotaxis+in+linear+and+complex+gradients+of+interleukin-8+formed+in+a+microfabricated+device.">Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device.</a> Nat. Biotechnol. 20, 826-830 (2002).</li><li>Lin, F., Nguyen, C. M., Wang, S. J., Saadi, W., Gross, S. P., 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=Effective+neutrophil+chemotaxis+is+strongly+influenced+by+mean+IL-8+concentration.">Effective neutrophil chemotaxis is strongly influenced by mean IL-8 concentration.</a> Biochem. Biophys. Res. Commun. 319, 576-581 (2004).</li><li>Chung, B. G., Flanagan, L. A., Rhee, S. W., Schwartz, P. H., Lee, A. P., 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=Human+neural+stem+cell+growth+and+differentiation+in+a+gradient-generating+microfluidic+device.">Human neural stem cell growth and differentiation in a gradient-generating microfluidic device.</a> Lab Chip. 5, 401-406 (2005).</li><li>Saadi, W., Wang, S. J., Lin, F., 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+parallel-gradient+microfluidic+chamber+for+quantitative+analysis+of+breast+cancer+cell+chemotaxis.">A parallel-gradient microfluidic chamber for quantitative analysis of breast cancer cell chemotaxis.</a> Biomed. Microdevices. 8, 109-118 (2007).</li><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></ol>

细胞暴露于稳定的表皮生长因子的浓度梯度，在向着更高浓度的迁移一个微流体装置。细胞跟踪分析，细胞迁移，趋化指数，细胞迁移运动的方向定位进行了调查。因此，该梯度产生的微流体平台可用于研究癌细胞转移，胚胎发育和轴突导向。

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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>Dextran-FITC</td>
<td>Reagent</td>
<td>Sigma Aldrich</td>
<td>FD10S </td>
<td>Fluorescein isothiocyanate (FITC) conjugated-dextran (10kD)</td>
</tr>
<tr>
<td>hr-EGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13247-051 </td>
<td>human recombinant Epidermal growth factor </td>
</tr>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>K.R. Anderson Co. Inc.</td>
<td>2065622 </td>
<td>Poly(dimethylsiloxane) (PDMS), Dow Corning Sylgard 184 (8.6 lb)</td>
</tr>
<tr>
<td>Negative photoresist </td>
<td>&nbsp;</td>
<td>Microchem</td>
<td>SU-8 50</td>
<td>&nbsp;</td>
<tr>
<td>Si wafer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>silicone wafer, 4 inch</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene tubing </td>
<td>&nbsp;</td>
<td>Becton Dickinon</td>
<td>PE 20</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 </td>
<td>cell-line</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>fibroblast cells</td>
</tr>
<tr>
<td>Inverted microscope</td>
<td>&nbsp;</td>
<td>Nikon </td>
<td>TE 2000</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
					</div>

a gradient-generating microfluidic device for cell biology

的制造和研究细胞行为的一个梯度产生的微流体装置的操作说明。微流体平台是一个有利的实验工具，因为它可以精确地操纵流体流动，使高通量的实验，并产生稳定的水溶性的浓度梯度。相比传统的梯度发生器，聚二甲基硅氧烷（PDMS）的基于微流体装置，可以产生生长因子具有良好定义的配置文件稳定的浓度梯度。在这里，我们开发了三个独立的进气口简单的梯度产生的微流体装置。三微合并成一个微产生浓度梯度。 isothyiocyanate荧光素（FITC）-葡聚糖的分子量类似表皮生长因子（EGF），生长因子梯度的稳定和形状被证实。我们表明，使用这种微流体装置，暴露表皮生长因子的浓度梯度成纤维细胞对高浓度的迁移。细胞迁移和细胞迁移运动的方向定位，定量评估细胞跟踪分析。因此，该梯度产生的微流体装置，可能是有益的研究和分析细胞迁移的行为。

Watch this Scientific Journal Video about A Gradient-generating Microfluidic Device for Cell Biology at JoVE.com

A Gradient-generating Microfluidic Device for Cell Biology

我们描述了一个协议梯度产生的微流体装置，可以产生良好定义的微环境中的空间和时间的梯度微细。在这种方法中，梯度产生的微流体装置可用于研究细胞定向迁移，胚胎发育，伤口愈合，癌症转移。

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 ...

a-gradient-generating-microfluidic-device-for-cell-biology

Research

JoVE Journal

Biology

15.2K 次观看.  Brigham and Women's Hospital. 我们描述了一个协议梯度产生的微流体装置，可以产生良好定义的微环境中的空间和时间的梯度微细。在这种方法中，梯度产生的微流体装置可用于研究细胞定向迁移，胚胎发育，伤口愈合，癌症转移。

视频: A Gradient-generating Microfluidic Device for Cell Biology

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 Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

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 ...

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process 1. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port 2. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.

We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.

脑片的刺激，利用微流控网络和标准灌注腔

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

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 ...

Micro-scale Engineering for Cell Biology

细胞生物学的微观尺度工程

Cell Capture Using a Microfluidic Device

细胞捕获使用一个微流体装置

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

PDMS微流体装置的廉价制造的非等离子键合

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

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 ...

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

微接触印刷的蛋白细胞生物学

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

微探针：操作和使用的局部表面处理

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...