作者大大承认PICT IBiSA平台在居里研究所（CNRS UMR144）。这项工作是由来自补贴：欧洲研究理事会AM.LD（Strapacemi 243103），该协会国立倒拉RECHERCHE（ANR-09-PIRI-0027-PCVI），该InnaBiosanté基金会（Micemico）到MP和PM。 LD和紧急救济协调员Strapacemi年轻的研究者授予AM.LD.

重要提示：本协议假定已经被制成含形状所需的微模具。在模具的制备方法的进一步信息已经被发表10。该协议还假定骨髓DCs培养是已知的。 1。芯片制造<ol><li>混合的PDMS油和固化剂在塑料杯中的重量比为10:1。调匀两种化合物。 </li><li>投组合在模具轴承微通道。的总高度应该为0.5-1厘米之间。 </li><li>中1小时，在真空罐钟除去气泡。 </li><li>在模具中硬化的PDMS由在65℃下放置后在烘箱中2小时</li><li>一旦PDMS是在室温下，切断周围的结构一大块用手术刀片和从模具中（ 图1A）剥开。 </li><li>钻孔，其中的细胞会被注入（通常为2毫米）和调整的PDMS用手术刀片，以适应大小的玻璃底培养皿中的迁移进行评估（ 图1B）。在继续之前，使用擦镜纸盘子去除残留的灰尘。这有利于PDMS到在步骤1.10中描述玻璃的结合。 </li><li>通过结构面粘连，剥离胶带清理含有渠道调整的PDMS芯片。 </li><li>超声处理的PDMS片30秒70％乙醇中以除去灰尘和小颗粒的PDMS。吹清洁的空气干他们很快之后。 </li><li>中30秒，在300毫乇通过空气（或氧气）激活的PDMS（结构向上）和培养皿等离子体处理。 </li><li>放置两个活性表面在接触到永久粘PDMS到衬底上。如果需要的话，可以使用金属镊子轻压在PDMS的顶部，迫使聚合物和菜（ 图1C）的玻璃之间的接触。 </li><li>孵育在烤箱芯片在65℃下FO，R 1小时，以加强约束力。 </li></ol> 2。微通道的涂层<ol><li>由空气等离子体激活整个结构在300毫乇，持续1分钟。这将促进液体进入该通道的下一个步骤。 </li><li>快速填充纤连蛋白芯片的入口孔在10μg/ ml的水。其它衬底，如PEG可用于修改细胞黏附到通道壁上。注意使液体扩散在整个结构。这可以通过眼睛的定期光或使用常规的亮视野显微镜检查容易。 注意：在非常小的结构，在这种扩散是困难，在通道入口的液体可以通过至少15分钟内将所述结构在真空罐钟被迫。以验证荧光底物可用于涂料的效率（ 图2）。 </li><li>孵育1小时，在室温下，以允许吸附在纤连蛋白，或任何其它涂层实名词率的信道的壁上。 </li><li>洗了3倍的结构用PBS以除去未结合的基板。 </li><li>洗涤后，进行协议3或4°C的存储芯片为以后使用（24小时最大值）。 </li></ol> 3。电池装入<ol><li>从板块中删除的PBS，并填写与微细胞培养基。让孵育1小时，以饱和的通道与平台。 注：对于涉及药物如分子抑制剂的实验，它是应垫上预温育的渠道与含有药物在合适的浓度的媒介。此外，一些药物往往在PDMS结构从而降低了有效浓度溶解。有些解决方案已经被提出来抵消这一问题11-12。 </li><li>除去浮区议会，并通过与培养液冲洗恢复半贴壁细胞。使用血细胞计数器计数细胞。 注：对于核成像中，赫斯特33342染色可以通过事先取得中在30分钟内用染料cubating 2×10 6细胞在完全培养基200毫微克/毫升。通过离心洗两次持续的协议之前，以去除多余的染料。 </li><li>在5分钟（时间和速度可以不同，这取决于细胞类型）离心所述细胞以300×g离心并弃去培养基。稀释沉淀，达到20×10 6细胞/ ml的浓度。 </li><li>从板块中删除多余的介质，并使用微量清空PDMS结构的入口孔。填写中的条目与5微升的细胞溶液，以达到在每个条目中孔1×10 5个细胞的量。 注：高细胞密度必须有利于细胞与渠道的接触。低细胞密度可能导致内部通道和实验失败的细胞数量低。 </li><li>孵育微芯片进行30分钟，在37℃的培养箱中培养。加入2 ml完全培养基的实验菜。 注意：整个PDMS结构必须bë覆盖，以避免在实验过程中的细胞干燥。如果2毫升是不够的，添加更多的介质来完全覆盖PDMS结构。 </li></ol> 4。成像<ol><li>把板在显微镜下才清洁与镜头清洁纸餐具外底面。 </li><li>来分析大量细胞的迁移，可使用10倍放大倍率和宽视场照明，在CO 2和温度搭载视频显微镜（ 图3）。以促进细胞的跟踪，Hoechst染色和UV光，可以使用（参见步骤3.2）。移民可以同时分析利用共聚焦显微镜才能迁移过程中获得更高分辨率的细胞结构。 </li><li>对于时间推移显微镜在10X，选择根据预期的细胞的速度（一般为2分钟为树突状细胞与5微米/分钟的速度迁移）时间频率。 注：这里所描述的协议确实ÑOT包括细胞迁移参数的分析。有几点列在讨论建议读者就如何着手从定时短片中提取信息。 </li></ol>

<ol>
	<li>Lauffenburger, D. A., Horwitz, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+a+physically+integrated+molecular+process.">Cell migration: a physically integrated molecular process.</a> Cell. 84 (3), 359-369 (1996).</li><li>L&#228;mmermann, T., 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=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Schor, S. L., Allen, T. D., Harrison, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+through+three-dimensional+gels+of+native+collagen+fibres:+collagenolytic+activity+is+not+required+for+the+migration+of+two+permanent+cell+lines.">Cell migration through three-dimensional gels of native collagen fibres: collagenolytic activity is not required for the migration of two permanent cell lines.</a> J. Cell. Sci. 41, 159-175 (1980).</li><li>Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decisions+about+dendritic+cells:+past,+present,+and+future.">Decisions about dendritic cells: past, present, and future.</a> Annu. Rev. Immunol. 30, 1-22 (2012).</li><li>Faure-Andr&#233;, 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=Regulation+of+dendritic+cell+migration+by+CD74,+the+MHC+class+II-associated+invariant+chain.">Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain.</a> Science. 322 (5908), 1705-1710 (2008).</li><li>Renkawitz, 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=Adaptive+force+transmission+in+amoeboid+cell+migration.">Adaptive force transmission in amoeboid cell migration.</a> Nat. Cell Biol. 11 (12), 1438-1443 (2008).</li><li>Jacobelli, 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=Confinement-optimized+three-dimensional+T+cell+amoeboid+motility+is+modulated+via+myosin+IIA-regulated+adhesions.">Confinement-optimized three-dimensional T cell amoeboid motility is modulated via myosin IIA-regulated adhesions.</a> Nat. Immunol. 11 (10), 953-961 (2010).</li><li>Irimia, D., Charras, G., Agrawal, N., Mitchinson, T., 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=Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels.">Polar stimulation and constrained cell migration in microfluidic channels.</a> Lab Chip. 7 (12), 1783-1790 (2007).</li><li>Moreau, H., 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=Dynamic+in+situ+cytometry+uncovers+T+cell+receptor+signaling+during+immunological+synapses+and+kinapses+in+vivo.">Dynamic in situ cytometry uncovers T cell receptor signaling during immunological synapses and kinapses in vivo.</a> Immunity. 37 (2), 351-363 (2012).</li><li>Heuz&#233;, M. L., Collin, O., Terriac, E., Lennon-Dum&#233;nil, A. M., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+in+confinement:+a+micro-channel-based+assay.">Cell migration in confinement: a micro-channel-based assay.</a> Methods Mol. Biol. 769, 415-434 (2011).</li><li>Ren, K., Zhao, Y., Su, J., Ryan, D., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convenient+method+for+modifying+poly(dimethylsiloxane)+to+be+airtight+and+resistive+against+absorption+of+small+molecules.">Convenient method for modifying poly(dimethylsiloxane) to be airtight and resistive against absorption of small molecules.</a> Anal. 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作者什么都没有透露。

这里，我们描述了微通道构成，以研究大量的细胞中单个实验的迁移特性的方法的设备。该实验系统由模拟内源性游走细胞在组织中发现的密闭环境的制约。然而，通过强制迁移在单个维度，它有利于细胞自动跟踪和衡量指标的提取（ 图5）。我们还表明，我们的设备是用荧光显微镜兼容的，因此可以适用于细胞运动的不同阶段（ 图4）来研究细胞器的定位。 <p class="jo...

迁移是一个复杂的细胞功能，是许多生理过程中的多细胞生物体，包括发育，免疫应答，和组织再生很重要。此外，某些病理情况下，如肿瘤的侵袭和转移依赖于细胞运动1。由于这些原因，细胞迁移已成为研究的一个主要领域中的基本和平移研究的背景下， 在体内 ，大部分组织的特征在于丰富的细胞外基质和高细胞密度。细胞迁移，因此，在生理条件下，发生在一个复杂的密闭环境中。古典的，很有可能是由于历史的原因和技术的限制，细胞迁移进行了研究，2D平面系统不重现许多组织，如坐月子中发现的环境属性。此外，因素如细胞粘附，这对于运动中的2D必不可少的，最近已表明，以不necessarily需要在体内或内部凝胶迁移，这表明该规则的运动细胞在二维和在其他环境中的机制是不同的2。几个系统已经发展到模仿的组织，最有名的是胶原蛋白凝胶，在扼要的细胞外基质成分3的性质，目的的复杂性。在这里，我们提出了微通道作为一个简单的补充方法，它允许一个密闭的环境下，在一个维度研究细胞迁移。 在这个系统中的细胞沿微通道进入，他们自发地进入迁移。迁移细胞然后获得的信道的形状，采用管状的几何形状，最有可能增强其极性。该细胞在信道的线性运动使自动跟踪单元和从实验定量参数的提取。从技术角度来看，这个系统很容易和灵活。该金属 -克的通道壁可以被操纵，所述通道的尺寸和形状可以适应，并有大量的细胞可以在单一实验中进行分析。该系统还可以按比例放大进行参与细胞运动的分子中程屏幕分析。这里所描述的协议已经使用的树突状细胞（DC）作为细胞模型已经标准化。这些细胞是关键的免疫系统，因为它们参与的特异性免疫反应的引发和维持4。 在体外 ，议会已经显示自发迁移在密闭的环境中，因此，研究细胞运动中的微通道5,6的良好模型。重要的是，该系统可以扩展到分析的任何其他能动的细胞类型如T淋巴细胞，中性粒细胞，或肿瘤细胞7-9的迁移。

<table border="0" cellpadding="0" cellspacing="0" width="947">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:273px;">PolyDimethylSiloxane (PDMS)</td>
			<td style="width:131px;">GE Silicones</td>
			<td style="width:139px;">RTV615</td>
			<td style="width:405px;">Package of 90% silicone base and 10% curing agent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Core sample cutter</td>
			<td>Ted Pella Int.</td>
			<td>Harris Uni-Core&#160;</td>
			<td>Diameter 2,5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass-bottom dish</td>
			<td>WPI</td>
			<td>Fluorodish FD 35-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasonic cleaner</td>
			<td>Branson Ultrasonics</td>
			<td>Branson 200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC 32 G</td>
			<td>For small samples (35 dishes). A bigger version is also available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin from bovine plasma</td>
			<td>Sigma Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PolyLysine grafted PEG (Pll-g-PEG)</td>
			<td>Susos</td>
			<td>PLL(20)-g[3.5]-PEG(5)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y27632</td>
			<td>TOCRIS</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

study of cell migration in microfabricated channels

这里所描述的方法允许在一个维度在封闭条件下细胞迁移的研究。它是基于使用微加工渠道，通过物理约束施加极化表型的细胞。一旦进入通道，细胞只有两种可能：向前或向后移动。这个简化的迁移，其中方向性限制有助于细胞的自动跟踪和量化参数来描述小区移动的提取。这些参数包括运动过程中细胞的速度，改变方向，并暂停。微通道也与使用荧光标记兼容，因此适合在高分辨率细胞迁移过程中，研究细胞内的细胞器和结构的定位。最后，该通道的表面可以用不同的基底进行官能化，允许的信道或haptotaxis的研究的粘合性能的控制。总之，本系统HERE的描述是为了分析大细胞数目的迁移中，其中两个的几何形状和对环境的生化性质受到控制的条件下，方便的独立实验的正常化和可重复性。

在每个实验中的PDMS的表面涂有适于研究的感兴趣的分子， 图2示出了通道涂覆有荧光分子，PLL-G-PEG，前和洗涤（步骤2.4）之后。这样的实验可以在通道中的涂层的均匀性的控制。 后细胞加载，视频显微镜可以执行遵循细胞迁移。 图3A示出的微通道的DC迁移至适当的浓度来跟踪小区的例子。既，相衬和Hoechst染色可以用于此目的（见讨论）。该技术也用?...

Watch this Scientific Journal Video about Study of Cell Migration in Microfabricated Channels at JoVE.com

Study of Cell Migration in Microfabricated Channels

研究细胞的自发迁移的一维密闭微环境的定量方法。此方法采用微加工通道的优势，并可以用来在单个实验中在不同条件下研究了大量的细胞迁移。

研究微加工通道细胞迁移的

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

study-of-cell-migration-in-microfabricated-channels

Research

JoVE Journal

Bioengineering

11.8K 次观看.  Institut Curie. 研究细胞的自发迁移的一维密闭微环境的定量方法。此方法采用微加工通道的优势，并可以用来在单个实验中在不同条件下研究了大量的细胞迁移。 

视频: Study of Cell Migration in Microfabricated Channels

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

同时捕获实时图像在两个发射通道采用了双摄像头发射分光系统：应用到细胞粘附

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

独特的毛细管作用通过微通道骨般的模板可以增强细胞的招募大骨缺损修复

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...