This work was supported by the Fritz-Bender-Foundation, Munich, Germany

1，准备迁移商会<ol><li>制备石蜡/凡士林（1:1）的混合和加热，直到混合物熔化。使用油漆刷和绘制2-3层的石蜡/凡士林（1:1）在玻璃载片的中间混合，根据图1B-1D。 注意:我们使用的是普通玻璃载玻片（76 * 26 * 1.0-1.5毫米（W / D / H）） </li><li>迅速涂抹于载玻片上融化的石蜡/凡士林混合，以避免固化而绘制。确保石蜡/石油冻层的长度为约2-2.5厘米和0.3〜0.5厘米的宽度。石蜡/石油凝胶层的厚度应为约0.1〜0.15厘米。 </li><li>加盖玻片，以凝固的石蜡/凡士林混合，并用两到三个层次石蜡/凡士林混合（ 图1E，1F）密封。使用4-8迁移室为一个共同的细胞迁移实验。地方迁移室íN的机架直立位置。 </li></ol> 2，制备胶原蛋白悬浮细胞混合的<ol><li>收获（ 例如 ，用0.25％胰蛋白酶/ EDTA）和计数感兴趣的细胞。将细胞重悬于含胎牛血清（FCS）的完全培养基，抗生素和建议的补充。制备4-81.5毫升反应管和含4-6×10 4个细胞（细胞的总数量取决于细胞的类型来进行分析）20微升细胞悬浮液与填充到50微升用完全培养基。 注:其它化合物（ 例如 ，生长因子，趋化因子，抑制剂等 ）被添加到细胞悬浮液。使用适当的储备溶液，使得细胞悬浮液的最终体积不超过50微升。 </li><li>准备452微升最终胶原蛋白悬浮液总量四细胞迁移实验。加入50μl的10×MEM（pH值5.1-5.5）和27微升7.5％的碳酸氢钠溶液（pH 9.0-9.5）到1.5 ml反应管中调匀。观察从橙黄色，以强烈的紫色（pH值9.0-9.5）溶液颜色转。加入375微升液体胶原蛋白悬浮液的10倍MEM /碳酸氢钠溶液调匀。最终的胶原悬浮液的pH值应为约7.5（由光紫颜色表示）。 注意:检查7.5％碳酸氢钠溶液的pH值。如果pH值约7.5的地方有一个开放的盖子的碳酸氢钠溶液在恒温箱（37℃，5％CO 2）的饱和的CO 2，增加了pH值（约9.0 - 9.5）。的胶原蛋白悬浮液的细胞混合物的pH为胶原网络的稳定性是至关重要的。如果它太低了胶原格将细胞迁移实验过程中坍塌。 注:在本协议中，使用从牛后腿液体胶原蛋白（pH值1.9-2.2）（2.9-3.3毫克/毫升的胶原蛋白，95％的胶原蛋白I型，5​​％的胶原蛋白IV型）。通过进一步的胶原蛋白晶格的制备协议的示例I型胶原，从其他来源，如猪或老鼠，给出了32,33。不要更改使用方案的体积，因为这将改变1.67毫克/毫升的晶格最终胶原蛋白的浓度。较高或较低的胶原浓度对胶原纤维的密度和伴随细胞迁移行为34在它们之间的平均距离的影响。 </li><li>加入100μl最后的胶原蛋白悬浮液50μl的细胞悬浮液，混匀thoroughly.The最后的胶原蛋白悬浮液（1.67毫克/毫升胶原蛋白）稍粘稠。传输的正确量（100微升），将其转移至细胞悬浮液之前吸取最终的胶原溶液一次向上和向下。 注意:一旦胶原悬浮细胞混合物结合的10倍MEM /碳酸氢钠溶液将pH值已经改变到7.5胶原纤维立即开始聚合。 </li><li>传输距离日最后的胶原蛋白悬浮细胞结构ê反应管的迁移室。轻轻拍打迁移室均匀分布的胶原蛋白悬浮细胞结构的迁移室（ 图1H）的底部。将迁移室中在机架直立位置并孵育约30分钟（37℃，5％CO 2），以允许胶原纤维的聚合。 </li><li>请注意，聚合胶原蛋白晶格稍有混浊，但仍是浅紫色。填补迁移室与完全培养基或完全培养基与利益补充剂和密封腔迁移石蜡/凡士林混合（ 图1I，1J）。 </li></ol> 3，录音和细胞迁移分析本节将通过时间推移视频显微镜和细胞迁移分析人工细胞追踪介绍细胞迁移的记录。 <ol><li>交换机上的显微镜和显微镜载物台HEA之三。调整温度至37℃。使用10倍的目标。放置迁移室在显微镜下和在视场聚焦大约50个细胞以上。 </li><li>采用多摄像机视频监控软件应用在延时模式记录细胞的迁移。保存细胞迁移的电影以适当的格式， 例如 ，"* .AVI"或"*。传送"。链接的细胞迁移的电影文件到含有辅助信息，如信元类型和实验条件的数据库。 注意:我们正在录制的淋巴细胞和HSPCs使用的1:80时间推移因子，表示0.75秒中的时间推移等于实时1分钟至2小时。肿瘤细胞是缓慢移动的细胞，并用1时间推移因素因而录得至少16小时:1,800（0.5秒的延时等于实时15分钟）。 </li><li>分析使用适当的细胞追踪应用软件，如ImageJ的软件插件细胞迁移的电影（概述在18给出）。对于一个无偏分析是至关重要的细胞将被随机选择的不知情的细胞是否是迁移活性或没有。 注意:我们使用的是自主研发的应用软件通过鼠标光标（这是定位于细胞核）准确地跟随移动的细胞手动跟踪每个实验至少30个细胞的路径。同时跟踪的跟踪单元的x，y坐标，根据对所用的时间推移模式是自动确定的。淋巴细胞/ HSPCs x，y坐标被确定每0.75秒（等于1分钟实时），而对于肿瘤细胞中的x，y坐标被确定每0.5秒（等于15分钟实时）。 </li><li>确定总每单元60 x，y坐标为典型的细胞迁移实验。这是等于1小时实时为淋巴细胞/ HSPCs和15小时实时为肿瘤细胞。 A"，"表示一个单元格并没有台湾之间移动O时间点，而"数值"表示"像素"的小区有2个时间点（ 图2A）之间移动的距离。 注:手动电池跟踪需要一定的经验。特别是快速迁移细胞难以跟踪和不同的细胞类型展览，可能辅以因素引发了不同的迁徙行为。如果同时跟踪细胞分裂，随机选择了继续跟踪一个女儿细胞。在迁移出视线场或死亡，同时跟踪是不容忽视的，但被认为是分析细胞。 </li></ol> 4，数据分析<ol><li>通过使用适当的软件应用程序， 比如 ，电子表格程序分析细胞的跟踪数据。 <ol><li>通过复制单元格的跟踪原始数据（ 图2A），到自行设计的电子表格模板分析细胞的跟踪数据。乘"数值"的总和，占总距离的细胞已迁移了修正系数转换"像素"为"微米"。 </li><li>确定两个细胞迁移参数:运动器官的活性（相当于迁移率）和主动运动（ 图2C，2D）的时间。 </li><li>确定已迁移更低超过25微米（阈值电平，以减少假阳性的细胞数）的细胞作为不移动的细胞。替换"数值"，这些细胞与"，"。 注意:参数"运动器官的活性"（或迁移率）表示追踪细胞群的细胞的两个时间点（ 图2D）之间已经移动的百分比。 A"的数值"被定义为一个移动单元，而"，"被定义为一个不移动的细胞。参数"主动运动的时间"（时间活性）表示的总时间的细胞中再已迁移的百分比特征研到观察期（ 图2C）的时间帧。 A"的数值"表示的单元已经移动，而"，"指示的小区还没有移动。该参数被进一步用于确定未移动的细胞数。 </li></ol></li><li>计算统计学意义，显示细胞迁移数据。 <ol><li>用Mann-Whitney U检验计算细胞（迁移率）的平均运动器官活动的统计显着性。考虑p值&lt;0.05为显著。 </li><li>显示细胞作为XY-图，箱线图，或为条形图图的平均运动器官的活性。显示单个细胞为基础的数据，如主动运动或速度的时间，为条形图图或柱状图。 注:如果选择电子表格软件应用程序不包含一个箱线图或柱状图工具网上搜索应该为寻找合适的TU进行torials。 </li></ol></li></ol>

The authors declare that they have no competing financial interests.

迁移的能力，是肿瘤细胞4的标志。不脱离原发肿瘤分离并通过周围的结缔组织肿瘤细胞迁移将不能播种继发性病变，这是几乎所有的癌症患者死亡的主要原因的能力。因为这种关系的许多研究都集中于肿瘤细胞的迁移。这些研究的目的是新颖的目标分子和靶通路，有效地阻断肿瘤细胞的迁移，从而损害或减慢转移形成的识别。对于这样的努力可能是β-阻断剂，它已显示出抑制转移性乳腺?...

如细胞融合（查看概述见1,2）细胞迁移是涉及过多的生理过程，包括胚胎发育，伤口愈合和免疫反应（综述见3）的另一生物学现象。然而，迁移的能力也是一个前提肿瘤细胞转移（综述见3,4）。 细胞迁移是一个复杂的，而不是由多个信号转导途径的相互作用的各种配体（ 例如 ，细胞因子，趋化因子，生长因子，激素，细胞外基质组分）的受体（ 例如 ，受体酪氨酸激酶的启动指示尚未完全理解的过程，趋化因子受体，整合素）相互作用5，最终导致肌动蛋白细胞骨架随之而来的重组与设计器和黏着物重组和整合素介导的信号6。 体外和体内细胞迁移测定已经开发了在过去的几十年，包括Boyden小室/ Transwell小室测定法7，划痕试验/伤口愈合测定8-10，三维（ 3D）胶原基质迁移实验11以及活体成像/镜（综述见12）。这些细胞迁移实验都有优点和缺点， 例如 ，关于成本和需求的设备，处理或获得的数据的可靠性。 两个Boyden小室/ Transwell小试验和划痕试验/伤口愈合测定法是容易的，低成本的和发达的测定法来测量细胞迁移的体外 7-10。在Boyden小室/ Transwell小室测定法的细胞接种在含插入孔（约8微米的直径）的顶部-即所谓的上隔室7。可选的，该插入件可以涂覆有细胞外米atrix的组件， 例如 ，纤连蛋白，胶原蛋白等 ，以模仿多个生理环境。同样，内皮细胞可以生长在刀片的顶端，从而模拟内皮细胞屏障13。在定义的时间间隔到下隔室窝藏媒体和补充剂，如生长因子和趋化因子的过程中已通过的孔的那些细胞，被用作读出，以定量细胞迁移（或外渗）。 在划痕试验/伤口愈合测定细胞接种在平板中，并生长至融合10。在实验设置板的依赖性可以预先涂覆有细胞外基质成分，例如纤维连接蛋白。创建一个从无到有/伤口从划痕的每一侧刮细胞单层单细胞后/伤口可以迁移到了国内空白，从而填补/修复它10。的刮伤/伤口的两侧之间的距离被确定中的时间依赖性和作为读出的单元10的迁移活性。然而，细胞增殖之间（这也可能导致划痕/伤口填充/愈合）和细胞迁移的鉴别时，建议在测定随时间推移的视频显微镜和单细胞追踪10结合。 然而，无论是Boyden小室/ Transwell小室法和划痕法/伤口愈合实验，是相当不完善有关生理状细胞环境。在Boyden小室/ Transwell小室测定细胞具有迁移穿过塑料的孔，而在划痕试验/伤口愈合测定细胞接种于一个二维的预包被的塑料板。同样地，它是公认的迁徙行为明显不同的二维和三维环境3之间。例如，成纤维细胞的三维矩阵​​粘连从特征上两个焦点和纤维状粘连不同维基质中α5β1和素αvβ3整合素，桩蛋白，其它细胞骨架成分，和粘着斑激酶14的酪氨酸磷酸化的内容。同样，电池嵌入在3D环境中也显示改变的迁徙行为15。因此，为了更准确地分析细胞迁移的迁移测定法，建议允许测量中的三维生理或生理状环境单细胞的迁移。 活体成像/显微镜是黄金标准的3D生理范围内测量细胞的迁移。这并不只属于胞外基质-细胞相互作用，而且还对不同类型的细胞，如淋巴结17内外渗16或淋巴细胞贩卖期间肿瘤细胞和内皮细胞之间的相互作用，迄今为止，是可能的，因为改进的荧光显微镜技术，例如双光子共聚焦激光扫描显微镜，使用重要的荧光染料和转基因小鼠品系表达荧光蛋白的衍生物12,16,17。此外，活体成像/显微镜可以与手动和自动跟踪电池18的总和。由于2光子共聚焦激光扫描显微镜的需要以及动物（和相应的转基因动物模型）然而，活体成像/显微镜是一种相当高成本的技术。 克服了boyden小室/ Transwell小室试验和划痕试验/伤口愈合测定法的局限性和分析中的三维胶原蛋白基质迁移实验被开发11,19 3D环境中不同细胞类型的迁移。由此，迁移的细胞被嵌入在三维胶原纤维网络，其中更酷似到体内的情况。共同地，由于时间推移的视频显微镜真实细胞迁移测定允许determi国家的几迁移参数以及其响应于亲洄游因子或抑制剂的改变。各种细胞类型可以使用这种技术，包括淋巴细胞和白细胞11,20，造血干/祖细胞21-24，和肿瘤细胞5,25-29进行分析。除了 ​​单个细胞也将细胞簇或球状体，可以嵌入在胶原基质伴随单个细胞从细胞团/球体进入胶原移民的分析中晶格30,31。 该协议提供了关于一个简单但功能强大的技术分析中的3D环境的不同类型细胞的迁徙行为的概述- 体外方法产生的结果是接近的体内情况。

<table border="0" cellpadding="0" cellspacing="0" width="722">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Name of Material/ Equipment</td>
			<td style="width:192px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Leica DM IL inverted microscope</td>
			<td style="width:192px;">Leica, Wetzlar, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Microscope stage heater</td>
			<td style="width:192px;">Distelkamp Electronic, Kaiserslautern, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">JVC C1431 video camera</td>
			<td style="width:192px;">JVC, Bad Vilbel, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Axis 241Q video server</td>
			<td style="width:192px;">Axis communication GmbH, Ismaning, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Mac G5 Computer</td>
			<td style="width:192px;">Apple Macintosh</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">iMac</td>
			<td style="width:192px;">Apple Macintosh</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">FileMaker Pro</td>
			<td style="width:192px;">FileMaker GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Multi-camera video surveillance software(Security Spy)</td>
			<td style="width:192px;">Bensoftware, London, UK</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Runtime Revolution Media 2.9.0</td>
			<td style="width:192px;">RunRev Ltd., Edinburgh, UK</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Paraffin</td>
			<td style="width:192px;">Applichem GmbH, Darmstadt, Germany</td>
			<td style="width:119px;">A4264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Petrolatum jelly</td>
			<td style="width:192px;">local drug store</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Purecol (liquid collagen)</td>
			<td style="width:192px;">Nutacon BV, Leimuiden, The Netherlands</td>
			<td style="width:119px;"></td>
			<td>contains 2.9-3.3 mg/ml bovine collagen (95% collagen type I, 5% collagen type IV)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">10x MEM</td>
			<td style="width:192px;">Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">M0275</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">7.5% Sodium Bicarbonate solution</td>
			<td style="width:192px;">Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">S8761</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">EGF</td>
			<td style="width:192px;">Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">E9644</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">U73122</td>
			<td style="width:192px;">Merck Millipore, Darmstadt, Germany</td>
			<td align="right" style="width:119px;">662035</td>
			<td>dissolve first in CHCL3; reconstitute in DMSO just prior to use</td>
		</tr>
	</tbody>
</table>

analysis of cell migration within a three-dimensional collagen matrix

迁移的能力，是各种细胞类型的标志，并在一些生理过程，包括胚胎发育，伤口愈合，免疫反应起着至关重要的作用。然而，细胞迁移也是使这些肿瘤细胞从原发肿瘤分离开始转移性扩散的关键机制在癌症。在过去几年内各种细胞迁移测定已开发来分析不同类型的细胞的迁移行为。因为细胞的运动器官的行为明显的二维（2D）之间的不同和三维（3D）环境中，它可以假设该被嵌入在三维环境中细胞迁移的分析将产生更显著细胞迁移数据。所描述的三维胶原蛋白基质迁移测定的优点是，细胞包埋于胶原纤维代表的细胞外基质的主要成分的一种生理三维网络。由于时间推移的视频显微镜真实细胞迁移测定，允许响应于亲洄游因子或酶抑制剂的几种迁移参数以及它们的变化的判断。各种细胞类型可以使用这种技术，包括淋巴细胞/白细胞，神经干细胞，和肿瘤细胞进行分析。同样地，还细胞簇或球状体可以在胶原基质伴随单个细胞从细胞群/球体进入胶原晶格移民的分析中被嵌入。我们的结论是3D胶原基质迁移实验是一种通用的方法来分析在一个生理的3D环境中细胞的迁移。

所使用的三维胶原蛋白基质迁移测定结合的时间推移的视频显微镜和计算机辅助细胞跟踪允许对各种细胞的迁移参数包括基于人群的参数（ 例如 ，平均运动器官的活性）和单细胞为基础的参数的确定（ 例如 ，主动运动，速度，距离时间迁移）。所获得的细胞追踪数据集，数据处理和数据显示的一个例子示于图2中 ，一个小区的跟踪数据文件类似的表（ 图2A）。</s...

Watch this Scientific Journal Video about Analysis of Cell Migration within a Three-dimensional Collagen Matrix at JoVE.com

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

细胞迁移是涉及过多的生理的是，如伤口愈合和免疫反应，和病理生理过程，如癌症的生物现象。在3D胶原基质迁移试验是一种多用途的工具内的三维生理状环境分析不同类型的细胞的迁移特性。

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 ...

analysis-of-cell-migration-within-a-three-dimensional-collagen-matrix

Research

JoVE Journal

Bioengineering

23.6K 次观看.  Witten/Herdecke University. 细胞迁移是涉及过多的生理的是，如伤口愈合和免疫反应，和病理生理过程，如癌症的生物现象。在3D胶原基质迁移试验是一种多用途的工具内的三维生理状环境分析不同类型的细胞的迁移特性。 

视频: Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

科研

生物工程

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

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 ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

平面梯度扩散系统的调查趋化三维胶原基质

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

制备及分析<em&gt;体外</em&gt;三维乳腺癌代理项

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.