Name: concentric gel system to study the biophysical role of matrix microenvironment on 3d cell migration
Uploaded: 2015-04-03T15:00:00
Description: Watch this Scientific Journal Video about Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration at JoVE.com

作者感谢W. Sun和K·詹森的批判性的讨论，并承认支持纳米生物力学实验室，在新加坡国立大学。 NAK承认支持由居里夫人IIF奖学金。

1.细胞收获<ol><li>得到的MD-MBA-231细胞从37℃，5％CO 2的培养箱中培养。分离，以0.5％胰蛋白酶-EDTA溶液从组织培养板上的细胞。使用1个毫升胰蛋白酶-EDTA溶液用于在T25培养瓶中的细胞进行培养。 </li><li>沉淀细胞在15ml锥形管中，离心，在200×g下4分钟，吸出上清液，并悬浮细胞在5ml培养基。 </li><li>计数细胞密度，ρ，使用血球。 注意：为了制备细胞接种内凝胶，将细胞悬液稍后?...

<ol>
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Cell Biol. 201 (7), 1069-1084 (2013).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nat. Rev. Mol. Cell Biol. 10 (7), 445-457 (2009).</li><li>Vedula, S. R. K., Ravasio, A., Lim, C. T., Ladoux, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration:+a+mechanistic+perspective.">Collective cell migration: a mechanistic perspective.</a> Physiology. 28 (6), 370-379 (2013).</li><li>Petrie, R. J., Doyle, A. D., Yamada, K. 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In this protocol we describe an in vitro assay to study the 3D migrational behavior of cells in matrix environments that topologically resemble ECMs encountered in vivo. There are three main strengths of this assay as compared to other currently available methods. First, this assay allows one to simultaneously examine the cell migration mechanisms at both population level and individual cell level. This opens up possibilities of studying collective cell migration13, which has to date been lar...

细胞迁移起着各种生理反应，如胚胎发育，止血，和免疫反应，以及在病理过程，如血管疾病，炎症，癌症和1的关键作用。解剖底层细胞迁移的生物化学和生物物理因素，因此从根本上重要的是不仅要了解的细胞功能的基本原则，又能促进各种生物医学应用，例如在组织工程，抗转移和抗炎药物开发。由于体内观察是技术上的挑战，有很多的努力一直专注于体外再演细胞迁移。 体外方法来研究细胞迁移已经基本上被设计为检测在二维（2D）表面上，最显着的划伤或伤口愈合测定法2。这些试验提供了简单的实验装置，方便在线 -细胞成像，并提供有益的见解底层细胞迁移的各种生化机制。但是，这些测定法不占外基质（ECM）架构和重塑，这是在理解体内迁移关键方面。最近，人们已越来越多地认识到，3D培养模型，常常在基于胶原的基质3中，提供了更好的类似于体内情况的一个平台。事实上，细胞表现出的迁徙动力学是从那些在2D表面明显，特别是由于不同的维数的环境4。此外，基体的生物物理和机械性能灵敏影响细胞迁移5，包括在肿瘤细胞侵袭6的上下文中。 在这里，我们提出研究三维细胞迁移行为的ECM与生物物理特性，可以轻松地用不同的制备条件的方法。细胞是接种在“内凝胶”和被允许逃逸到并侵入最初无细胞“外凝胶”。该方法依赖于细胞的能力来识别的存在，并倾向成，无细胞迁移区域中的外凝胶，这是密切相关的细胞mechanosensing 7。在这项研究中，我们采用的胶原蛋白网络，通过高度侵袭性​​乳腺癌细胞，MD-MBA-231入侵的ECM。机械性能和两者的内部和外部的凝胶的微结构可以被调谐8和9，其特征实现生理学相关的条件。重建单元轨道和分析允许在两个群体水平和个体细胞水平的时空迁移行为的详细的定量检查。重要的是，同心凝胶系统的设置模仿面临迁移细胞，尤其是侵入癌细胞，从而提供了重要的见解的体内组织的拓扑对细胞迁移和转移的物理机制。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cell culture incubator</td>
			<td>Fisher Scientific Pte Ltd</td>
			<td>Model: 371, S/No 318854-6055</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon A1R</td>
			<td></td>
			<td>Inverted confocal laser scanning microscope equipped with incubator chamber</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent CellTracker dye CMTMR</td>
			<td>Life Technologies</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-bottom dish</td>
			<td>IWAKI Cell Biology</td>
			<td>3931-035</td>
			<td>35 mm diameter dish with 12 mm diameter glass-bottom well</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>iN CYTO</td>
			<td>DHC-N01 (Neubauer Improved)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprocessor pH meter</td>
			<td>Hanna Instruments</td>
			<td>pH 211</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutragen Collagen</td>
			<td>Advanced BioMatrix</td>
			<td>#5010-D</td>
			<td>Acid-solubilized bovine collagen type I (stock pH ~ 2)</td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td></td>
			<td>CFI Super Plan Fluor ELWD ADM 20XC, W.D. 8.2-6.9mm, NA 0.45.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Sartorius</td>
			<td>S/No 29153352</td>
			<td>Basic pH Meter PB-11</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
	</tbody>
</table>

concentric gel system to study the biophysical role of matrix microenvironment on 3d cell migration

细胞迁移的能力是在各种各样的细胞功能，在整个从胚胎发育寿命和伤口愈合肿瘤和肿瘤转移的关键。尽管强烈的研究努力，细胞迁移的基本生物化学和生物物理原理仍然没有完全了解，特别是在生理相关的三维（3D）的微环境。在这里，我们描述了在体外试验设计允许的三维细胞迁移行为定量检查。该方法利用了细胞的mechanosensing能力和倾向迁移到先前未被占据的细胞外基质（ECM）。我们使用高度侵袭性​​乳腺癌细胞的侵袭，MDA-MB-231，在胶原凝胶作为模型系统。细胞群的传播和单个细胞的迁移动力学数周的文化可以用活细胞成像进行监测和分析，以提取spatiotemporally分辨数据。此外中，该方法很容易适应于不同的细胞外基质，从而提供了一种简单而功能强大的方法来研究的生物物理因素对细胞迁移的微环境中的作用。

使用高度侵袭性乳腺癌细胞进行这里提出的同心凝胶测定中，MDA-MB-231，用2.4毫克/毫升的内胶原凝胶和= 2×10 6个细胞/ ml的细胞接种密度，作为一个例子。 如图2中 ，典型地经过培养几天，细胞违反内外凝胶接口，并开始侵入外凝胶。该细胞群主要传播径向向外。 外凝胶的聚合条件可被修改以研究对细胞迁移特性的矩阵的密度和机械性能的作用。 图3...

Watch this Scientific Journal Video about Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration at JoVE.com

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

机械性能和细胞外基质的组织强烈地影响细胞的三维迁移。 的体外方法来研究在生物物理变量的环境中，在人口和单个细胞水平的时空细胞迁移行为，进行说明。

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 ...

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

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Study of Cell Migration in Microfabricated Channels

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 ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

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 ...

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

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 ...

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

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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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...