Name: tissue engineering of tumor stromal microenvironment with application to cancer cell invasion
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Description: Watch this Scientific Journal Video about Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion at JoVE.com

APS是由黛布拉国际和英国皮肤基金会的支持。邓迪合作伙伴博士课程的大学 - YZN由A * STAR的支持。

这项研究是根据赫尔辛基原则宣言进行，批准了相应的伦理委员会。 1。媒体和试剂的制备<ol><li>的L-抗坏血酸-2 - 磷酸的库存200x中制备<ol><li>溶解29毫克每5ml的Dulbecco改良的Eagle培养基（DMEM）溶液中L-抗坏血酸-2 - 磷酸，并通过0.22μm的膜过滤器进行过滤。存储在-20℃下0.25毫升无菌分装</li><li>加0.25 ml等分试样的200倍的L-抗坏血酸-2 - 磷酸的库存，以每50毫升成纤维细胞培养基?...

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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-derived+dermal+matrix+drives+development+of+aggressive+cutaneous+squamous+cell+carcinoma+in+patients+with+recessive+dystrophic+epidermolysis+bullosa.">Fibroblast-derived dermal matrix drives development of aggressive cutaneous squamous cell carcinoma in patients with recessive dystrophic epidermolysis bullosa.</a> Cancer Res. 72 (14), 3522-3534 (2012).</li><li>Larouche, D., Paquet, C., Fradette, J., Carrier, P., Auger, F. A., Germain, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15.">Chapter 15.</a> Methods Mol. Biol. 482, 233-256 (2009).</li><li>Pouliot, R. 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由于本实验的性质，所需的完成的总时间可长达两个月。在这段时间里，悉心照顾和无菌组织培养的做法必须被用来防止微生物污染。 除了 ​​其作为羟脯氨酸和胶原蛋白的羟赖氨酸的合成辅助因子的作用，维生素C能刺激成纤维细胞中特定的22胶原mRNA表达。抗坏血酸选择这里是更稳定的L-抗坏血酸-2 -磷酸23。投喂建议每周三次，但会总?...

在三维组织培养中使用的生物材料，使研究人员在生理条件更类似于体内环境比1概括的2D粘附和塑料衬底的下研究细胞行为在实验室中。特别是，阔步前行已与采用三维培养方法在气-液界面1-4建模层上皮。这种技术忠实地模仿角质形成细胞分化和肿瘤细胞的侵袭实现更高的灵活性和保真度的研究人员在研究这些过程。生物材料基板的选择模仿基质环境主要涉及使用I型胶原，Engelbreth - 霍尔姆 - 群小鼠肉瘤矩阵和去epidermized真皮。例如，癌相关成纤维细胞已经显示出通过间质上皮相互作用6,7朝向癌侵袭5，起始，和无进展促进磨片Ñ​​生长在这样的基底。 的金标准模仿基质环境中的肌肤，使用这种技术的最大和最广泛研究的分层上皮细胞，被视为被撤销epidermized人真皮（DED）。 DED的制备涉及经胰蛋白酶消化或物理结社从人类尸体皮肤3,4去除表皮。然而，进入这样的皮肤是非常困难的与临床相关机构实验室和患病真皮几乎是不可能获得的。作为替代方案，实验室经常使用的类型的组合I型胶原（从大鼠尾隔离）和/或Engelbreth - 霍尔姆 - 群小鼠肉瘤矩阵。 后发现在1927年由Nageotte 8，胶原蛋白可以很容易地用醋酸和盐沉淀中分离出来，其应用组织培养是由Huzel随后首创LA和他的同事9。胶原蛋白涂层被证明是优于玻璃为29株和组织外植体作为审问埃尔曼和Gey中9细胞培养。目前，胶原蛋白在组织培养中所使用的主要类型是从鼠尾腱中分离，并通常从商业来源购买。然而，其缺点为忠实基板概括的是，鼠尾胶原是不相同的人类胶原或人类真皮，其中I型和III型胶原蛋白是存在作为主要成分，并分离的大鼠尾胶原必然是分散。 Engelbreth -霍尔姆-群小鼠肉瘤矩阵是一种胶状蛋白质混合物通过培养Engelbreth -霍尔姆-群小鼠肉瘤细胞分泌的10。的主要成分是层粘连蛋白，IV型胶原蛋白，硫酸肝素蛋白多糖，巢蛋白和巢蛋白和这些蛋白质的精确比例将随每一批。除了结构上的亲teins，该矩阵也包含显著的生长因子如转化生长因子β，表皮生长因子，胰岛素样生长因子1，牛成纤维细胞生长因子，血小板衍生的生长因子，它会改变细胞行为11,12的水平。指向Engelbreth -霍尔姆-群小鼠肉瘤矩阵的庞大的复杂性，共有1,851蛋白质被确定在最近的蛋白质组学研究13。鉴于此矩阵的丰富性和复杂性，谨慎已经解释，并与它的使用11比较不同实验时，被告知。 我们的实验室有遗传性皮肤疾病，尤其是那些有倾向发展皮肤鳞状细胞癌（CSCC）14出浓厚的兴趣。在隐性营养不良型大疱性表皮松解（RDEB），严重的水泡病种系突变的COL7A1基因的情况下<s向上&gt; 15-17，我们已经确定，在这些患者中皮肤微环境是肿瘤促进18。在这个研究过程中，我们无法评估肿瘤促进嵌入胶原蛋白I / Engelbreth - 霍尔姆 - 群小鼠肉瘤矩阵和调查评估细胞自身，原生基质的方法皮肤成纤维细胞的特性。为了实现这一目标，我们修改了以前的技 ​​术，从露西圣日耳曼的实验室工作对人的皮肤替代物19,20。耳门的技术是能够重建人的皮肤使用原代人角质形成细胞和成纤维细胞培养物中不存在的合成或尸体支架的组织良好的基底膜。 在本文中用来概括的皮肤肿瘤基质微环境（原生基质）中的步骤，直接从体外初级基质细胞衍生的描述18。本地矩阵p由成纤维细胞的长期培养roduced被用作皮肤相当于测定为CSCC细胞侵袭。我们使用无论是从细胞外基质由RDEB成纤维细胞（短少在Ⅶ型胶原（C7）），或从RDEB成纤维细胞逆转录病毒转导与Ⅶ型胶原表达构建和展示对肿瘤单胶原蛋白的深刻影响分泌而得本地矩阵显示数据细胞的侵袭。

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	<tbody>
		<tr height="64" style="height:48.0pt">
			<td class="xl63" height="64" style="height:48.0pt;width:66pt" width="88">L-Ascorbic acid 2-phosphate</td>
			<td class="xl63" style="width:66pt" width="88">Sigma</td>
			<td class="xl63" style="width:66pt" width="88">A8960</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl65" height="42" style="height:31.5pt;width:66pt" width="88">DMEM with l-glutamine,</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88">Life Technologies</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88">11995-073</td>
			<td class="xl67" rowspan="2" style="border-bottom:1.0pt solid black;
 border-top:none;width:66pt" width="88"></td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td class="xl63" height="106" style="height:79.5pt;width:66pt" width="88">4,500 mg/L d-glucose, 110 mg/L sodium pyruvate</td>
		</tr>
		<tr height="85" style="height:63.75pt">
			<td class="xl63" height="85" style="height:63.75pt;width:66pt" width="88">100x Penicillin-Streptomycin</td>
			<td class="xl63" style="width:66pt" width="88">Life Technologies</td>
			<td class="xl63" style="width:66pt" width="88">15070</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Vaseline</td>
			<td class="xl63" style="width:66pt" width="88">VWR</td>
			<td class="xl63" style="width:66pt" width="88">PROL28908.290</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Clonal cylinders</td>
			<td class="xl63" style="width:66pt" width="88">Sigma</td>
			<td class="xl63" style="width:66pt" width="88">Z370789</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="148" style="height:111.0pt">
			<td class="xl63" height="148" style="height:111.0pt;width:66pt" width="88">Nylon Net Filter Disc Hydrophilic 100um 25um diameter 100/pk</td>
			<td class="xl63" style="width:66pt" width="88">Millipore</td>
			<td class="xl63" style="width:66pt" width="88">NY1H02500</td>
			<td class="xl64" style="width:66pt" width="88"></td>
		</tr>
		<tr height="127" style="height:95.25pt">
			<td class="xl63" height="127" style="height:95.25pt;width:66pt" width="88">Bent stainless steel wire mesh support</td>
			<td class="xl63" style="width:66pt" width="88">Made in house</td>
			<td class="xl63" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88">Dimensions were made so that the mesh would fit into 6-well plates</td>
		</tr>
	</tbody>
</table>

tissue engineering of tumor stromal microenvironment with application to cancer cell invasion

上皮细胞上嵌有间充质细胞的基质三维器官型培养物广泛用于研究上皮细胞分化和侵入。鼠尾I型胶原蛋白和/或基质从Engelbreth-的Holm-群小鼠肉瘤细胞衍生传统上被用作底物的模型矩阵或基质微环境在其中间充质细胞（通常是成纤维细胞）来填充。虽然使用这种矩阵实验是非常丰富的，可以说，由于一个蛋白质（如I型胶原）或基底膜成分和生长因子（如矩阵来自小鼠含量高的压倒一切的存在肉瘤细胞），这些基材都不最好的反映基质组成的基质细胞本身所作出的贡献。学习由初级真皮成纤维细胞分离自患者的肿瘤易发生，遗传起泡症（隐性营养不良产生的原生矩阵大疱性表皮松解），我们已经适应了现有的本地矩阵协议，研究肿瘤细胞的侵袭。成纤维细胞诱导产生自身的矩阵是长期的文化。这个天然基质，然后从培养皿中脱落细胞和上皮细胞被接种到它之前的整个共培养升至空气 - 液体界面。细胞分化和/或侵入然后可以评估随着时间的推移。这种技术提供了评估在3D设置的上皮 - 间充质细胞相互作用而不需要合成的或外国矩阵的唯一缺点是时间的长期需要产生的天然基质的能力。这里我们描述了该技术的应用来评估单个分子的由成纤维细胞所表达的能力，VII型胶原蛋白，来抑制肿瘤细胞的侵袭。

<html>这种技术开辟了探讨，并根据不同的三维基质环境比较肿瘤细胞的侵入行为（在这种情况下CSCC）的可能性。使用这种技术，可以产生不仅如此概括的RDEB真皮环境的C7-缺陷型原生的矩阵，但也已被基因工程化以过表达C7 18附加的矩阵。就像在图2中 ，入侵RDEB CSCC角质形成细胞是显著滞后于C7-过表达的天然基质相比，C7-缺陷RDEB控制。这个侵入是通过标准组织学方法，其中所?...

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Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

肿瘤微环境基质与应用癌细胞侵袭组织工程

组织工程化的成纤维细胞衍生的天然基质是一种新兴的工具来生成支持上皮细胞增殖和分化的基质衬底。这里应用此方法，以评估不同的基质细胞类型的肿瘤细胞生物学的影响的协议被提出。

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Research

JoVE Journal

Bioengineering

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

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

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 ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Tissue Engineering of a Human 3D in vitro Tumor Test System

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

肌成纤维细胞和约束的应用工程纤维蛋白的​​组织结构和应变诱导细胞和胶原蛋白（RE）组织

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

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

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 ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

在生理体外模型和病理血流应用的血管细胞重塑调查

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...