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（DMEM，1％L-谷氨酰胺和10％胎牛血清）中所要求的天，对于0.1毫米L的终浓度-抗坏血酸2 - 磷酸酯。 </li></ol></li><li>角质细胞生长培养基的制备<ol><li>隔离初级SCC角质形成细胞和文化先前已经21所述。角质形成细胞生长培养基的制备描述其中为好。 </li><li>简单地说，准备介质如下： 300毫升的DMEM和100ml Ham氏F-12补充有10％FBS的 0.4毫克/毫升氢化可的松 5毫克/毫升的胰岛素 10纳克/毫升的EGF 5毫克/毫升的转铁蛋白 8.4纳克/毫升霍乱毒素 13纳克/毫升三碘甲状腺氨酸 1X青霉素 - 链霉素溶液</li></ol></li></ol> （2） 在成纤维细胞来源的原生矩阵中的L-抗坏血酸-2 -磷酸补充培养基体外构建<ol><li>每孔种200000的成纤维细胞在6孔板中在补充有L-抗坏血酸-2 -磷酸的成纤维细胞培养基（20000个细胞/ cm 2）。 REFEED每2-3天用2-5毫升媒体。 （注：再投喂频率和音量可调节，以适应不同的单元格的个性化需求，请参见“讨论”）。 </li><li>一厚层细胞嵌入在细胞外基质的形成将在6周后，可见以肉眼（端<stro纳克&gt;图1）。从组织培养板中释放这一层，轻轻刮去矩阵的周围用1毫升微量移液器尖端，然后按矩阵的边缘朝着井中心。这层细胞和基质（本机矩阵）应当从板表面容易分离，并且现在将浮在介质。散布本机矩阵列于媒体，以避免它折叠起来到自身。 </li><li>让本机矩阵flo​​at和改造了5天，不断变化的媒体每2-3天。由于拉伸强度和内矩阵固有重塑，基质将收缩急剧而成为降低到一个较小的，但是更厚的天然基质，并且准备好被用于入侵检测。 </li></ol> 3。入侵检测与肿瘤鳞状细胞癌角质形成细胞<ol><li>拿起钝钳本地矩阵轻轻地转移到尼龙网。一旦上了尼龙网，传播矩阵轻轻地用1毫升微量说谎尽可能平坦尖而钝钳。 </li><li>通过涂抹在一端有少量无菌凡士林准备无菌克隆缸。 </li><li>将克隆缸上的天然基质，用凡士林面朝下。这是为了确保本机矩阵和克隆环之间的密封。 </li><li>添加CSCC细胞克隆缸（250,000细胞在100μl角质细胞生长介质）。 </li><li> 6小时后取出克隆气瓶时，CSCC细胞已落户在本机矩阵。 </li><li>提起尼龙网与本地矩阵和宫颈鳞癌细胞对气 - 液界面上弯曲的不锈钢丝网的支持。 </li><li>添加补充有抗坏血酸的角质细胞生长直到介质电平触发的天然基质的底部。 </li><li>更换介质每2-3天收获7和14天后播种CSCC细胞。 </li></ol> 4。三维培养收获和组织学准备<ol><li>修正了在4％看齐aformaldehyde在室温下过夜。 </li><li>平分在蜡样品和嵌入于福尔马林固定的石蜡包埋块，与切割表面朝外。 </li><li>另外，嵌入平分样本OCT和液氮冷冻的组织块单元立即冻结。 </li><li>切4微米的部分上切片脱蜡和（如有必要）。染色使用标准的苏木精和曙红。以可视化的CSCC细胞，对角蛋白14鼠单克隆抗体（LL001，内部），可在免疫组化染色中使用。 </li></ol>

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作者什么都没有透露。

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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			<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>
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			<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;
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		</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?...

Watch this Scientific Journal Video about Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion at JoVE.com

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

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。

肿瘤微环境基质与应用癌细胞侵袭组织工程

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

tissue-engineering-tumor-stromal-microenvironment-with-application-to

Research

JoVE Journal

Bioengineering

9.7K 次观看.  University of Dundee. 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。 

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

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

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

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

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

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 ...

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

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 study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

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

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

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

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

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

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

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

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or 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 ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

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

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 vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

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

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

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 ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...