Name: engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization
Uploaded: 2013-10-28T21:45:00
Description: Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

（BMM）的生物医学材料研究所的研究方案进行了这项研究。 BMM是由荷兰经济事务，农业和创新部共同资助。感谢购买！Hartstichting的财务贡献。

1。文化人类腔小隐细胞<ol><li>隔离腔小隐蚤，根据二次使用材料的指引从供体获得，根据协议等施耐尔9细胞，并储存在液氮中。从2×2毫米在6孔板培养一个捐助者的裁片的部分小隐静脉蚤。使用2枚，每口井。一般来说足够的细胞可以得到以填补约3瓶0.25×10 6个细胞在液氮中。的HVSCs的特点是肌纤维母细胞，显示波形蛋白，不表达结蛋白和亚群表达α平滑肌肌动蛋白<s...

<ol>
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M., 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=Optimal+cell+source+for+cardiovascular+tissue+engineering:+venous+vs.+aortic+human+myofibroblasts.">Optimal cell source for cardiovascular tissue engineering: venous vs. aortic human myofibroblasts.</a> Thorac. Cardiovasc. Surg. 49, 221-225 (2001).</li><li>Mol, A., 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=Autologous+human+tissue-engineered+heart+valves:+prospects+for+systemic+application.">Autologous human tissue-engineered heart valves: prospects for systemic application.</a> Circulation. , I152-I158 (2006).</li><li>Ahmann, K. A., Weinbaum, J. S., Johnson, S. L., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+degradation+enhances+vascular+smooth+muscle+cell+proliferation+and+matrix+deposition+in+fibrin-based+tissue+constructs+fabricated+in+vitro.">Fibrin degradation enhances vascular smooth muscle cell proliferation and matrix deposition in fibrin-based tissue constructs fabricated in vitro.</a> Tissue Eng. Part A. 16, 3261-3270 (2010).</li><li>John, J., Quinlan, A. T., Silvestri, C., Billiar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boundary+stiffness+regulates+fibroblast+behavior+in+collagen+gels.">Boundary stiffness regulates fibroblast behavior in collagen gels.</a> Ann. Biomed. Eng. 38, 658-673 (2010).</li><li>Rubbens, M. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+Collagen+Organization+at+the+Micro-level+in+Fibrin-based+Engineered+Tissue+Constructs.">Strain-induced Collagen Organization at the Micro-level in Fibrin-based Engineered Tissue Constructs.</a> Ann. Biomed. Eng. 41, 763-774 (2013).</li><li>Merryman, W. D., 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=Correlation+between+heart+valve+interstitial+cell+stiffness+and+transvalvular+pressure:+implications+for+collagen+synthesis.">Correlation between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen synthesis.</a> Am. J. Physiol. 290, (2006).</li><li>Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cellular+mechanotransduction+to+biologically+inspired+engineering:+2009+Pritzker+Award+Lecture,+BMES+Annual+Meeting+October+10,+2009.">From cellular mechanotransduction to biologically inspired engineering: 2009 Pritzker Award Lecture, BMES Annual Meeting October 10, 2009.</a> Ann. Biomed. Eng. 38, 1148-1161 (2009).</li><li>Sander, E. A., Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+fiber+alignment+pattern+alters+extracellular+matrix+synthesis+in+fibroblast-populated+fibrin+gel+cruciforms+and+correlates+with+predicted+tension.">Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension.</a> Ann. Biomed. 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描述的模型系统研究细胞和胶原细胞填充的纤维蛋白结构具有很大的潜力（重新）组织（德容格等15），如用于组织工程的目的。通过使用作为初始细胞载体的血纤维蛋白，纤维蛋白降解后，组织细胞和内源性矩阵只创建。以这种方式，细胞受到刺激进行反应的应变，无论是静态的或环状的性质，通过施加收缩力16,17，检测边界12，刚度或显示菌株避免的。 <p class...

心血管组织有一个突出的承载功能。在特定的内容和组织中的细胞外基质的胶原纤维向承重性能和支配整体组织强度1。在组织工程机械空调的构造被使用-通常包括（循环）使劲方案是-以增强组织的组织和机械性能2,3。充分的了解应变诱导胶原蛋白组织创建与预定义的胶原蛋白结构的组织的复合组织的几何形状还没有被实现。这主要是由于我们有限的知识，在发展组织的胶原蛋白的重塑。现有的车型主要使用静态应变4-6胶原重塑，最终净结果信息。在这里，我们提供了一个高度可调的模型系统，它允许在一个实时的方式，在三维胶原蛋白（重）组织的研究，受到影响的情况下的静态或环状的应变。组织结构的纤维蛋白为主，确保所有构造中的胶原蛋白是内源性的。在构建体的细胞和胶原组织的可视化，和一个内部参考系统使我们能够移居时间推移分析细胞和胶原结构。在这个协议中，我们将描述人类腔小隐的细胞（HVSCs），因为这些细胞被称为增强细胞外基质生产和改造能力在工程心血管组织7矩阵和我们既定的用途，根据使用的模型系统的容格等[8]的工作

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture plastic</td>
			<td>Greiner</td>
			<td></td>
			<td>Includes culture flasks and pipettes</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Greiner</td>
			<td>758075</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastomer and curing agent</td>
			<td>Dow Corning Corporation</td>
			<td>3097358-1004</td>
			<td>Silastic MDX 4-4210#</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>Regular store</td>
			<td></td>
			<td>You can buy this at a regular store, only use the soft side</td>
		</tr>
		<tr>
			<td>Bioflex culture plates</td>
			<td>Flexcell Int</td>
			<td>BF-3001U</td>
			<td>Untreated</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphatase</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>&#949;-Amino Caproic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>D7754</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine thrombin</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 syringe filter</td>
			<td>Whatmann (Schleicher and Scheul)</td>
			<td>10462100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres</td>
			<td>Invitrogen</td>
			<td>F-8829</td>
			<td>Blue fluorescent, 10 &#956;m diameter</td>
		</tr>
		<tr>
			<td>Flexcell FX-4000T</td>
			<td>Flexcell Int</td>
			<td></td>
			<td>Includes rectangular loading posts</td>
		</tr>
		<tr>
			<td>Cell Tracker Orange</td>
			<td>Invitrogen Molecular Probes</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA35-OG488</td>
			<td></td>
			<td></td>
			<td>Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td>Needed for cell isolation</td>
		</tr>
	</tbody>
</table>

engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization

在开发胶原组织的胶原蛋白含量和组织可以影响局部组织的菌株和组织约束。组织工程师的目标是利用这些原则来创建与预定义的胶原蛋白结构组织。胶原重构，以控制最终的组织结构的确切相关的过程的充分理解，但是，是缺乏的。特别是（重新）胶原纤维的方向响应在组织机械负荷条件的变化知之甚少。我们开发了一个体外模型系统，包括双向约束肌成纤维细胞的种子纤维蛋白结构，进一步阐明胶原蛋白（重）响应我的方向）恢复双轴单轴静载荷条件及ii）循环单轴载荷双向约束在装载方向，使用FLEXCELL FX4000T装载装置之前和之后的变化的构造。时间推移共聚焦成像到visualize胶原蛋白（重新）方向，一个非破坏性的方式。 细胞和胶原组织中的构造可以进行实时可视化，和一个内部参考系统使我们能够移居时间推移分析细胞和胶原结构。可以调整的模型系统的各个方面，如健康和患病细胞的细胞来源或使用。可以使用添加剂，以进一步阐明相关机制胶原重构，例如，通过添加蛋白酶或阻断整合。该构建体的形状和大小可以很容易地适应特定需要的，从而导致在一个高度可调的模型系统来研究细胞和胶原蛋白组织（重）。

这个模型系统允许种子培养成肌纤维细胞纤维蛋白凝胶。 图1A显示了一个组织首先培养静态双轴约束下的。组织的约束通过切割纤维蛋白凝胶从两个约束被释放，以创建单轴静态约束，和组织压块和重塑之后（ 图1A）。对于循环应变，组织培养以及静态双轴约束下。 5天之后，可以应用于周期性单轴拉伸（ 图1B）。为了诱导胶原蛋白的重新取向，单轴应变的?...

Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

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

这个模型系统开始从填充的成肌纤维细胞的血纤维蛋白凝胶，可用于研究内源性的胶原蛋白（重）非破坏性的方式组织实时。系统的模型是非常可调谐的，因为它可以用不同的细胞来源，培养基添加剂，可以容易地适应具体需要。

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 ...

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高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

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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

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