Name: observing and quantifying fibroblast-mediated fibrin gel compaction
Uploaded: 2014-01-16T13:45:00
Description: Watch this Scientific Journal Video about Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction at JoVE.com

我们感谢乔治·朱迪斯和史蒂文·埃利亚森捐赠人类皮肤成纤维细胞和拉梅什·拉古帕蒂帮助与应变跟踪算法。这项工作由美国教育部国家需要研究金领域的研究生援助（GAANN P200A120071）提供。

1. 模具准备
在 Parafilm 上准备一个模具，按照所需的几何形状（图 1A 和 1B）布局每个解释的位置。每个除外植物的空间相距约1-2毫米。此距离相当于在外植物之间生成纤维对齐的理想间距。将模具附在盖玻璃区域下方，用胶带准备样品。
2. 绝育
使用 70% 乙醇彻底清洁生物反应器的所有组件，并在实?...

<ol>
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制定此协议的目的是观察和量化参与细胞介质 ECM 改造的机制。这些过程是一些生物现象的基础，对工程组织2，22，减少疤痕1，23，并了解病理组织重塑12，24有重要影响。使用延时 DIC 显微镜可以解决和量化细胞牵引力导致的纤维纤维的位移和对齐。这里观察到的重组是改造过程的第一阶段，它遵循胶原蛋白11中观察到的组织模式。重组之后是纤维素降解和 ECM 合?...

研究细胞-基质相互作用的一个重要工具是细胞填充胶原蛋白凝胶1，2。凝胶提供了一个3D环境，更接近组织的 体内 特征，更适合理解细胞行为比传统的2D文化3提供。早期研究发现，成纤维细胞在胶原蛋白凝胶中均匀分布，细胞会迅速巩固胶原纤维并压缩凝胶4，5。自由浮动凝胶中的收缩成纤维细胞，然后在凝胶完全达到压实1，6，7后不久过渡到静止状态。在边界上受约束的凝胶成纤维细胞保持在一个活跃的合成状态8，它们产生纤维对齐的方式依赖于凝胶几何和外部约束5，9。细胞活动的差异似乎是内部张力（或缺乏紧张）的结果，这种张力是细胞通过凝胶中的胶原纤维上的内质素施加牵引力而形成的。
这项技术的一个变种包括将成纤维细胞去除植物（即细胞团）放置在胶原蛋白凝胶中相距一段距离，观察细胞-基质相互作用，以及将外植（有时称为韧带状带）10-12之间的纤维对齐逐渐发展。去植系统的主要优点是，它允许将细胞排列成简单的几何图案，从而更容易可视化和探索细胞驱动的纤维重新调整背后的机制。这些对齐模式（主要取决于细胞牵引力、细胞空间分布、凝胶几何学和凝胶上的机械约束之间的相互作用）非常重要，因为它们在全球组织组织、机械功能和局部机械环境中发挥着核心作用。
在组织工程领域，生产机械功能、工程组织的战略之一是控制细胞压实形成的纤维对齐模式，使工程组织具有类似于原生组织14，15的纤维对齐。这种对齐被认为是工程组织复制原生组织复杂机械行为所必需的。这种策略的修改是用纤维蛋白凝胶16取代胶原蛋白凝胶。纤维蛋白凝胶在压实过程中与胶原蛋白凝胶形成类似的对齐模式。随着时间的推移，纤维蛋白会降解，代之以遵循初始纤维纤维对齐模式的细胞合成 ECM。与胶原蛋白凝胶衍生结构17相比，由此产生的工程结构显著提高了机械性能。
纤维凝胶中的对齐过程和随后的改造事件以复杂且不为人知的方式进行。为了更好地描述这些相互作用及其对细胞行为和 ECM 重塑的影响，我们开发了一种基于去种植法的程序。在这种方法中，成纤维细胞去植以不同的几何图案放置在纤维蛋白凝胶上。凝胶保持在环境控制，显微镜安装的生物反应器18，压实和纤维重新调整的过程监测与延时差干扰对比度（DIC）显微镜。位移字段使用自定义算法进行量化。从这些实验中获得的数据对许多过程有着广泛的影响，包括优化组织工程策略、改善伤口愈合和治疗病理组织重塑。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sigma-Aldrich</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Invitrogen</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lonza</td>
			<td>DE14-701F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular Probes</td>
			<td>F8858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>A10483-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>11430-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisher-Scientific</td>
			<td>SS264-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A3428-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Biotense Bioreactor</td>
			<td style="width:85px;">ADMET</td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ti-Eclipe Microscope</td>
			<td>Nikon</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Bottom Petri Dish</td>
			<td>MatTek</td>
			<td>P35G-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Top Petri Dish</td>
			<td>MatTek</td>
			<td>P35GTOP-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Luer fittings, PVC tubing with Luer ends</td>
			<td>Cole-Parmer</td>
			<td>30600-65</td>
		</tr>
	</tbody>
</table>

observing and quantifying fibroblast-mediated fibrin gel compaction

嵌入胶原蛋白和纤维蛋白凝胶中的细胞在凝胶的纤维上连接并施加牵引力。这些力量可导致凝胶微观结构的局部和全球重组和重组。这个过程以复杂的方式进行，部分取决于细胞的位置、凝胶的几何形状和凝胶上的机械约束之间的相互作用。为了更好地了解这些变量如何产生全球纤维对齐模式，我们使用延时差干涉对比 （DIC） 显微镜与环境控制的生物反应器一起观察几何间隔的外植（成纤维细胞群）之间的压实过程。然后使用自定义图像处理算法分析图像，以获取应变的地图。从这项技术获得的信息可用于探索各种细胞-基质相互作用的机械生物学，这对理解伤口愈合、疾病发展和组织工程应用过程中的流程具有重要意义。

组织重塑是一个复杂的过程，部分是由细胞和周围矩阵之间的相互物理相互作用推动的。细胞重组周围的纤维，在光纤网络中产生张力。纤维与机械环境的对齐反过来控制细胞行为，使细胞和基质在全球范围内重组，产生改造后的组织。在这个实验中，外植的细胞，最初在形态上是圆形的，开始延伸到凝胶中，并附着在纤维纤维（图3）上。细胞施加牵引力，通过凝胶传播，并诱导纤?...

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Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

观察和量化纤维细胞介导的纤维素凝胶压实

延时显微镜和图像处理技术用于观察和分析成纤维细胞介导凝胶压实和纤维素纤维在48小时内在环境控制的生物反应器中的重新调整。

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Research

JoVE Journal

Bioengineering

Preparation of Complaint Matrices for Quantifying Cellular Contraction

投诉矩阵量化细胞收缩的准备工作

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

科研

生物工程

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

微组织使用胶原蛋白的模块的制备凝胶含有细胞

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

心肌在心脏组织工程中的纤维蛋白凝胶的封装

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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 ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

白细胞 - 富血小板纤维蛋白，一种新型生物材料的表征

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

形成用凝胶辅助补液巨型的聚合物囊泡

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

在生物环境的新技术的产生和观测化学发光

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

用数字全息显微镜 (DHM) 定量化低浓度微生物

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...