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，并在实验前在紫外线下消毒 2-3 小时。如果使用替代容器代替生物反应器，则应使用适当的灭菌技术。有关使用玻璃底培养皿的评论，请参阅第 4.12 步。
3. 纤维凝胶制备
生物反应器的组件消毒后，在盖玻璃表面投下薄薄的纤维蛋白凝胶层。在桑德 等人中描述了为制造6.6毫克/毫升纤维蛋白凝胶而准备纤维蛋白原和血栓库存解决方案的细节。13叶 等人的类似协议。19 制作 3.3 毫克/毫升纤维蛋白凝胶也可以在 JoVE 网站上查看。
<ol><li>在 DMEM 中以 1000 万颗珠子/毫升的浓度准备荧光微珠溶液。珠子将用于帮助跟踪凝胶位移。为了达到这种浓度，将 0.017 毫升微珠库存溶液和 0.149 毫升 DMEM 组合成微中管。</li>
	<li>将此悬架暂停 10 分钟，以分散珠子并使溶液均匀化。</li>
	<li>纤维素解决方案-在15毫升c管中，将0.22毫升纤维蛋白原库存溶液与0.44毫升20m HEPES缓冲区混合。加入 0.1667 毫升 DMEM，加入步骤 3.1 中创建的微珠。</li>
	<li>血栓解决方案 - 在单独的 15 毫升 c 管中，混合在一起 0.0328 毫升血栓库存解决方案、0.131 毫升 20 m HEPES 缓冲器和 0.00246 毫升2M CaCl 2 。</li>
	<li>通过上下管道 5-10 倍，小心地将血栓溶液（步骤 3.4） 与纤维蛋白原溶液（步骤 3.3）混合，直到溶液均匀分布。尽量避免引入气泡。要减少产生的气泡量，在混合时要小心不要完全放电移液器。</li>
	<li>加入血栓会导致溶液快速凝胶（约30秒）。尽快将混合溶液输送到盖玻璃上。允许凝胶在RT聚合。</li>
	<li>密封生物反应器，插入加热块，并将热电联连接到温度控制器。在 37 °C 下孵化凝胶 15-30 分钟。</li>
</ol>4. 细胞外植准备
<ol><li>从含有人类皮肤成纤维细胞的T-75烧瓶中取出介质。</li>
	<li>小心地用大约5毫升磷酸盐缓冲盐水（PBS）冲洗表面，去除血清蛋白。加入1毫升的肌氨酸-EDTA，孵育3分钟，或直到细胞升高。</li>
	<li>细胞被提起后，在离心机中旋转悬架200 x g 5分钟。去除超自然物，将颗粒重新用在 DMEM 的体积中，最终浓度为 2000 万个细胞/毫升。</li>
	<li>当离心机中的细胞向下旋转时，生物反应器与加热块和热联体断开。将生物反应器转移到生物安全柜中，并小心地取下盖子，采用怀疑技术。</li>
	<li>按照模具上的图案，将细胞悬浮的0.3μl输送到聚合纤维蛋白凝胶上，从而产生去移植物。每个解释应包含大约6，000个细胞。确保使用小容量微皮条客提示（0.1-10 μl）。</li>
	<li>允许细胞在37°C下在纤维素基质上沉淀并附着在1小时。</li>
	<li>生物反应器仍然开放，加入约5毫升的DMEM补充10%胎儿牛血清（FBS），1%青霉素链霉素，0.1%氨基丙胺B，和10毫克/毫升阿普罗汀直接进入生物反应室。DMEM是碳酸氢碳酸酯缓冲，需要5%的二氧化碳2来保持中性pH。由于生物反应器未提供 CO2，在使用前 2-3 小时在孵化器中用 5% CO2调节介质。阿普罗汀是一种血清蛋白酶抑制剂，广泛用于降低纤维蛋白降解率20。</li>
	<li>重新seal生物反应器。使用注射器通过入口端口上的带刺配件提供额外的 5 毫升 CO2条件介质。缓慢分配介质，并确保生物反应器室的整个体积被填充。小心去除生物反应器中形成的气泡。</li>
	<li>在整个实验过程中，向生物反应器提供新鲜、5% 的二氧化碳条件介质，以维持pH、供应营养物质和清除废品。设置一个注射器泵与10毫升或30毫升注射器充满了5%的CO2条件介质。将注射器直接连接到生物反应器盖上的入口端口，并配有Luer-lock安装的无菌管。取出男性配件，并将管子连接到生物反应器上的带刺配件上（见图 1C）。</li>
	<li>将灌注率设置为 0.01 毫升/分钟。将经过改装的管子连接到两个出口端口，并将两端放入 100 毫升烧嘴中以收集废物。</li>
	<li>使用实验室插孔设置入口和插座馈送的高度，以便在生物反应器中不会出现压力差（请参阅 图 1D 以供参考）。</li>
	<li>如果没有生物反应器，用玻璃顶部在 35 mm 玻璃底培养皿中准备样品。使用具有盖片大小的盖片大小，该尺寸可针对要使用的特定目标集进行优化。在培养皿中准备的样品应保持在37°C和5%CO2的孵化器中。 注意：聚苯乙烯去极化光，并会干扰DIC成像，所以玻璃顶部应该使用，如果DIC成像将进行。相位对比是一种合适的替代成像模式。在孵化器和显微镜之间传输菜肴会使图像注册变得困难。如果图像没有正确注册，则第 6 节计算的应变将不准确。</li>
</ol>5. 延时成像
<ol><li>样品准备完毕后，重新安装生物反应器，重新连接加热块和热电联，并将生物反应器温度设置为37°C。</li>
	<li>将生物反应器设置在安装在显微镜上的电动舞台上。将 20X DIC 目标定位在视图端口下。较低的放大目标是可以接受的，特别是如果没有精确的机动化阶段。确保偏振器、分析仪和棱镜都到位。或者，也可以使用相对比度对照样品。</li>
	<li>打开成像软件。</li>
	<li>将目标集中在三个解释植物之间的区域。</li>
	<li>在成像软件中保存此位置的坐标（x、y 和 z）。</li>
	<li>为了对解释层之间和周围的整个区域进行成像，选择获取大图像并指定区域大小的选项。这将允许在指定区域周围获取多个图像，并创建一个平铺图像，表示样本的较大区域。</li>
	<li>将暴露时间和光强度设定为尽可能低的值，以避免因光毒性导致的细胞死亡，同时仍然提供足够的分辨率来区分细胞、微珠和纤维纤维。</li>
</ol>6. 应变跟踪
有关应变跟踪软件（图2）的详细信息和说明，请参阅拉古帕蒂 等人。21该算法是一个自定义的 MATLAB 代码，可以从 <a href="http://www.license.umn.edu/default.aspx">http://www.license.umn.edu/default.aspx</a>下载。请注意，DIC 图像通常有足够的纹理用于应变跟踪。微珠被包括在内，作为对计算菌株场的检查。如果要进行应变跟踪，则在完全相同的位置拍摄所获得的图像以便对图像进行注册至关重要。未注册的图像会产生虚假的菌株。

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未声明任何利益冲突。

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?...

Watch this Scientific Journal Video about Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction at JoVE.com

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

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。

观察和量化纤维细胞介导的纤维素凝胶压实

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 ...

observing-and-quantifying-fibroblast-mediated-fibrin-gel-compaction

Research

JoVE Journal

Bioengineering

5.9K 次观看.  University of Iowa. 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。

视频: Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

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

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial 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

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

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

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 inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

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

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 ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

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

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

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

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

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

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

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 ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

凝胶序列: 一种同时测序库的方法用水凝胶基质制备 DNA 和 RNA

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...