这项工作是由美国心脏协会Predoctoral的奖学金JLI和国家机构的希思授予R01AR056624支持。

答：单聚合物静电<ol><li>准备电的解决方案之前，在光引发剂0.5％的溶液，Irgacure 2959（I2959），去离子水溶解在37 ° C几天。这一步是没有必要的，如果光反应的聚合物不被使用。 </li><li>结合methacrylated透明质酸（梅阿，看到伯迪克合成等 ），聚（环氧乙烷）（PEO，900 kDa的），在去离子水I2959准备与终浓度为2％，3％梅阿的解决方案私立教育机构，和0.05 wt％的I2959。使用一个旋涡混合解决方案，直到它被清除。聚合物的类型和浓度，以及使用的溶剂可以修改的，这取决于所需的脚手架属性的步骤。 </li><li>转移到一个注射器的解决方案，并附加18号，6英寸长，钝端针到底。 </li><li>计划注射泵在1.2毫升/小时的速度弹出。插入设备的注射器和针头。 </li><li>将一个高压电源的接地导致的收集装置。为了收集随机分布的纤维使用一个平面的金属板，或使用〜10米/秒旋转的芯棒收集对齐纤维。将带正电的铅针。静电仪器的原理 ， 参见图1。 </li><li>调整针或收集装置，有两者之间的15厘米的距离。 </li><li>启动注射泵的流量。当流体可视化的针尖上，打开电源，22千伏的电压设置。 </li><li>收集完成后（从几分钟薄膜，较厚的垫长达24小时），删除从收集设备的支架，并存储在真空条件下过夜，以确保完全去除的溶剂。 </li><li>可视化的纤维形态，利用扫描电子显微镜（SEM），有代表性的支架对齐和不结盟的结构如图1所示。 </li></ol>注：样品流速，收集装置的距离，和电压依赖于聚合物和溶剂的组合，并为每个系统，通常是通过扫描电镜观测支架形态，必须优化。 B.光交联和Photopatterning <ol><li>从支架垫，切出5mm x 5mm的样品。 </li><li>交联准备通过配售每个支架上覆盖玻片陪衬，直接放在支架上光罩，用干净的玻片上覆盖长尾夹夹闭两端。注：四个样本，可一次完成，并没有一个模式的透明度，可用于交联，如果不想要一个模式。 </li><li>清除脚手架在氮气室成立。重要的是要保持氧气，它可以抑制交联的构造。 </li><li>放置支架安装在氮气室下〜10毫瓦/厘米2 365纳米的光，用5分钟的准直适配器。参见原理图如图 2 。 </li><li>删除每个支架，并在12孔板地点。 </li><li>添加到每口井，封口膜板式蒸发的水和地点，以防止在37 ° C 24小时2 mL去离子水。换水三次。 </li><li>光学显微镜下可视化孔形成（参见图2中的例子） 。现在准备使用的支架。 </li></ol>注：聚合物的种类很多，但是photopatterning只能用于光反应的聚合物，光交联是没有必要的。 C.双聚合物静电荧光纤维可视化<ol><li>准备在90％乙醇的私立教育机构（200 kDa的）5 wt％的解决方案。在搅拌700转50 ° C时至少2个小时之前，以静电。 </li><li> PEO的解决方案转移到一个注射器和添加电液中的终浓度为10 mg / ml浓度的DAPI。用铝箔保护光注射器。 </li><li>准备除也为电液中25微米的终浓度添加methacryloxyethyl thiocarbamoyl罗丹明B（MeRho 683.24克/摩尔），在步骤A2相同的解决方案。用铝箔保护光注射器。 </li><li>小心地用透明胶带，以确保methacrylated盖玻片（见Khademhosseini等 。）芯棒表面。注：纤维将成为束缚methacrylated盖玻片。 </li><li>一个5毫米的长片硅胶管，带Luer锁附件注射器的一端连接和附加针的另一端。插入注射泵的注射器和针头通过孔的电风扇。重复与其他注射器和芯棒对面站点的电风扇。 fanners翻译的L芯棒ength和使用，以确保双方聚合物的平等分配造成脚手架。参见原理图如图 3 。 </li><li>在一个适当的按表1部分所述的调整参数。确保针尖芯棒为中心。 表1。双聚合物静电参数 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 注射器 </td><td> 电风扇针尖（厘米） </td><td> 针尖芯棒（厘米） </td><td> 流量（毫升/小时） </td><td> 应用电压（kV） </td></tr><tr><td>梅阿</td><td> 6 </td><td> 15 </td><td> 1.2 </td><td> 22 </td></tr><tr><td>私立教育机构</td><td> 6 </td><td> 10 </td><td> 1.2 </td><td> 15 </td></tr></tbody></table> </li><li>一旦一切都正确对齐，关灯和芯棒和注射器泵打开。取下注射器铝箔。当流体是两针的技巧上可见，同时打开电源插头在fanners。 </li><li>当收集完成后，关闭电源和芯棒和拔掉fanners。小心地取出使用刀片的盖玻片和磁带。 </li><li>可视化的纤维，使用配备过滤罗丹明和DAPI荧光显微镜。看到图3的例子。注：荧光纤维是最清楚，以查看时短一段时间（&lt;3分钟）静电，但是，这个过程可以延长时间的无限量，以创造较厚的结构，尤其是不使用盖玻片， 。 </li></ol> D.在棚架的种子细胞<ol><li>将每个盖玻片连接到个人，以及一个适当大小的孔板与静电聚合物。注：与纤维细胞相互作用到methacrylated玻片上电，很容易可见。此外，MeRho可以再次用于光纤可视。 </li><li>孵育过夜，以确保完全去除溶剂和副产品的任何可能浸在PBS的棚架。 </li><li>从井中取出的PBS。 </li><li>消毒支架，放置在一个30分钟的层流罩下的杀菌灯。如果一个完整的支架正在播种，下一个30分钟的杀菌灯翻转的脚手架和地方。 </li><li>执行标准细胞培养，并准备在需要的细胞密度集中的细胞悬液（例如，6000细胞-2厘米）。例如，使用22毫米x 22毫米盖玻片的细胞悬液100毫升。在孵化器中放置1小时。 </li><li>细胞培养基中添加适量的每口井。进入孵化器放置的支架。 </li><li>使用市售的活/死套件染色的细胞。注：如DAPI（细胞核）和荧光标记的鬼笔环肽（肌动蛋白压力纤维），其他类型的细胞染色，可用于可视化的细胞。 </li><li>可视化细胞和纤维用荧光显微镜配备过滤TRITC和FITC。参见图4的例子。 </li></ol>代表性的成果： <img src="/files/ftp_upload/1589/1589figure1tmb.jpg" border="0" alt="图1" /> 图1。不结盟脚手架形成（上）和对齐支架形成（底部）的设备安装示意图说明。示例扫描电子显微镜图像，每种类型棚架所示。比例尺=5μm的。请<a href="https://www.jove.com/files/ftp_upload/1589/1589figure1.jpg">点击这里</a>看到图1的放大版本。 <img src="/files/ftp_upload/1589/1589figure2tmb.jpg" border="0" alt="图2" /> 图2。图案方法的原理。放置在光交联之间的光源光掩膜和支架，然后洗去未反应的聚合物所形成的模式。光罩和扫描电镜图像的孔隙的形成和冻干后支架。比例尺= 100微米。请<a href="https://www.jove.com/files/ftp_upload/1589/1589figure2.jpg">点击这里</a>看大图图2。 <img src="/files/ftp_upload/1589/1589figure3tmb.jpg" alt="图3" border="0" /> 图3多聚合物静电设置和必要的加工参数的原理图，以及一个代表了两种纤维的人群（例如：红梅阿和蓝色PEO）混合物的荧光图像，同时静电，形成多聚合物支架。比例尺= 100微米。请<a href="https://www.jove.com/files/ftp_upload/1589/1589figure3.jpg">点击这里</a> ，看到一个较大的version的图3。 <img src="/files/ftp_upload/1589/1589figure4tmb.jpg" border="0" alt="图4" /> 图4。一个例子活/死人类间质干细胞和它们之间的相互作用与静电纤维染色的形象。比例尺= 100微米。图4看到一个更大的版本， <a href="https://www.jove.com/files/ftp_upload/1589/1589figure4.jpg">请点击</a>这里。

<ol>
	<li>Burdick, J. A., Chung, C., Jia, X., Randolf, M. A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>Baker, B. M., Gee, A. O., Metter, R. B., Nathan, A. S., Marklein, R. A., Burdick, J. A., Mauck, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29, 2348-2358 (2008).</li><li>Ifkovits, J. L., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Photopolymerizable+and+degradable+biomaterials+for+tissue+engineering+applications.">Review: Photopolymerizable and degradable biomaterials for tissue engineering applications.</a> Tissue Engineering. 13, 2369-2385 (2007).</li><li>Khademhosseini, A., Eng, G., Yeh, J., Fukuda, J., Blumling, J., Langer, R., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromolding+of+photocrosslinkable+hyaluronic+acid+for+cell+encapsulation+and+entrapment.">Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment.</a> J. Biomed Mater Res A. 79A, 522-532 (2006).</li><li>Mauck, R. L., Baker, B. M., Nerurkar, N. L., Burdick, J. A., Li, W. J., Tuan, R. S., Elliott, D. M., M, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+on+the+Straight+and+Narrow:+The+Mechanics+of+Nanofibrous+Assemblies+for+Fiber-Reinforced+Tissue+Regeneration.">Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration.</a> Tissue Engineering B. 15, 171-193 (2009).</li><li>Sill, T. J., Von Recum, H. a. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+fibrous+scaffolds+with+multi-scale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multi-scale and photopatterned porosity.</a> , (2009).</li></ol>

静电是用来准备从聚合物纤维支架。 Photocrosslinkable支架的基础上被用来作为一个例证，在光线照射是需要交联透明质酸。随着反应macromers的使用，如梅阿，先前已证明增强细胞分布的通道被纳入棚架光交联过程中使用口罩，以形成宏观和微观多孔支架。此外，两个不同的聚合物，同时静电使用我们自定义的仪器。两种截然不同的纤维人口的存在，验证与荧光染料除了每个静电解决方案，后来用荧...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DAPI</td>
<td> Reagent</td>
<td>Invitrogen</td>
<td>D1306</td>
<td>&nbsp;</td>
<tr>
<td>I2959</td>
<td>Reagent</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEO 200 kDa</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>17503</td>
<td>&nbsp;</td>
<tr>
<td>PEO 900 kDa</td>
<td>Reagent</td>
<td>Sigma</td>
<td>189456</td>
<td>&nbsp;</td>
<tr>
<td>Methacryloxethyl thiocarbamoyl rhodamine B</td>
<td>Reagent</td>
<td>Polysciences</td>
<td>23591-100</td>
<td>Prepare stock solution in DMSO</td>
</tr>
<tr>
<td>Live/Dead Stain Kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>L3224</td>
<td>Contains Calcein (stains live cells green) and ethidium homodime (stains red dead cells)</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>Equipment</td>
<td>KD Scientific</td>
<td>KDS100</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Power Source</td>
<td>Equipment</td>
<td>Gamma High Voltage</td>
<td>ES30P-5W</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Motor</td>
<td>Equipment</td>
<td>Triem Electric Motors, Inc</td>
<td>0132022-15</td>
<td>Must attach to a custom built mandrel</td>
</tr>
<tr>
<td>Tachometer</td>
<td>Equipment</td>
<td>Network Tool Warehouse</td>
<td>ESI-330</td>
<td>Use to monitor mandrel speed</td>
</tr>
<tr>
<td>Omnicure UV Spot Cure System with collimating adapter</td>
<td>Equipment</td>
<td>Exfo Life Sciences Division</td>
<td>S1000</td>
<td>&nbsp;</td>
<tr>
<td>Silicone Tubing</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51135K151</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Female Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K293</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Male Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K143</td>
<td>&nbsp;</td>
<tr>
<td>Needles</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>14-825-16H</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>Equipment</td>
<td>Corning</td>
<td>2875-22</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrospinning fibrous polymer scaffolds for tissue engineering and cell culture

作为组织工程领域的的发展，是一个巨大的需求，生产更合适的材料和加工技术，以解决更复杂的器官和组织的要求（如机械和血管）。静电是一种流行的技术来创造，模仿原生细胞外基质的结构和大小规模的纤维支架。这些纤维支架也有用的，因为细胞培养基质的纤维可以被用来直接细胞的行为，包括干细胞的分化（见广泛的审查由莫克<em&gt;等。</em&gt;和西尔<em&gt;等。</em&gt;了解更多信息）。在这篇文章中，我们描述了静电纺丝聚合物的一般过程，作为一个例子，electrospin一个反应的光线照射交联透明质酸（见Ifkovits<em&gt;等。</em&gt;审查photocrosslinkable材料）。我们还介绍了进一步的处理能力，如photopatterning多聚合物支架形成。 Photopatterning可用于创建渠道和多尺度孔隙支架，以增加细胞浸润和组织分布。多聚合物支架是有用的，以便更好地调整棚架的属性（力学和退化），其中包括专为细胞浸润的孔隙度。此外，这些技术可以扩展到包括广泛的聚合物和反应macromers的创建提供必要的线索，开发成功的组织工程结构的复杂支架。

Watch this Scientific Journal Video about Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture at JoVE.com

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

静电聚合物组织工程和细胞培养的过程中，在这篇文章中解决。具体来说，静电photopatterning多聚合物静电额外的处理能力的光反应macromers描述。

静电纤维聚合物支架用于组织工程和细胞培养

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

electrospinning-fibrous-polymer-scaffolds-for-tissue-engineering-cell

Research

JoVE Journal

Biology

21.4K 次观看.  University of Pennsylvania. 静电聚合物组织工程和细胞培养的过程中，在这篇文章中解决。具体来说，静电photopatterning多聚合物静电额外的处理能力的光反应macromers描述。

视频: Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Experimental Approaches to Tissue Engineering

组织工程的实验方法

科研

生物学

Micro-scale Engineering for Cell Biology

细胞生物学的微观尺度工程

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

专访：干细胞研究的生物反应器和三维支架材料表面改性

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

组织工程细胞在三维凝胶封装

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

工程细胞透水蛋白

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

增强生物安全与聚合物复合物Ecotropic慢病毒的人体细胞的转导

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...