我们感谢德博拉·STUDER的帮助与bioprinter。 这项工作是由欧盟第七框架计划（FP7/2007-2013）根据赠款协议Ñ°NMP4-SL-2009-229292。

1。下游的泊洛沙姆407的解决方案如果可用，泊洛沙姆溶液进行准备在寒冷的房间（4℃）。如果没有可用的，一个玻璃瓶放置在烧杯中，用冰冷的水填充。在较高的温度下，将泊洛沙姆凝胶点以上，因此无法正确地溶解。 <ol><li>加入60毫升冰冷的PBS溶液倒入玻璃瓶中，并用磁力搅拌器剧烈搅拌。 </li><li>称取24.5克泊洛沙姆，并将其添加少量的冷PBS。等待，直到泊洛沙姆部分溶解后再加入更多的。 </li><li>搅拌溶液，直到所有的泊洛沙姆溶解。 </li><li>达到，直到最终体积为100ml，加入冷PBS。的最终浓度为24.5％（重量）/ v的</li><li>停止搅拌溶液，让它休息在4℃下，直到在溶液中的气泡和泡沫已经消失。被困在凝胶内的气泡，将被转移到的p子Rinter盒，将导致印刷牺牲模具缺陷。 </li><li>过滤器（0.22μm的滤膜）的溶液以去除任何不需要的颗粒可能堵塞针直接进入打印墨盒。滤波步骤应在冷室中进行（或如果不与冷却的提示，过滤器等 ），以避免在过滤器中的泊洛沙姆凝胶化。加载的墨盒保持在4℃，直到在实验前30分钟。 </li></ol> 2。三维打印机的下游在这项工作中所用的3D打印机从regenHU“生物工厂”。挤出系统的一部分，由几部分组成。下压在上面的盒被安装到连接器通过一个路厄锁定适配器。该连接器的桥梁在盒的出口和入口的电磁阀之间的空间。在出口处的电磁阀，具有不同直径的针都可以使用。素材是挤压到一个子施特拉特真空移动阶段举行。 图1中所示的系统的主要部分。其他挤出的系统可用于打印过程中，需要做的优化过程中，为每个系统。 <ol><li>电磁阀（喷嘴直径0.3毫米）和针（内径0.15毫米）放置在单独的1.5毫升试管中充满超纯水，将它们放置在一个加热的超声波浴中清洗30分钟。冲洗阀用乙醇清洗和干燥氮气枪。 </li><li>安装在打印机中的阀和针以及一个空的，干净的墨盒。 </li><li> 3巴的压力系统应用和安装阀针用压缩空气吹出来的任何残余液体。对于小直径的针，它是建议有一个过滤器（共同的注射器过滤器，孔径0.45μm），以避免安装在出口处的压缩空气的小颗粒可能堵塞针的条目。 </lI&gt; <li>关闭压力和安装墨盒装有泊洛沙姆。墨盒应该从冰箱取出墨盒安装前约30分钟，所以泊洛沙姆可以达到室内温度和凝胶。 </li><li> 3巴的压力下应用的系统和分配泊洛沙姆，直到它到达针尖和被挤压在一个连续的链。 </li></ol> 3。印刷参数的优化要创建精确的三维结构，在印刷过程中进行优化选择的材料和浓度。根据每种材料的粘度和三维打印系统将产生一个特定的分注量和线的厚度为一组固定的参数。 <ol><li>有了一个合适的CAD软件（能够从图纸创建ISO文件），绘制一个线长度大致相同的结构，你打算打印。 </li><li>放置在显微镜载玻片25毫米×75毫米×1毫米或在打印机中的任何另一方的基板，然后将它固定，打开真空。 </li><li>在打印机软件中，设置高频率为50赫兹，并设置3巴的高压电磁阀。 </li><li>打印一层的单个行，速度为300mm /分钟的阶段。 </li><li>降低压力，直到达到所需的线路宽度。您也可以控制音量，通过阀门的开口时，被挤出。 </li><li>降低频率的阀，直到没有连续的线可以被再印刷。选择的频率高于此值。 </li></ol>注意：一旦所需的线的宽度和连续线实现的，确定最佳的阶段的速度和针的升程，即后一个印刷层的层厚度。 <ol start="7"><li>打印彼此顶部几层，经过多次印刷层，如果针是在正确的位置上一层以上。调节层的厚度（针阀升程），以使每一层被印在顶部的下一个（ 图3）。 </li><li>降低的阶段的阶段速度300 mm / min的阶梯状，使挤压层的以前的相同的位置上（ 图4）的开始和结束。过高的阶段速度导致舞台移动的挤压材料之前已经触及上一层。 </li><li>打印的支柱结构遵循的步骤3.1.-3.8，但是，而不是绘制单行绘制一个点。参数，把重点放在打印时，支柱的压力（调节层的厚度和泊洛沙姆的柱直径），阀的打开时间（挤出量），并在打印头中的停留时间应存放的支柱上面的位置。 </li><li>当参数进行了优化，打印几层线应该导致在一个坚实的墙，或在案件之分，一个支柱。减参数，供以后使用。 </li></ol>使用过程中发现从这点上的优化过程参数。 <ol><li>一个显微镜载玻片上打印的内部结构（这里是一个支柱阵列），并让它干通宵。这是一个）降低的结构的尺寸和厚度，和b）提供了更好的结构和衬底之间的粘附，因此提离回填期间可避免。 </li><li>随着CAD软件绘制的结构，由外壁周围的结构，你打算洗脱，充满。打印与泊洛沙姆的结构。墙上的印刷将采取6分钟。 </li></ol>注意：要打印的壁具有至少为3.5毫米的内部结构，由于针的尺寸相差的。否则，印刷的外壁会破坏内部结构<ol start="3"><li>准备解决方案，您要回填SACR的ificial模具（这里是在去离子水中的1％琼脂糖）。琼脂糖溶液应具有在35℃和45℃之间的温度下下方此温度下，在琼脂糖固化速度太快，高于此温度下，它可能会破坏印刷支柱，因为的泊洛沙姆结构将软化。 </li><li>慢慢填补牺牲模具回填的解决方案，用吸管。这应该缓慢地进行，以避免破坏内部的壁的结构。 </li><li>让的回填溶液凝胶或交联，这取决于所用的聚合物。在琼脂糖固化的情况下发生在4℃下10分钟。 </li><li>将回填牺牲模具中，在冰浴中10分钟，以洗脱泊洛沙姆结构。 </li><li>结构回填用纸巾吸干，并把它放在一个新的显微镜载玻片。按该结构，小心地将显微镜载玻片之间的空间内的第三水凝胶的空隙，以避免漏回填结构和玻璃显微镜载片上。 </li></ol> 5。填补空隙<ol><li>为了填补留下的洗脱的泊洛沙姆的空隙，填充到配备了30 G针的注射器预期的聚合物溶液。在这个例子中，我们使用的是1％的海藻酸钠甲基丙烯酸酯在0.15M NaCl的溶液中，加入0.05％w / v的锂苯基-2 ,4,6-trimethylbenzoylphosphinate的（LAP）和2.5％（体积/体积）的Alexa-488的共轭纤维蛋白原。 Alexa的-488共轭纤维蛋白原增加可视化的目的。 </li><li>光聚合的聚合物具有高强度UV灯（100瓦，365纳米，与衬底的距离是3.5厘米）5分钟，图像构造使用的落射荧光或共聚焦显微镜。 </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+collagen+scaffolds+with+predefined+internal+morphology+made+by+solid+freeform+fabrication.">Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication.</a> Biomaterials. 24, 1487-1497 (2003).</li><li>Lee, W., 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=On-demand+three-dimensional+freeform+fabrication+of+multi-layered+hydrogel+scaffold+with+fluidic+channels.">On-demand three-dimensional freeform fabrication of multi-layered hydrogel scaffold with fluidic channels.</a> Biotechnology and bioengineering. 105, 1178-1186 (2010).</li><li>Turturro, M., Christenson, M., Larson, J., Papavasiliou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+(MMP)+sensitive+PEG+diacrylate+(PEGDA)+hydrogels+with+spatial+variations+in+matrix+properties+direct+vascular+cell+invasion.">Matrix metalloproteinase (MMP) sensitive PEG diacrylate (PEGDA) hydrogels with spatial variations in matrix properties direct vascular cell invasion.</a> J. Tissue. 6, 302-302 (2012).</li><li>Butterworth, A., Garcia, M. D. L., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photopolymerized+poly(ethylene)+glycol+diacrylate+(PEGDA)+microfluidic+devices.">Photopolymerized poly(ethylene) glycol diacrylate (PEGDA) microfluidic devices.</a> Roy. Soc. Ch. , 4-6 (2005).</li><li>Shachar, M., Tsur-Gang, O., Dvir, T., Leor, J., Cohen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+immobilized+RGD+peptide+in+alginate+scaffolds+on+cardiac+tissue+engineering.">The effect of immobilized RGD peptide in alginate scaffolds on cardiac tissue engineering.</a> Acta biomaterialia. 7, 152-162 (2011).</li><li>Jeon, O., Bouhadir, K. H., Mansour, J. M., Alsberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocrosslinked+alginate+hydrogels+with+tunable+biodegradation+rates+and+mechanical+properties.">Photocrosslinked alginate hydrogels with tunable biodegradation rates and mechanical properties.</a> Biomaterials. 30, 2724-2734 (2009).</li><li>Mauck, R. L., 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=Functional+tissue+engineering+of+articular+cartilage+through+dynamic+loading+of+chondrocyte-seeded+agarose+gels.">Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels.</a> J. Biomech. Eng-T Asme. 122, 252-260 (2000).</li><li>D'Arrigo, G., 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=Hyaluronic+acid+methacrylate+derivatives+and+calcium+alginate+interpenetrated+hydrogel+networks+for+biomedical+applications:+physico-chemical+characterization+and+protein+release.">Hyaluronic acid methacrylate derivatives and calcium alginate interpenetrated hydrogel networks for biomedical applications: physico-chemical characterization and protein release.</a> Colloid Polym. Sci. 290, 1575-1582 (2012).</li><li>Pescosolido, L., 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=Hyaluronic+Acid+and+Dextran-Based+Semi-IPN+Hydrogels+as+Biomaterials+for+Bioprinting.">Hyaluronic Acid and Dextran-Based Semi-IPN Hydrogels as Biomaterials for Bioprinting.</a> Biomacromolecules. 12, 1831-1838 (2011).</li><li>Guo, Y., 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=Hydrogels+of+collagen/chondroitin+sulfate/hyaluronan+interpenetrating+polymer+network+for+cartilage+tissue+engineering.">Hydrogels of collagen/chondroitin sulfate/hyaluronan interpenetrating polymer network for cartilage tissue engineering.</a> J. Mater. Sci-Mater. M. 23, 2267-2279 (2012).</li></ol>

作者无关申报。

这里，我们提出，在第一次，使用牺牲模，可在冷水中迅速溶出，由于约20℃的泊洛沙姆凝胶 - 溶胶转变温敏聚合物快速创建生物大分子结构不能有足够的分辨率打印速度的全过程，使泊洛沙姆有趣。这里描述的技术可以用于图案形成的水凝胶，水凝胶或在另一个先前已报告的其他材料35的微流体通道的创建。泊洛沙姆作为牺牲模具的优点是，它可以被印在任意的几何形状为固体层由层构?...

组织工程方法已经在过去几年取得了很大的进步，对于人体组织和器官的再生19,20。然而，到现在为止，已组织工程的焦点往往是有限的组织，具有简单的结构，或小的尺寸，如膀胱21,22或皮肤23-25。人的身体，但是，包含许多复杂的三维的组织，细胞和细胞外基质被布置在空间定义的方式。为了制造这些组织中，这种技术是必需的，可以放置在指定位置上的一个三维的结构内的细胞和细胞外基质的脚手架。生物印刷有潜力成为这样一种技术，制造复杂的三维组织的愿景，可以实现10,11,26-28。 生物印刷被定义为“材料转让过程使用的图案和组装生物相对埃文特材料-分子，细胞，组织，和可生物降解的生物材料-规定的组织来完成一个或多个生物功能“，它包含了几种不同的技术，工作在不同的分辨率和长度尺度，从亚微米分辨率的两个光子聚合29分辨率为150微米至420微米挤压印刷1,12,30。不是单一材料或材料组合将满足要求每个方法31。挤出印刷，关键参数是粘度和凝胶时间32，高粘度和快速凝胶化是可取的。 3D打印是一种技术，它允许创建复杂的几何形状30,33,34牺牲模具很容易的创建。这个过程是基于模具的建设，利用快速原型技术，如挤压bioprinter。使用创建的牺牲模具难以印刷由于其低的粘度和凝胶时间慢的材料，这些材料以形成复杂的结构。这里介绍的方法涉及到创建一个牺牲模具的材料组成的，在低温下迅速溶解，并可以准确地被挤压。的嵌段共聚物聚（乙二醇）99-聚（丙二醇）67-聚（乙二醇）99（也被称为的Pluronic F127或泊洛沙姆407）满足这些要求。它已经被用于在挤压印刷1中的修改后的版本，但据我们所知，从未被使用未修改的版本，由于它的不稳定性，在液体环境中的打印。泊洛沙姆407还示出了逆热响应行为18 即它从凝胶在冷却时的溶胶。最重要的是，它可以打印任意弯曲成复杂的结构具有非常高的保真度。这允许创建一个结构化的水凝胶从低粘度的材料，在这种情况下慢胶凝琼脂糖，该溶液通过移液到印刷的牺牲模具。具有高的保真度和快速洗脱从铸造的结构化的水凝胶，使得它快速和灵活的方法来创建不同的几何形状的模具，无需使用的平版印刷方法，因为它往往是必需的掩模或邮票打印的牺牲模具的组合。铸造后的结构化的水凝胶，可以进一步与另一种材料是不适合用于挤出印刷由于其低粘度填充。这是在我们的情况下，一个低粘度海藻酸钠丙烯酸甲酯的解决方案。在这里，我们提出的方法反向温敏凝胶图案牺牲模具使用支柱数组的例子。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Poloxamer (Pluronic F127)</td>
 <td>Sigma</td>
 <td>P2443</td>
 <td ></td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td><a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=10010015" target="_blank">10010-015</a></td>
 <td ></td>
 </tr>
 <tr>
 <td>CAD software</td>
 <td>regenHU</td>
 <td>BioCAD</td>
 <td ></td>
 </tr>
 <tr>
 <td>Alginate methacrylate</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Fibrinogen From Human Plasma, Alexa Fluor 488 Conjugate</td>
 <td>Invitrogen</td>
 <td>F13191</td>
 <td ></td>
 </tr>
 <tr>
 <td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)</td>
 <td>Innovent e.V Technologieentwicklung Jena</td>
 <td></td>
 <td >Synthesized by Innovent for the FP7 Project Nr NMP4-SL-2009-229292</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Lonza</td>
 <td>50004</td>
 <td ></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Bioprinter</td>
 <td>regenHU</td>
 <td>Biofactory</td>
 <td ></td>
 </tr>
 <tr>
 <td>Valve</td>
 <td>regenHU</td>
 <td>300 &mu;m Nozzel Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>regenHU</td>
 <td>150 &mu;m Inner Diameter</td>
 <td ></td>
 </tr>
 <tr>
 <td>Zeiss Axioobserver with ApoTome</td>
 <td>Zeiss</td>
 <td></td>
 <td ></td>
 </tr>
 <tr>
 <td>UV Light Source</td>
 <td>UVP</td>
 <td>Blak-Ray B-100AP High Intensity UV Lamp</td>
 <td>100 W</td>
 </tr>
 </tbody>
</table>

printing thermoresponsive reverse molds for the creation of patterned two-component hydrogels for 3d cell culture

生物印刷是一项新兴技术，在快速原型制造行业有它的起源。不同的印刷工艺可分为接触生物印刷1-4（挤出，蘸笔，软光刻技术），非接触式生物印刷5-7（激光的正向转 ​​移，喷墨沉积）和基于激光的技术，例如双光子光聚合8。它可用于许多应用，如组织工程9-13，14-16生物传感器的微细加工作为一种工具，回答基本的生物学问题，如影响共培养不同的细胞类型17。与常见的光刻或软光刻的方法不同的是，挤出生物印刷具有的优点在于，它不要求一个单独的掩模或印戳。使用CAD软件，可以快速地改变结构的设计和调整，根据运营商的要求。这使得生物印刷更灵活，比基于光刻的做法。 在这里，我们展示了印刷的牺牲模具来产生多材料的三维结构内的水凝胶，例如，使用一个数组支柱。这些支柱可以代表一个血管网或管内神经导向导管的空心结构。所选择的材料的牺牲模具是泊洛沙姆407，温度敏感型聚合物具有优异的印刷性能，这是液体在4℃，固体其胶凝温度以上〜20℃时为24.5％w / v的溶液18。这个属性允许基于泊洛沙姆牺牲模具洗脱需求在缓慢溶解的固体材料，尤其是狭窄的几何形状具有优势。泊洛沙姆印在显微镜载玻片创建牺牲模具。琼脂糖移液到模具中并冷却，直至凝胶化。泊洛沙姆在冰冷的水洗脱后，在琼脂糖模具的空隙被填充与藻酸盐丙烯酸甲酯藻IKED FITC标记的纤维蛋白原。填充空隙，然后交与UV落射荧光显微镜成像和结构。

代表性的结果表明的倒模技术（ 图2中所示）将创建结构化的第二材料，可填充有凝胶。在每次印刷过程中的开头的印刷参数首先优化。分步进行调整参数将导致在描绘在图3和图4单行打印时打印的多层构造。如果层厚度（后一个印刷层的针阀升程）过低时，人们会发现，针触摸前述层。如果针太高，出现波纹的表面上印刷的构造，将出现。此可以看出，在<st...

Watch this Scientific Journal Video about Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture at JoVE.com

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

àbioprinter被用于创建图案的水凝胶的基础上牺牲的模具。泊洛沙姆模具与第二水凝胶回填，然后洗脱，留下空隙充满了三分之一的水凝胶。此方法使用快速的洗脱和良好的印刷泊洛沙姆产生复杂的生物大分子结构。

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

printing-thermoresponsive-reverse-molds-for-creation-patterned-two

Research

JoVE Journal

Immunology and Infection

15.0K 次观看.  Cartilage Engineering & Regeneration. àbioprinter被用于创建图案的水凝胶的基础上牺牲的模具。泊洛沙姆模具与第二水凝胶回填，然后洗脱，留下空隙充满了三分之一的水凝胶。此方法使用快速的洗脱和良好的印刷泊洛沙姆产生复杂的生物大分子结构。

视频: Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

科研

免疫与感染

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

类器官的模型传染病：人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

流式细胞仪为基础的检测方法的NK细胞功能的监控

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

小鼠和人源性胃上皮单层培养对再生的研究

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

巨型囊泡内单细胞层的细菌细胞培养

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

反向转录循环介导的等温放大（RT-LAMP）测定，用于对罗非鱼湖病毒进行特定和快速检测

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...