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><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</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>&lt;u&gt;&lt;a href=&quot;http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;amp;productID=10010015&quot; target=&quot;_blank&quot;&gt;10010-015&lt;/a&gt;&lt;/u&gt;</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 colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Bioprinter</td><td>regenHU</td><td>Biofactory</td><td></td></tr><tr><td>Valve</td><td>regenHU</td><td>300 &amp;#956;m Nozzel Diameter</td><td></td></tr><tr><td>Needle</td><td>regenHU</td><td>150 &amp;#956;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

Universidad de Cartagena UDEC

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

构建模块化水凝胶片的微模式化宏观缩放3D细胞结构

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

用于可重复模式形成的混合凝胶立方体器件中的初始三维单元群集控制

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

光调谐生物打印水凝胶，用于探测细胞对 ECM 硬化的反应

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

作者聚焦：使用 3D 打印光可调谐水凝胶了解慢性肺病 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

3D生物打印光可调水凝胶研究成纤维细胞活化

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...

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

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用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

用于可重复模式形成的混合凝胶立方体器件中的初始三维单元群集控制

趋化梯度多细胞响应的三维分析

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

3D生物打印光可调水凝胶研究成纤维细胞活化

3D生物打印光可调水凝胶研究成纤维细胞活化