Name: light-mediated formation and patterning of hydrogels for cell culture applications
Uploaded: 2016-09-29T15:00:00.000+00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications at JoVE.com

这项工作是由药物发现和由机构发展奖从美国国立卫生研究院（分别为P20GM104316和P30 GM110758-01，）的将军医学科学研究所资助的高级生物材料，皮尤慈善信托基金特拉华COBRE方案的支持（00026178），国家科学基金事业奖（DMR-1253906），在宝来惠康基金（1006787），并在特拉华大学的美国国家科学基金会IGERT SBE2程序（奖学金到L. Sawicki）。作者感谢在特拉华大学特拉华生物技术研究所生物成像中心培训，并获得共聚焦显微镜，凯瑟琳·威利女士在视频拍摄过程中的帮助，马修Rehmann先生慷慨地提供从骨髓，克里Ĵ教授孤立的hMSCs 。Kloxin和Stephen马先生慷慨地提供光掩膜和威尔弗雷德陈教授对于使用自动酶标仪的。 </p&gt;

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The authors have nothing to disclose.

这里介绍的过程演示技术通过硫醇 - 烯点击化学凝胶形成内photoencapsulate细胞，随后photopattern与生化线索凝胶。利用光的最初形成水凝胶可以在聚合之前在聚合物溶液内均匀混合和细胞的悬浮液中。快速聚合"锁"在定义的模具的形状的凝胶，并封装悬浮在水凝胶网络中的细胞。凝胶还可以模塑成根据所需的最终应用许多不同的形状（ 例如 ，玻璃载玻片或注射器提示）。例如，作为光衰减的薄样品内限定在附连到玻璃载玻片水凝胶包封的用于3D培养细胞可用于成像应用特别有用。注射器的模具可用于细胞的快速封装，允许在很短的时间内制备的样品更大数目（相对于载玻片s）表示可用于诸如流式细胞仪或定量PCR需要大量的细胞的实验。随后，这些凝胶然后可以与生化线索以引发所需的细胞反应，例如分化或侵入构图。30,31 为生存力和代谢活性测定表明呈现细胞供料系统和构图条件的存活。注意，代谢活性，直至3个天非可降解的凝胶监测（RGKGRK肽交联），以评估聚合和构图条件对细胞功能的初始作用。此外，在1和6天封装后在可降解肽交联（GPQGIWGQ）制定凝胶支持细胞保持活力，并在1周文化传播的hMSCs的膜完整性检测（现场/死可行性/细胞毒性染色）。的额外的细胞系的存活力已被报道用于32类似于光图案的条件此处使用，并且能够用于使用活/死染色和代谢活性试验中所描述的水凝胶系统进行评估。虽然我们没有观察到使用这种材料系统和相关的程序（4.2和4.3）的细胞活力的问题，一些类型的细胞可以是用自由基和/或光照射敏感。在这种情况下，用户可能33考虑使用非光引发材料系统，如叠氮化物-炔烃，12 FXIII，31或根据阿尔德-狄尔斯水凝胶形成化学。 轻便的技术来检测和凝胶还呈现（3.1和3.2）中的量化生化线索图案化。埃尔曼的测定法是特别感兴趣的，因为试剂是可商购的并且无需额外的合成处理步骤或更昂贵的试剂（ 例如 ，荧光标记的肽）。埃尔曼的测定可用于精确地确定与下各色生化线索的游离硫醇的变形nt的光图案化的条件下，以及迅速可视化模式。用于定量肽掺入之前和图案化后的硫醇官能团的浓度，作为肽掺入的定量测量，直接评估用埃尔曼的测定。虽然这种类型的定量可以用荧光标记的肽来完成，34基于成像的量化，需要耗费更多的时间处理和分析的步骤（ 例如 ，校准曲线的荧光标记的肽和产生的合成涉及荧光肽浓度利用图像分析）。用于成像的肽掺入Ellman试剂可直接应用于样本，并立即可视化。而限定于X和Y平面为图案的可视化，该技术可作为一个简单的常规方法来确定是否含有游离硫醇基的基质已经形成图案。要注意的是埃尔曼的测定不是C是很重要的onsidered细胞相容性，因此，虽然它也可以用于观察和量化photopatterns，它不能在细胞存在下进行。在三个维度和在细胞的存在成像图案，荧光肽的水凝胶基质中的共轭仍然是一个功能强大的和广泛使用的方法。图案的分辨率可以在x进行评价，y和使用共聚焦显微镜z轴架，并且该方法是细胞相容性，使得图案化的区域或非图案区域内的细胞可以被识别。总之，埃尔曼的检测和基于成像技术是研究人员评估在数量上和质量上的材料体系内生化线索的光图案的补充工具。 Photoclick，或者更广泛地光引发，在细胞的存在的水凝胶的形成和改性化学是很多的。这里介绍的成型，封装和图案化技术是不限量资讯科技教育所描述的材料系统，并且可以应用到替代基于光的化学品，如硫醇炔，35叠氮化物-炔烃，4和其它硫醇-烯化学（ 例如 ，巯基降冰片烯），10,13以及与不同的光引发剂，如的Irgacure 2959，曙红Y，和樟脑醌。注意，用户可根据需要调整工艺参数（ 例如 ，孵育时间，聚合时间，细胞密度），以确保条件保持细胞相容性，这些其他系统。由于图案化工艺要求的alloc修饰肽（多个）扩散到水凝胶（2.2.3-2.2.5），此过程可能证明对于增加整联蛋白结合部分（ 例如 ，肽或细胞外基质蛋白的最有用片段）的水凝胶，其中需要用于由细胞和完全整合激活产生牵引力的配体与网络的附着。36注，对生物分子可是类似活性在溶液中或在固定（ 例如 ，生长因子或细胞因子），对部分扩散（〜1小时）温育步骤进入水凝胶可能导致信令卷积构图结果事件。其他方法已建立了生长因子的固定或本地封存其图案化。37-39此外，图案的分辨率是通过在曝光控制支配。这里，光掩模允许通过凝胶深度，并在X和Y平面图案创作;然而，用凝胶内生化线索的图案化较大的空间控制可以与照射的替代方法，如使用双光子共焦显微镜来产生在x模式，y-和z-平面来实现。34,40最后，要注意的是，虽然此过程中使用的材料系统只最初与生化线索改性，正交photoclick化学可以用于允许阿尔特是很重要这里提出在基体性能口粮随着时间的推移12的过程和技术添加多样性到当前的方法为具有良好限定和spatiotemporally控制属性创建合成基质。特别是，该过程中使用的试剂和材料的商业可用性将是广泛的兴趣，在使用基于水凝胶的生物材料用于在受控细胞培养应用的研究是有用的。

点击化学在对众多的生物医学应用，包括药物递送，组织工程，和受控的细胞培养材料的设计越来越多地使用的，由于其选择性的，有效的，并且经常细胞相容性的反应性。这利用光触发1-3 Photoclick化学或引发反应（ 例如 ，叠氮化物-炔，4-硫醇-烯，5和四唑-烯烃6）是用于生物材料的形成或修改特定的兴趣。温和的条件和何时以及控制那里发生的光使这些反应非常适合于细胞的存在的生物材料性能的用户控制的控制。-7,8-具体地说下快速率，硫醇-烯photoclick化学已被用于以生成具有强大的机械性能5,9和用于多种细胞类型，包括，但封装基于水凝胶的生物材料不电讯有限公司于，人类间质干细胞（hMSCs），成纤维细胞，软骨细胞和胰腺癌细胞，用细胞培养和交付承诺。-10,11-此外，这些化学品已被用于生化线索的空间图案来模仿的关键方面天然细胞微环境和促进适当的细胞-基质相互作用，包括粘附，分化和侵入。3,12 用于与光硫醇-烯水凝胶，含有半胱氨酸（硫醇）肽通常是具有与细胞相容性的条件下快速，光引发聚合的丙烯酸酯或降冰片烯（&#39;烯&#39;）官能化的聚合物进行反应。建设13展开该工具箱，我们试图建立对于水凝胶的形成与所需的最少的合成处理或者是对他们的合成细胞外基质广泛使用市售新品多功能和可访问的构建块的方法。14具体来说，肽用氧羰修改（的Alloc）保护赖氨酸:一个吊坠，整联蛋白结合组来促进细胞粘附[K（ALLOC）GWGRGDS = Pep1Alloc]或两个非降解或细胞降解的交联[K（ ALLOC）RGKGRKGK（ALLOC）G或KK（ALLOC）GGPQGIWGQGK（ALLOC）K = Pep2Alloc，分别]。与这些序列中，分别建立了快速反应（1-5分钟）四臂硫醇改性聚使用细胞相容性剂量长波长UV光（10毫瓦/ cm 2的在365nm处）和（乙二醇）（PEG4SH）的条件下光引发剂的锂苯基-2,4,6- trimethylbenzoylphosphinate（LAP）。所得水凝胶是数周的细胞培养条件下是稳定的。以使细胞驱动的降解和重塑，酶可切割的肽凝胶交联（ 即 ，GPQGIWGQ），15和模型的初级细胞，人类间质干细胞（hMSCs）内引入，仍然封装和都灵后高度可行这些矩阵内摹文化。此外，肽已被空间地这些材料中图案化，和的hMSCs保持存活和光图案化的条件下代谢活性。备用挂件肽序列，这里未示出（ 例如 ，IKVAV，YIGSR，GFOGER 等 ），也可以矩阵内掺入探测与周围的微环境额外细胞相互作用。这些结果是有希望的为这些基于水凝胶的材料的应用为三维（3D）细胞培养和输送来研究和直接的细胞 - 基质相互作用为多种细胞类型。 此处，方法photoencapsulate细胞和所提出的水凝胶系统内随后photopattern生化线索呈现（ 图1）。技术来观察，也量化这些photopatterns被证明:值得注意的是，ⅰ）的定量和定性用埃尔曼的测定法来确定modifica的图案的基板和ii）互补的定性使用荧光肽（AF488Pep1Alloc）的范围内的免费硫醇重刑在三个层面观察这些模式。此外，分析以确定存活力（活/死活力/细胞毒性染色）和代谢活性（MTS; 3-（4,5-二甲基-2-基）-5-（3-羧基甲氧基）-2-（4-磺苯基） -2H四唑）呈现，使用户可确定为水凝胶基质中的不同细胞系photoencapsulation和光图案化条件下细胞相容性。虽然展示了一个浅显基于光的photoclick水凝胶系统中的协议，该技术可以在细胞的存在可以应用于对photoencapsulation和光图案许多其它自由基引发的水凝胶系统。

<table><tbody><tr><td>Custom Peptides</td><td>Various Vendors</td><td>----</td><td>Peptides may also be synthesized via standard SPPS techniques with materials from vendors including ChemImpex and ChemPep.</td></tr><tr><td>4-arm PEG Thiol, MW 20k</td><td>JenKem USA</td><td>4ARM-SH</td><td>Listed under Multi-arm Homofunctional PEGs. PEG4SH may also be synthesized as previously referenced.</td></tr><tr><td>Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate</td><td>Colorado Photopolymer Solutions</td><td>Li-TPO</td><td></td></tr><tr><td>Dimethyl Phenylphosphonite</td><td>Sigma Aldrich</td><td>149470</td><td>Caution, causes severe skin burns and eye damage. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>2,4,6-Trimethylbenzoyl Chloride</td><td>Sigma Aldrich</td><td>682519</td><td>Caution, causes severe skin burns and eye damage. Wear protective gloves, clothing, and eye protection.</td></tr><tr><td>Lithium Bromide</td><td>Sigma Aldrich</td><td>213225</td><td></td></tr><tr><td>2-Butanone</td><td>Sigma Aldrich</td><td>360473</td><td></td></tr><tr><td>Flask, Round Bottom, 100&amp;nbsp;ml</td><td>Chemglass</td><td>CG-1506-05</td><td></td></tr><tr><td>80 ml Filter Funnel, Buchner, Medium Frit</td><td>Chemglass</td><td>CG-1402-L-02</td><td>Filter paper inside a regular glass funnel may be used if desired.</td></tr><tr><td>Magnetic Stir Bars</td><td>Various Vendors</td><td>----</td><td></td></tr><tr><td>Magnetic Stirring and Hot Plate</td><td>Various Vendors</td><td>----</td><td></td></tr><tr><td>Vacuum Dessicator</td><td>Various Vendors</td><td>----</td><td></td></tr><tr><td>Deuterium Oxide</td><td>Acros Organics</td><td>16630</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>ThermoFisher Scientific</td><td>14190-250</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>ThermoFisher Scientific</td><td>15070-063</td><td></td></tr><tr><td>Fungizone</td><td>ThermoFisher Scientific</td><td>15290-018</td><td></td></tr><tr><td>Trypsin-EDTA (0.5%), no phenol red</td><td>ThermoFisher Scientific</td><td>15400-054</td><td>Select appropriate medium and trypsin depending on cell type.</td></tr><tr><td>Microcentrifuge tubes</td><td>Various Vendors</td><td>----</td><td>1.5 or 2 ml sizes, sterile.</td></tr><tr><td>BD Syringe with Slip (Luer) Tips (Without Needle)</td><td>Fisher Scientific</td><td>14-823-16H</td><td>Product number listed here is for a 1 ml syringe (16H), Various sizes are available (14-823-XX).</td></tr><tr><td>Fisherfinest Premium Plain Glass Microscope Slides</td><td>Fisher Scientific</td><td>12-544-1</td><td></td></tr><tr><td>High-Purity Silicone Rubber, 0.010&quot; Thick, 6&quot; x 8&quot; Sheet, 55A Durometer (Gasket)</td><td>McMaster Carr</td><td>87315K62</td><td></td></tr><tr><td>Ethanol, 200 Proof</td><td>Decon Labs</td><td>2701</td><td></td></tr><tr><td>RainX, Original</td><td>Amazon</td><td>800002243</td><td>May be purchased from other vendors.</td></tr><tr><td>Sigmacote</td><td>Sigma Aldrich</td><td>SL2</td><td></td></tr><tr><td>Photomasks</td><td>Advance Reproductions Corporation</td><td>----</td><td>Photomask Division, different designs may be printed as desired.</td></tr><tr><td>DTNB; Ellman&#039;s Reagent; 5,5-dithio-bis(2-nitrobenzoic acid)</td><td>ThermoFisher Scientific</td><td>PI-22582</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma Aldrich</td><td>S5136</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma Aldrich</td><td>E6758</td><td></td></tr><tr><td>Sodium Hydroxide, Pellets/Certified ACS</td><td>Fisher Scientific</td><td>S318</td><td></td></tr><tr><td>Orthophosphoric Acid</td><td>Alfa Aesar</td><td>33266</td><td></td></tr><tr><td>Cysteine Hydrochloride Monohydrate</td><td>Sigma Aldrich</td><td>C7880</td><td></td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td><td>ThermoFisher Scientific</td><td>L-3224</td><td></td></tr><tr><td>CellTiter 96 Aqueous One Solution Assay</td><td>Promega</td><td>G3582</td><td></td></tr><tr><td>Centrifuge</td><td>Various Vendors</td><td>----</td><td>Capable of speeds at 90-110 x g.</td></tr><tr><td>Multiwell Plate Reader</td><td>Various Vendors</td><td>----</td><td>Capable of reading absorbance at 405 nm in a 96-well plate.</td></tr><tr><td>Epifluorescent or Confocal Microscope</td><td>Various Vendors</td><td>----</td><td>To visualize peptide patterns and cells within hydrogels.</td></tr><tr><td>Omnicure Exfo Series 2000</td><td>Excelitas Technologies</td><td>----</td><td>Alternate light systems may be used to polymerize hydrogels.</td></tr><tr><td>Zeiss Zen Lite Software</td><td>Zeiss</td><td>----</td><td>Available at zeiss.com; compatible with images taken on Zeiss microscopes</td></tr><tr><td>ImageJ</td><td>NIH</td><td>----</td><td>Available at imagej.nih.gov; applicable for general image analysis</td></tr></tbody></table>

light-mediated formation and patterning of hydrogels for cell culture applications

点击化学已被研究用于大量生物材料的应用，包括药物递送，组织工程和细胞培养。特别是，光介导的点击反应，如光引发硫醇 - 烯和硫醇炔反应，得到过的材料特性的时空控制，并允许系统的设计具有高度用户控制的特性的控制。制造和使用由光以及由这些硫醇-X photoclick化学提供的多功能性，得到精度基于水凝胶的生物材料的变形是越来越大的兴趣，特别是对于细胞的明确定义的，仿生的微环境中的培养。在这里，我们描述了细胞，并使用由硫醇 - 烯photoclick聚合反应灵活，模块化的构造块水凝胶基质内生化线索的后续光图案的photoencapsulation方法。具体地讲，一种方法提出了共从烯丙氧基羰nstructing水凝胶（的Alloc）-functionalized肽交联和挂件肽部分和硫醇官能化的聚（乙二醇）（PEG），在锂acylphosphinate光引发剂和细胞相容性剂量长波紫外（UV）光的存在下迅速聚合。轻便的技术来可视化光图案和量化描述加入的肽的浓度。此外，方法，是为包封细胞，特异性人类间质干细胞，并确定其活性和活性。而形成与硫醇的alloc水凝胶的初始图案显示在这里，这些技术广泛地可应用于其他一些光以及自由基引发材料系统（ 如 ，巯基降冰片烯，硫醇-丙烯酸酯）的生成图案化衬底。

设置和步骤photoencapsulate细胞和含有包封的细胞随后photopattern凝胶在图1中所描绘的，和用于制备储液的例子，以形成10重量％的凝胶在表1中提供。使用表1中 ，单体的量（PEG4SH ，Pep2Alloc，±Pep1Alloc）和聚合的水凝胶需要光引发剂（LAP）的计算。基于这些计算，PEG4SH，Pep1Alloc，Pep2Alloc和LAP的储备溶液制备并有和没有Pep1Alloc用于形成和分别光图案凝胶，（ 图1A）混合。接着，将细胞从平板收集，计数，并以适当的量离心封装（ 图1B）。将细胞沉淀重新悬浮在凝胶形成溶液（在PBS中的肽/聚合物/ LAP），和细胞/单体混合物转移到玻璃或注射器摩尔DS。细胞被在光应用包封在水凝胶内（1-5分钟10毫瓦/厘米2在365纳米的）（ 图1C）。对于光图案（ 图1D），凝胶浸泡Pep1Alloc和LAP 30分钟到1小时和30分钟，使肽和引发剂扩散到聚合基质。这些肽载货凝胶覆盖有具有期望图案的光掩模，并暴露于光（1分钟）的第二剂量，以在基质内缀合的肽游离硫醇。挂件系绳仅共价连接到该凝胶在暴露于光的区域，有利于适当的细胞-基质相互作用和模仿朝向探测细胞功能和命运体外天然细胞微环境的关键机械和生物化学特性。 埃尔曼的检测提供了一种简便且廉价的方法来量化photopatte内凝胶修改和肽掺入rned基板。图案化和未图案化的凝胶（ 即 ，具有或不具有肽修饰凝胶）在埃尔曼的反应缓冲液中后聚合浸泡。接着，半胱氨酸标准和在缓冲液浸泡过的凝胶放置在48孔板并用Ellman试剂（ 图2A，左）温育。 1小时30分钟后，将样品的等分试样放置在96孔板的各个孔中，并在吸光记录在405nm处。从半胱氨酸标准校正曲线（ 图2A，右）绘制（吸光度VS浓度，线性拟合），并且可基于其稀释因子来确定的游离硫醇在凝胶中的量。这些游离硫醇浓度为10％（重量）的凝胶的各种聚合和光图案化条件示于图2B。无Pep1Alloc 1或5分钟聚合（蓝条，I = 1分钟和第II = 5分钟）凝胶具有统计学类似的游离巯基含量，表明Ť帽发生快速反应，凝胶化是1分钟（双尾t-检验，p&gt; 0.05）内完成。因此，附加的暴露于光（2-5分钟）不会导致的官能团的进一步转化。无Pep1Alloc 1分钟聚合凝胶与Pep1Alloc浸泡（3毫克/毫升）和LAP（2.2毫摩尔）30和90分钟（绿色棒，II = 30分钟，IV = 90分钟），并暴露于光的第二剂量1分钟。在游离硫醇的减少（60-80％相对于1分钟条件）表示在这些条件下挂件线索凝胶的有效修饰。如果较高的修饰是期​​望的，增加的Pep1Alloc溶液浓度可以用作Pep1Alloc的无障碍游离硫醇在更稀肽溶液可限制转换;例如，我们已经发现，浓度高达20毫克/毫升Pep1Alloc产生&gt; 90％的游离硫醇的转换。 独特的是，肽的图案添加到该水凝胶可用Ellman试剂（ 图3A，下5分钟）被快速成像。然而，图案的可视化失去了由于通过凝胶黄色TNB 2-二价阴离子的扩散时间（大于5分钟）。为了提高成像分辨率并在三个维度（x轴，y和z平面）和在细胞存在下观察的图案，荧光肽加成（AF488Pep1Alloc）可被利用。在图3B中x，y和用共焦显微镜拍摄的图像栈的z突起示出，表明图案分辨率（微米尺度）。 约（94±2）％和可降解凝胶（Pep2Alloc = GPQG↓外高桥）内包封的hMSCs（94±1）％保持存活（绿色细胞体）的封装后1天和6天，分别用几个死细胞（红核）观察到（ 图4A）。此外，hMSC的铺展6天后封装（观察<st<html>荣&gt;图4A，插图），这表明细胞可重塑和与整联蛋白结合RGDS改性这些MMP降解基质相互作用。封装在非可降解凝胶（Pep2Alloc = RGKGRK）细胞进行代谢活性测定法1和3天的封装后（ 图4B）提供细胞活力的第二测量并证明细胞保持与测试的各种photoencapsulation和光图案化条件活性埃尔曼的测定（ 图2B）。特别是，有代谢活性30分钟和90分钟的孵育之间与Pep1Alloc + LAP（条件III和IV）和初始封装（条件I和II）无显著差异，这表明该过程适合于在光图案的应用包封细胞的存在（双尾t-检验，p&gt; 0.05）。 2fig1.jpg"/&gt; 图 1: 设置为水凝胶中封装的细胞，随后与生化线索光图案 （A）准备大分子单体和光引发剂的储备溶液混合（PEG4SH =骨干，Pep2Alloc =交联，Pep1Alloc =挂件粘接部分（RGDS），LAP =光引发剂）。 （B）将细胞收集封装。 （C）的细胞用没有或整联蛋白结合肽大分子单体溶液（PEG4SH / Pep2Alloc / LAP或PEG4SH / Pep2Alloc / Pep1Alloc / LAP，分别）混合，并在暴露被封装到的光。 （D）的过程中凝胶形成含过量硫醇基凝胶（这里，PEG4SH / Pep2Alloc / LAP，有2mM过量硫醇形成的）可与生化线索通过随后加入与单一的alloc基（Pep1Alloc， 例如官能化肽的图案化， RGDS，IKVAV， 等），以促进细胞粘附内的凝胶的特定区域。在这里，用荧光标记的RGDS凝胶的图案所示。 <a href="https://www.jove.com/files/ftp_upload/54462/54462fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54462/54462fig2.jpg" /> 图 2: 定量埃尔曼的实验设置和结果来评估图案化水凝胶的变形例 （A）的凝胶和半胱氨酸标准与Ellman试剂48孔板温育。线性标准曲线绘制以确定凝胶样品中的半胱氨酸的浓度。 （B）多余的硫醇期间凝胶形成水凝胶内注册成立，并消耗后，图案带有侧ALLOC肽（I = 1分钟聚合; II = 5分钟聚合; III = 30分钟孵育Pep1Alloc; IV = 90分钟孵化无线<html>日Pep1Alloc;既III和IV聚合和配光图案的1分钟）。显示的数据说明了平均值（N = 3）误差条显示标准误差。 <a href="https://www.jove.com/files/ftp_upload/54462/54462fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54462/54462fig3.jpg" /> 图 3: 埃尔曼的测定和荧光图像来可视化photopatterned水凝胶 （A）的 Ellman试剂可用于快速检测在x图案（线）和y平面（黄色=未图案化区域，比例尺= 1毫米）。 （B）的荧光肽可用于在X观察的图案，y和z平面（绿色=图案化区域;200μm的比例尺; 10X水浸渍目标;实施例/ EM五百二十五分之四百八十八纳米）。large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54462/54462fig4.jpg" /> 图 4: 活力和不可降解的水凝胶photopatterned细胞内的代谢活性 （A）为例焦Z堆栈（Z-投影; 10X水浸渍目标）的活的（绿色;前/ EM五百二十五分之四百八十八NM）和死的hMSCs（红色;前/ EM580分之543nm）的封装内水凝胶后24小时封装（比例尺= 200微米）。细胞封装（插入图像，比例尺= 50微米）6天后这些水凝胶内扩散。 （B）细胞的代谢活性的1和3天（深色和浅色条，分别）封装后的各种聚合（I = 1分钟聚合; II = 5分钟聚合）和构图条件（III = 30分钟孵育Pep1Alloc ; IV = 90分钟incubatio类n Pep1Alloc;两个聚合和配光图案的1分钟）。显示的数据说明了平均值（N = 3）误差条显示标准误差。 <a href="https://www.jove.com/files/ftp_upload/54462/54462fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54462/54462tbl1.jpg" /> 表1: 示例设置来计算股票的解决方案体积制作水凝胶被蓝色高亮显示在电子表格中单元格表示用户定义的参数。的其它量是基于这些设定来计算。每个原液，使凝胶的最终卷突出橙色。白细胞包含用于计算基于所述用户定义的参数的最终体积的公式。需要注意的是2.2毫LAP的浓度相当于0.067％（重量）。

Watch this Scientific Journal Video about Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications at JoVE.com

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

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 ...

light-mediated-formation-patterning-hydrogels-for-cell-culture

Research

JoVE Journal

Bioengineering

12.9K 次观看.  University of Delaware. 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. 

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

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 ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

生物化学

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

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

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 ...

免疫与感染

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 ...

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 ...

生物学

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生物活性蛋白或多肽在水凝胶上的应用

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

超薄 Porated 弹性水凝胶作为双细胞培养的仿生基底膜

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

使用水凝胶图案化技术控制细胞粘附，用于牵引力显微镜的应用

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...