This research was supported by a National Leading Research Laboratory Program (Grant NRF-2013R1A2A1A05006378) through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning. The authors also acknowledge a KAIST Systems Healthcare Program.

1.制备微模式化模具和水凝胶的<ol><li>通过标准的两步光刻技术15,17用于铸造PDMS模具生产使用的SU-8光致抗蚀剂的硅晶片的表面上所希望的微观图案。示出的例子使用一个肝小叶状网状图案（ 图1）。 </li><li>称出PDMS和固化剂溶液与1:5的比例（即，12.5克PDMS和2.5克固化剂）。 </li><li>彻底混合15克的溶液，脱气气泡在真空室中，然后蔓延混合溶液到硅晶片的微图案化表面均匀内的箔铸造菜。 </li><li>放置在硅晶片上65℃下加热板90分钟在一个平面上以固化的PDMS。 </li><li>从铸造盘和硅晶片剥离固化的PDMS。 </li><li>切的PDMS的边缘，并且将其放置到一个100毫米直径的培养皿所述微图案的一面朝上。 </li><li>使用70％的乙醇和蒸馏水主灭菌培养皿洗涤微图案固化的PDMS。然后，完全进行10分钟干燥后在炉中在65℃。 </li><li>溶解并混合的O / N 3克在100毫升蒸馏水的粉末状非离子表面活性，产生3％（重量/体积）涂布液。 </li><li>放置微图案的PDMS模具在等离子体清洁器然后清洁处理1分钟（85瓦，0.73毫巴）以创建一个亲水性表面，以促进加入的含水液体。然后，外套与至少3小时（ORO / N）用实验室摇杆100 ml的表面活性剂溶液与PDMS的表面上。 </li><li>洗从PDMS模具表面活性剂溶液并完全干燥后在炉中在65℃下进行10分钟。然后，灭菌每个微图案模具通过暴露于紫外线（UV）辐射30多分钟。 </li></ol> 2.准备细胞悬浮在水凝胶前体<ol><li>至制备水凝胶前体，溶解在10ml磷酸盐缓冲盐水（PBS）的0.1克藻酸钠粉末，形成1％（重量/体积）藻酸盐前体。完全溶解粉末，孵化，然后将它们O / N。 </li><li>过滤通过连接至1ml注射器0.22微米过滤的溶液。 </li><li>培养的HepG2细胞在补充有10％胎牛血清和1％青霉素-链霉素上的常规组织培养皿直到70％Dulbecco改良的Eagle培养基（DMEM） -在5％CO 2的湿润培养箱80％汇合，在37℃ 。 </li><li>洗一次用PBS将细胞，然后加入1毫升0.05％的胰蛋白酶-EDTA 4分钟，在5％CO 2的湿润培养箱中在37℃trypsinize它们。离心机从培养皿收获在250×g离心3分钟，重悬，用1毫升的PBS之后除去上清液的细胞。 </li><li>用自动细胞计数单个分布式细胞在PBS中的数计数器。 </li><li>离心后，在250×g离心3分钟并去除的PBS，加入1毫升的1％（重量/体积）的藻酸钠溶液到其余的细胞沉淀，并使用移液混合它们平缓。细胞的最终接种密度应该是5×10 6 - 10 7细胞/毫升。孵育细胞/水凝胶悬浮液，在5％CO 2的加湿培养箱中在37℃。 </li></ol> 3.装载和交联的细胞/水凝胶暂停<ol><li>溶解1.47克氯化钙脱水在100毫升DDH 2 O的以生产交联试剂（ 即 100毫米氯化钙2·2H 2 O在DDH 2 O的）。 </li><li>冲洗使用70％的乙醇超声换能器加湿器的内部，并用200毫升的交联试剂的填充。加湿器为110毫米宽300毫米长和170毫米深（ 即 110毫米×300毫米×170毫米（宽x高x深））。 </li><li>放置微图案PDMS模具在等离子体清洁器然后清洁处理1分钟，在85瓦到创建一个亲水表面。 </li><li>稳步加载7.2微升充分混合细胞/水凝胶悬浮液的在模具中的微图案的边缘。示出的例子使用一个肝小叶状网状图案（ 图1）。悬浮液的体积取决于地形图案。 </li><li>为了实现细胞/水凝胶悬浮液的凝胶化，打开加湿器，并验证该加湿器产生的交联试剂的雾。喷交联试剂到水凝胶前体为5分钟，覆盖范围为5厘米之内的PDMS模具的表面地形。 注意:距离更长和更短于5厘米可能导致不完整的和不均匀的凝胶化，分别。确保该加湿器具有250毫升/小时的喷雾速度，生产在5分钟; 20毫升的交联试剂的雾。 </li><li>继交联过程中，关闭加湿器并填写PDMS莫LDS用PBS。 </li></ol> 4.处理单模块化水凝胶表<ol><li>通过用200微升枪头，轻轻吹打PBS周围的水凝胶片分离的微图案模具各硬化水凝胶片。 </li><li>拿起使用最终裁员1000微升吸管尖端每个浮动含水凝胶片。每个肝小叶状网状凝胶片具有8毫米×8.7毫米的尺寸，并且在100 - 200微米厚。 </li><li>使用水凝胶构造作为一个单元部件中的12孔板过了一个星期转移水凝胶片材的单层在1ml DMEM中在12孔板中，并培养所述细胞在体外以浮动方式，在5 ％CO 2的加湿培养箱中在37℃。交换培养基隔日1次。 </li></ol> 5.装配多层凝胶表<ol><li>重复步骤1.1至1.5，以生产出18毫米×18毫米x 4 mm尺寸的PDMS一个框架（宽x高×深），并且含有170μm的高柱结构在其下表面。使用42克PDMS和固化剂的混合物的，具有相同的比例如在步骤1.2。放置在硅晶片上一个专门的聚碳酸酯模的PDMS框架来创建具有8毫米×9mm的×2 mm尺寸的内部框架（宽x高x深）。 </li><li>消毒PDMS帧和180微米孔径的尼龙滤纸于121℃浸没在蒸馏水和镊子在高压釜中进行15分钟。 </li><li>放置在一个60毫米直径的陪替氏培养皿的灭菌的PDMS帧到四分之一一张尼龙滤纸（直径5厘米）的。 </li><li>传送一个模块化的水凝胶片成使用端切断1000微升枪头PDMS帧的内部。用于组装的水凝胶张应培养至少他们被制造后一天。 </li><li>使用空200μl的移液管尖对准每个模块化的水凝胶片与PDMS框架的边缘。 </li><li> 6层 - 使用模块化的水凝胶片材，以形成一摞4重复步骤5.4和5.5。 </li><li>通过在PDMS帧的底部流动它通过所述柱结构除去培养基。再加入2微升藻酸盐溶液（2％重量/体积）在多层构建体的一个角落。 </li><li>喷交联试剂进行30秒的雾到多层构建体附加的每个层的边缘与另一。使用2毫升交联剂的雾（在250毫升/小时的喷雾速度）。 </li><li>轻轻漂洗多层构建体用400μl的DMEM，并使用镊子除去PDMS帧。 </li><li>通过与挺杆继加入4ml DMEM中的细胞轻轻擦拭分离从滤纸上的多层构建体。 </li><li>转移的多层构造到6孔板装有3毫升DMEM中使用滤纸，及培养的细胞在体外以浮动方式，与水凝胶构建体在37℃下在6孔板在一个星期，在5％CO 2的加湿培养箱多尺度蜂窝脚手架。交换培养基隔日1次。 </li></ol>

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The authors have nothing to disclose.

这个协议提供了一种制造模块化的水凝胶片，并把它们组装，以形成三维的细胞支架的一个简单方法。 为了构建在短时间内鲜明图案化藻酸盐的结构，我们应确定一个交联的过程，可以创造足够刚性的结构，以从模具保持复杂微图案，以及维持细胞活力和新陈代谢。我们已经开发出一种交联方法，包括溶胶 - 凝胶转变，喷使用以雾状形式的加湿器的交联试剂。如果没有这种交联过程中，微规模图案不能在薄水凝胶的形式产生构造与有限扩散深度（100 - 200微米），它具有高扩散渗透率到的代谢物和营养物质。 60分钟，18 - - 20交联剂钛此外，与现有的扩散为基础的方法，这需要30相比我是相对较短（3 - 5分钟）。 此外，也有人有挑战性操纵薄水凝胶片材，其中只有在液体介质是机械稳定的。 如图1所示，薄水凝胶结构简单且稳定地处理，并用一个端切断常规叶尖为不同培养条件之间transferal操纵。使用产生和操作技术，我们可以提供不仅在用于药物试验的潜在应用浮动方式单层片也堆叠的多层结构，具有潜在的应用作为支架用于组织样构建。 多层蜂窝架构可以通过堆叠模块化单层片材的水凝胶（图3A）来生成。每个模块化水凝胶片材可以包含不同类型的细胞，并且可以暴露于不同的培养条件。这些多层构建体然后可以选择性地ASSEM流血。然而，简单地堆叠水凝胶片可导致细胞坏死的组装结构内时的总深度超过限定扩散深度（图3C）。为了克服这个问题，有必要对齐微筛孔图案，如肝小叶状网格中，当多层片材的水凝胶被组装。因此，我们需要具有相同的内部图案的PDMS帧作为水凝胶片的边界对齐堆叠片材的水凝胶。因此，在组装好的网状水凝胶片对准孔阵列微图案可用于形成膨胀管道结构，有利于更有效的养分运输改善细胞生存力（图3B）。 这种技术的一个限制是不存在的藻酸盐水凝胶的细胞 - 基质相互作用的。藻酸盐不能提供细胞粘附的配体在体内样细胞-基质相互作用; 21然而，它确实关闭器的机械和几何效应作为三维细胞包封支架与细胞 - 细胞相互作用。对于原代细胞和干细胞，藻酸盐可用于以下的修改与含RGD的肽，21或与胶原或明胶组合。14,18另一个限制是，一旦细胞包埋凝胶片被制造为低于低细胞密度5×10 6个细胞/ ml，建筑的多层结构的可导致水凝胶片之间弱的附件，因为结构仅边缘被固定通过使用附加藻酸盐。然而，这些层可以紧凑地堆叠经由排水构造与PDMS帧下一个支架。此外，细胞在水凝胶片材的更高密度可以增殖的表面，以及一天培养后的层，这将有助于层之间的附件，如图3A内。 水凝胶片工房这里描述和灰和操作技术可适于体外培养系统和组织样细胞模型，以提供在体内样的微环境的应用，包括细胞分析和工程人工脏器。这种技术可以适用于基于细胞的药物测定和需要不同几何三维蜂窝微型和结合了各种细胞或水凝胶类型macroenvironments生物学研究。

水凝胶是特别有前途的生物材料，并且预计将在基础生物学，药理学试验和医学重要1的水凝胶为基础的蜂窝构造的Biofabrication已经建议减少使用的动物实验中，2,3-取代移植组织，4和提高基于细胞的测定。5,6-含有水（加氢）的粘弹性材料（凝胶）允许进行封装和保持在支架结构，以控制3D细胞微环境的大量细胞。与微流体或微图案的技术的指导组合，该水凝胶结构的几何形状可以精确地在细胞比例控制。迄今为止，多种水凝胶的形状，包括颗粒，7 - 9纤维，10 - 12，片材，13 - 15已经被用作建筑物单元在自下而上的Appro酸痛到宏观规模的多细胞结构的制造。 既基于水凝胶的颗粒和纤维一直容易且迅速地制造为应用程序作为微尺度蜂窝环境中，使用微流体装置的流体控制。然而，如工程化组织的基本单位，它将是复杂的重新排列，并扩大其体积为宏观结构。16，是更难以实现宏观规模构建体相比，以产生微米级的基本模块。可用于水凝胶基的构建体的片状单元通过简单的组装过程来增加支架的体积。必然，水凝胶片材的堆叠层不仅提供一个容积增加，而且在一个三维空间中的几何延伸。 我们以前曾报道制造微图案化的水凝胶片材，13的方法- 15连同其组装成多捻ERED蜂窝架构。该技术使得复杂微图案和蜂窝结构的模块化设计通过多层结构的层叠过程。通过堆叠模块化水凝胶片，这是微图案的制造中，可以实现一个三维细胞培养系统具有受控的宏观尺度细胞微环境。此视频协议描述了一个简单而强大的制造方法，可用于构建模块化水凝胶片材，基于人肝癌细胞系（HepG2细胞）。我们证明本文简单操作这些图案化的模块化的水凝胶片材，和它们的组装成的多层结构。

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning Corporation</td><td>000000000001064291</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td>Powdered nonionic surfactant&amp;nbsp;</td></tr><tr><td>Alginic acid sodium salt, low viscosity</td><td>Alfa Aesar</td><td>B25266</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td></td></tr><tr><td>Ultrasonic humidifier</td><td>MediHeim</td><td>MH-2800</td><td>Modified equipment, Maximum sprayed rate: 250 ml/hr</td></tr><tr><td>Nylon net filter hydrofilic, 180 &amp;mu;m</td><td>EMD Millipore</td><td>NY8H04700</td><td></td></tr><tr><td>Polycarbonate mold</td><td></td><td></td><td>Customized mold for fabrication of a PDMS frame pattern</td></tr></tbody></table>

construction of modular hydrogel sheets for micropatterned macro-scaled 3d cellular architecture

水凝胶可以在使用微流体或微图案的技术，以提供在体内样的三维（3D）几何组织微观尺度进行图案化。所得三维水凝胶为基础的蜂窝结构已被引入作为替代动物实验为先进的生物研究，药理学试验和器官移植的应用程序。尽管水凝胶为基础的颗粒和纤维，可以容易地制造，这是很难操纵它们用于组织重建。在这种视频中，我们描述了一种制造方法，微图案化藻酸盐水凝胶片材，以及它们的组件，以形成一个宏观尺度三维细胞培养体系具有受控细胞微环境。 200微米，具有精确的微图案 - 采用钙胶凝剂薄雾形式，薄水凝胶片材易于与厚度在100〜生成。细胞然后可与水凝胶片材在几何指导培养独立的条件。此外，水凝胶片材可以容易地使用微量具有端切割尖端操纵，并且可以通过使用图案化的聚二甲基硅氧烷（PDMS）帧堆叠它们组装成多层结构。这些模块化水凝胶片，其可以使用一个浅显工艺制造，具有的体外药物分析和生物学研究，包括微观和宏观结构和组织重建的功能研究潜在的应用。

我们已经描述了制造和操纵独立的细胞水凝胶表。 如图1中，我们制备微图案化的PDMS模具，和包含细胞的水凝胶上样到这些模具的亲水性表面和交联使用增湿以产生胶凝剂的气溶胶雾。以下从模具释放，HepG2细胞都在独立的凝胶片材具有各种图案（图2）中培养。因此，该薄水凝胶片提供了一种三维培养环境。此外，模块化的水凝胶片材可以通过层叠片材使用具有端切断尖端微量移，并使用PDMS帧，这使得3D宏观尺度细胞培养物（ 图3）进行组装。 <img alt="图1" src="/files/ftp_upload/53475/53475fig1.jpg" /> 图1.流量 查RT显示水凝胶板和多层水凝胶片的制作。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53475/53475fig1large.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/53475/53475fig2.jpg" /> 图2. 独立式三维细胞培养中的各种水凝胶表。喷雾启用藻酸盐水凝胶的精确图案形成在微观尺度交联剂的制造方法。此外，所制造的水凝胶片材可以从模具被收获并在独立的条件下很容易地操纵。 HepG2细胞图案化，并在一平的水凝胶片 （A）中培养，水凝胶片带六角支柱（B）目（C）中，肝小叶状目（D）中，孔的阵列（E）和含多处Ëmicrocomb般的微纤维（F）。该比例尺如下:500微米（A，D，F），100微米（B，C），和2毫米（E） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53475/53475fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53475/53475fig3.jpg" /> 图3. 装配各类水凝胶薄片的微模式水凝胶片为宏观尺度蜂窝构造。可以通过层-层堆叠与对准装配，包括含HepG2细胞5水凝胶片沾上绿色荧光和NIH3T3细胞的组件沾满了红色荧光（A）。微图案化的水凝胶片材具有扩展流水线结构（B）的一个组件提供更高的细胞活力塔n表示非图案化的水凝胶片材（C）的7天后的。的存活率，通过染色HepG2细胞用钙黄绿素-AM（显示为绿色的活细胞）和乙锭同型二聚体-1（示出为红色的死细胞）进行评估。比例尺:500微米（A）和 200微米（B，C） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53475/53475fig3large.jpg" target="_blank">点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture at JoVE.com

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

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 ...

construction-modular-hydrogel-sheets-for-micropatterned-macro-scaled

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.2K 次观看.  Korea Advanced Institute of Science and Technology (KAIST). 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. 

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

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 ...

科研

生物工程

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

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

在单元格拉丹水凝胶中刺激细胞行为的应变梯度芯片

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

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 ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

一种简便快速制备磁响应元件的水凝胶基微型机械的添加剂制造技术

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

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 ...

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 ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

来自微凝胶棒的互连大孔3D支架

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...