作者要感谢实验室的教授安东尼Windebank DRG的清扫和文化分享他们的专业知识，以及Shaochen陈教授关于DMD的设置有益的讨论。这项研究是杜兰大学和路易斯安那州摄政委员会（补助部分资金由LEQSF [2009-10] - RD - A - 18）和美国国立卫生研究院（NS065374）。

1。 DMD的安装<ol><li>的DMD板，UV光导（准直器）和4倍物镜都是垂直安装隔振表。 </li><li>紫外线光导应进行调整，使照射角度为45 °的相对的镜子，镜子的平面低于24 °（图1）平面镜阵列。 </li><li>物镜安装物镜的距离，从DMD对应焦距的镜头。 </li><li>下面放置一个倒置显微镜的物镜，这样，从DMD的反射聚焦光线可以通过显微镜观察。从物镜的聚合表面的距离应约与镜头的工作距离。然后在显微镜阶段可以用来调整这个距离，以精细的重点图像。这个距离可能会有所不同，取决于选择的聚合表面。 </li></ol> 2。双水凝胶的结构，组织外植体培养 A.背根神经节外植体的附着力<ol><li> 6以及胶原蛋白涂层细胞培养插入（康宁）与雨- X的照顾，以避免膜本身，外壳的墙壁。 （另外，疏水性障碍笔可能被使用。） </li><li>一夜之间粘附在孵化器中的1.5毫升37 ° C和5％的CO 2水合物插入。附着力介质与10％的小牛血清，1％青霉素/链霉素，0.5mm的L -谷氨酰胺，和20微克/毫升的NGF neurobasal中等。 </li><li>收获胚胎背根神经节（DRG）从E - 15幼鼠。 DRG应接种到胶原蛋白涂层的6孔插入，插入多达4％。 </li><li>孵育2小时插入允许DRG的坚持膜。 </li></ol> B.动态遮罩聚合数字微镜器件（DMD）是一个1024 × 768阵列的个别镜子，投影电视，有选择性地反射光线的基础上镜的位置。对于我们而言，DMD是使用模式紫外线（UV）光到photocrosslinkable凝胶，建立在一个简单，快速的方式可指定凝胶几何。图1描绘了DMD和紫外线光路设置。虽然我们的DMD是一个独立的单元，也可以被集成设备与许多现有显微镜的使用。 <ol><li>从插入，删除所有多余的液体，添加500μL聚合介质内插入。聚合介质中含有10％PEG（分子量1000）和0.5％Irgacure 2959溶解在辅以20微克/毫升NGF的和1％笔/链球菌neurobasal中等。 </li><li>将下的雨- X的处理过的载玻片上DMD器件的插入。 </li><li>装入适当的黑白图像，作为“光罩”的DMD使用，通过使用所包含的ALP的3个基本的GUI程序。对于我们而言，选择一个分叉的形状，让突起的制导系统的实施。 </li><li>使用一个可视化的倒置显微镜，将与适当的位置使用一个DMD的反射的可见光源光罩组织植。 </li><li>开关紫外线光源的可见光源，照亮的PEG溶液，直到交联是足够的。 （和5.0瓦/厘米2 DMD的事件，交联，可在低至55秒完成的条件。），为所有四个插入DRG的重复。 </li><li>每个插入洗净，用无菌DPBS笔，链球菌和1％的三倍。 </li><li>添加下面的细胞培养插入1.5ml的生长介质，并允许在一个孵化器30分钟的平衡。生长介质neurobasal培养基中含有2％的B - 27，1％笔/链球菌，0.5 L -谷氨酰胺，和20μg/ mL的NGF的。 </li></ol> C.二次水凝胶 Puramatrix <ol><li>神经元的应用，1％Puramatrix是根据制造商的指示，在无菌的H 2 O的0.15％稀释，并辅以1微克/毫升，溶于层粘连蛋白。 </li><li>从PEG的空隙，其中包含DRG的外植体，用无菌棉尖撒施，Kimwipe，或微量，必须清除所有多余的媒体。 </li><li> Puramatrix添加聚乙二醇与微空隙，为了填补空的空间而不溢出。根据空隙率，通常〜1μL，每建造。 </li><li> Puramatrix开始与生理盐溶液接触后立即自组装的过程中，即肿的PEG，但1.5毫升培养液添加下面的插入，并在孵化器中放置一小时，以确保总胶凝。 </li><li>最初，Puramatrix是微酸性，所以一小时后改变媒体允许的pH值达到平衡。 </li><li>媒体的变化需要大约每48小时。要小心，所有的媒体是添加下面的插入，保护机械薄弱Puramatrix的完整性。 </li></ol>琼脂糖<ol><li>琼脂糖神经元的应用，是在生长培养基稀释至1％溶液，并放置在60℃水浴，直到琼脂糖完全溶解（约1小时）。解决的办法是再辅以1微克/毫升溶于层粘连蛋白。 </li><li>从PEG的空隙，必须清除所有多余的媒体。 </li><li>琼脂糖加入PEG的空隙，为了填补空的空间而不溢出。根据空隙率，通常〜1μL，每建造。 </li><li> 1.5毫升培养液预冷（8℃），在6孔板，琼脂糖转移到冷冻媒体的插入，并维持在8℃冰箱至少3分钟允许琼脂糖凝胶。 </li><li>最后，插入转移到1.5毫升（37℃）预热生长介质，并在孵化器保持在37 ° C，并要求每48小时的媒体的变化。 </li></ol> 3。双水凝胶的三维细胞封装双水凝胶封装是适当的时候使用任何自组装凝胶。 photocrosslinkable凝胶，在这种情况下挂钩，作为一个结构的自组装凝胶几何演示支持，例如Puramatrix或琼脂糖。一些方法，特别是凝胶和光掩膜选择的类型，将取决于特别是所需的应用程序。 <ol><li>对DMD加载一个合适的面具。对于我们的应用，细胞的生存，我们只是选择了一个圆柱形的Puramatrix介绍，以帮助在细胞成像。细胞信号转导的研究人员可能会在房室几何感兴趣，以便趋化分子的扩散。此外，动脉的粗略估计显示表示可能血管研究中的应用。 </li><li>治疗聚酯细胞插入降雨- X的文化，在DMD的地方有雨 - X涂层幻灯片的墙壁。 </li><li>加入500μL聚合介质插入。聚乙二醇交联诱导暴露于紫外灯55秒。 </li><li>洗净，用无菌DPBS笔，链球菌和1％的三倍。 </li><li>从PEG的空隙中删除所有多余的介质。 </li><li>离心所需的密度成颗粒细胞。必须小心去除细胞沉淀中混合前，Puramatrix开始接触后立即与盐溶液自组装。暂停细胞，在无菌的H 2 O稀释0.15％Puramatrix内，辅以10 ％的蔗糖。 </li><li>在PEG的空洞内注入Puramatrix /细胞/蔗糖的混合物。 </li><li>插入下面的中等增长1.5毫升，并允许一小时内孵化器的凝胶。 </li><li>一小时后更改的媒体，以后每48小时〜。 </li></ol> 4。单水凝胶的三维细胞封装一个单一的水凝胶封装将适用于任何其中一个photocrosslinkable水凝胶内的细胞，可以检查的情况。 <ol><li>对DMD加载一个合适的面具。对于细胞的存活研究，我们再次选择了一个基本的圆形面具代表一个圆柱体。口罩类似的方法4所示，可以再次申请相应的研究领域。 </li><li>治疗聚酯纤维细胞培养一个有雨- X的插入，在DMD的地方有雨 - X涂层幻灯片。 </li><li>在任何所需浓度的细胞，可直接添加10％的PEG溶液，混合，以确保均匀分布。 </li><li>加入500μL聚合介质插入。聚乙二醇交联诱导暴露于紫外灯55秒。 </li><li>洗净，用无菌DPBS笔，链球菌和1％的三倍。 </li><li>填写增长低于插入顶部的媒介，改变每1〜2天。 </li></ol> 5。薄膜水凝胶的聚合<ol><li>对DMD加载一个合适的面具。 </li><li>治疗胶原蛋白涂层聚酯雨 - X细胞培养插入，并有雨 - X涂层幻灯片。 </li><li>加入足够的聚合介质，以刚好盖住底部插入（〜6孔板插入250-300μL）。允许媒体渗透插入膜在室温为30-45分钟。 </li><li>删除多余的聚合介质，用微量或Kimwipe插入。添加足量的紫外线透明油完全覆盖插入底部。允许插入坐15-30分钟，在室温下，油，形成一个独特的层以上的聚合介质的饱和插入膜足够长的时间。 </li><li>将根据DMD的插入和幻灯片。由暴露在紫外线照射下诱导的聚乙二醇交联。由于交联的PEG层的薄，可以在短短15秒完成，在5.0 瓦 /厘米2事件对DMD。 </li><li&gt;洗净插入三次，用无菌DPBS笔，链球菌和1％。 （如果油性残留物的持久性是一个问题，一个温和的清洁剂，如吐温20（1％）可能被添加缓冲液洗。） </li><li>店在6孔组织培养板的插入。悬浮细胞生长介质中添加所需浓度和足够量的细胞悬液，细胞内的文化插入吸管，以获得所需的的细胞密度。然后，添加下面插入足够的生长介质，完全保持细胞活力（〜1.5毫升）。 </li><li>经过48小时后，用无菌DPBS和1％的笔链球菌插入三次驱逐任何unadhered细胞。变化，大约每48小时一次的媒体。 </li></ol> 6。代表性的成果双水凝胶的例子构造含有背根神经节植都在图2所示。注意，细胞迁移和突起的延伸是有限的，以双水凝胶的细胞宽松的地区兴建。图3描述了同样封装在双水凝胶结构的分离细胞。由于DMD的光掩膜的动态性质，是有限的，只有通过进行封装的几何尺寸和分辨率的光学。细胞封装，也有可能在一个单一的photopolymerizable水凝胶，聚乙二醇，并进行现场/死可行性测试图4证明。封装在PEG只是作为一个例子，如聚乙二醇，并不代表一个理想的环境，为神经细胞。因此，在我们的PEG构造实现细胞活力，这是可以理解的。最后，利用薄的PEG作为图案的限制性细胞粘附层细胞培养插入电影的例子，如图5所示。此外，提供可能的“坏”结果的例子在图6。 结果只占一小部分，在我们的实验室开发的方法可能使用。他们是为了证明我们的方法的易用性，通用性和可行性，并可以作为“证据原则”处理的研究人员开发出他们自己可能适应。 <img src="/files/ftp_upload/2636/2636fig1.jpg" alt="图1" /> 图1：用于光刻的光路示意图。插图：紫外灯照射角度45 °，24 °以下的平面反射镜阵列的DMD。 <img src="/files/ftp_upload/2636/2636fig2.jpg" alt="图2" /> 图2含双水凝胶结构在背根神经节标记的增长和扩散。 AD）的图像，把它与聚乙二醇结构（灰色）的Beta三微管蛋白（绿色），DAPI染色细胞核（蓝色）标记的突起。载于背根神经节植Puramatrix位于图案的圆形区域，日益走向分岔（S）的突起，。 <img src="/files/ftp_upload/2636/2636fig3.jpg" alt="图3" /> 图3，上午与钙黄绿素，活细胞标记，标记的细胞生长的培养基在48小时后的双水凝胶结构。 AD）的各种PEG的形状，充满含有分离DRG神经元（〜5 × 3细胞/ mL）Puramatrix。 <img src="/files/ftp_upload/2636/2636fig4.jpg" alt="图4" /> 图4：单水凝胶含有钙黄绿素AM（绿色）和乙啶二聚体- 1（红色）标记后，在生长中期的24小时（5X10 3细胞/ mL）死细胞标记的活细胞构造。 <img src="/files/ftp_upload/2636/2636fig5.jpg" alt="图5" /> 图5。细胞限制性聚乙二醇聚合使用“测试模式”，有选择性地坚持分离的细胞胶原膜的透水支持涂薄电影。 A，B）活细胞标记后48小时内与钙黄绿素AM（绿色），而乙啶二聚体- 1（红色）标记的死细胞。最小的细胞粘附在薄的聚乙二醇膜面积发生。 <img src="/files/ftp_upload/2636/2636fig6.jpg" alt="图6" /> 图6。不良的结果代表的图像。一）部分聚合，导致不可用聚乙二醇PEG的构造。在预聚物中，聚合介质的不足，紫外线照射不足或不当的光学重点的半月板的存在，可能是由于不正确的聚合。二）形象塑造的Beta三微管蛋白（绿色），DAPI染色细胞核（蓝色）标记的突起的聚乙二醇结构（灰色）。该突起能够成长以外的图案PEG渠道。这往往发生在条件挂在注射部分的顶部Puramatrix溢出。

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C., Roy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+digital+micro-mirror+device-based+system+for+the+microfabrication+of+complex,+spatially+patterned+tissue+engineering+scaffolds.">A digital micro-mirror device-based system for the microfabrication of complex, spatially patterned tissue engineering scaffolds.</a> Journal of Biomedical Materials Research Part A. 77, 396-405 (2006).</li><li>Naiser, T., Mai, T., Michel, W., Ott, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+maskless+microscope+projection+photolithography+system+and+its+application+in+light-directed+fabrication+of+DNA+microarrays.">Versatile maskless microscope projection photolithography system and its application in light-directed fabrication of DNA microarrays.</a> Review of Scientific Instruments. 77, 063711-063711 (2006).</li></ol>

本文所述的方法可用于任何寻求简单，重复性的细胞培养系统的调查。从理论上说，由于多种可用photopolymerizable凝胶，环境可定制允许使用任何类型的细胞，包括整个组织植。此外，双水凝胶系统可以改善的空间控制在介绍自我聚合水凝胶，往往会形成对自己的无定形的形状。由此产生的“2.5D”micropatterned水凝胶的结构提供了一个2D的配置，使便捷的微观评价中的神经生长的3D矩阵。的基板上的凝胶聚合，也可以是多种多样的，允许更大的控制实验设计。我们的方法是优化利用细胞培养透水支持，正如我们所看到玻片上（数据未显示）的聚合相比，提高生存能力（图4）。然而，其他聚合表面可能更适用不同的应用：在微流体实验或细胞的总单位，例如，使用在玻璃上制作幻灯片。 我们这些文化系统的经验，确定潜在的困难地区。首先，细心的技术要求，以保持构造的不育。由于笨重的DMD设置性质，它是难以操作的无菌条件下聚合的步骤。为了解决这个问题，描述的方法冲洗步骤是有益的，应在所有媒体使用抗生素。此外，最终聚合结构的厚度和形状是高度依赖的预聚物的混合物的流体行为，和半月板的存在可以导致凝胶结构，太薄或不完全聚合（图6）。可以采取两个步骤来减少形成的半月板内细胞培养插入。对于厚的水凝胶（&gt; 200微米），周围的内壁插入雨- X的简单的涂层是足够的。然而，简要介绍了上述结构（&lt;200微米）薄，油层是需要尽量减少半月板和否定氧自由基聚合淬火。决议被发现，厚度而定，在实现降低特征尺寸越来越较厚的凝胶，。该决议也各不相同，取决​​于功能是否代表在水凝胶的积极或消极的救济。但是，我们取得了足够高的分辨率与最小特征尺寸的构造上〜100只用显微镜目标聚焦光学微米的规模。 我们的实验表明，这里描述的双水凝胶结构的代表形成突起的生长和指导的基本体外模型奠定了良好的基础。缩微技术是一种适应18,19现有的方法，但我们设置强调一个简单的实现设计和生产细胞培养插入双水凝胶结构优化，提高细胞活力，以及重要的细胞培养插入以前壁组织外植体周围的交联。结果的范围是有限的，我们实验室的利益，但是，我们相信，在本出版物中描述的方法寻找一个相对便宜，快捷，易于使用的三维细胞培养制造方法的研究人员将证明是有益的的模型。

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Digital Micromirror Device</td>
<td>&nbsp;</td>
<td>Texas Instruments</td>
<td>DLPD4X00KIT</td>
<td>&nbsp;</td>
<tr>
<td>Collagen Coated Transwell Permeable Support </td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3491</td>
<td>Also referred to as Cell Culture Insert in manuscript</td>
</tr>
<tr>
<td>Polyester Transwell Permable Support</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3412</td>
<td>Also referred to as Cell Culture Insert in manuscript</td>
</tr>
<tr>
<td>Neurobasal Medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-036</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-164</td>
<td>&nbsp;</td>
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<td>Nerve Growth Factor</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13257-019</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
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<td>B-27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
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<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>&nbsp;</td>
<tr>
<td>Puramatrix</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354250</td>
<td>&nbsp;</td>
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<td>PEG 1000</td>
<td>&nbsp;</td>
<td>Polysciences, Inc.</td>
<td>15178</td>
<td>&nbsp;</td>
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<td>Irgacure 2959</td>
<td>&nbsp;</td>
<td>Ciba Specialty Chemicals</td>
<td>0298913AB</td>
<td>&nbsp;</td>
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<td>Oil</td>
<td>&nbsp;</td>
<td>Have used both canola oil and silicon oil</td>
<td>&nbsp;</td>
<td>Needs to be UV transparent, and minimize +/- meniscus formation</td>
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<td>OmniCure Series 1000</td>
<td>&nbsp;</td>
<td>Exfo</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Rain-X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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fabrication of micropatterned hydrogels for neural culture systems using dynamic mask projection photolithography

越来越多，图案的细胞培养环境，成为相关技术研究细胞的特点，和许多研究人员相信在3D 环境代表在体外实验中， 更好地模仿在1-3体内素质的需要。在诸如癌症研究4，5神经工程，心脏的生理6，和细胞基质的相互作用7,8等领域的研究表明，细胞的行为不同于传统的单层培养和三维结构之间的大幅。 水凝胶作为3D环境，因为他们的品种，多功能性和能力，量身定制，通过官能分子组成9-12。创建作为支持细胞矩阵结构，其中包括13静电，弹性体邮票14中存在大量的技术，喷墨印刷，添加剂photopatterning 16，静态的光掩模投影光刻17，动态遮罩microstereolithography 18 15。不幸的是，这些方法涉及到多个生产步骤和/或设备不容易适应传统的细胞和组织培养方法。在本议定书中所采用的技术调整后两种方法，使用数字微镜器件（DMD）创建动态交联几何特定的聚乙二醇（PEG）的水凝胶，通过紫外线发起自由基聚合诱导的光掩膜。由此产生的“2.5D”的结构提供了一个3D环境中，神经生长的制约。我们采用双水凝胶的方法，其中PEG作为一个细胞限制性地区供应结构，否则不成形，但宽容的细胞自组装Puramatrix或琼脂糖凝胶的。这个过程是一个快速简单的一个步骤制造，这是高度重现性好，与传统的细胞培养方法和基板的使用很容易地适应。 整个组织植如胚胎背根神经节（DRG），可以被纳入突起生长的实验检测，如双水凝胶结构。此外，分离的细胞可以被封装在photocrosslinkable或自我聚合凝胶，或选择性地坚持以渗透的支持膜细胞限制性photopatterning。使用的DMD，我们建立了水凝胶的结构〜1mm厚，但薄膜（&lt;200微米），PEG的结构是由氧自由基聚合反应的淬火有限。随后，我们开发出一种技术，利用聚合液，使薄的聚乙二醇结构聚合层以上的石油。 在这个协议中，我们描述了微的神经细胞和组织培养生产的三维凝胶系统迅速建立。双水凝胶结构表明，此处代表在体外模型，可能被证明在神经细胞的存活，迁移，和/或突起的生长和指导的研究涉及非常有用的多才多艺。此外，作为协议可以用于许多类型的水凝胶和细胞的潜在应用是千姿百态，具有广阔的。

Watch this Scientific Journal Video about Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography at JoVE.com

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

简单的技术是使用常规的细胞培养基质的动态遮罩投影光刻数字微镜器件的微型神经文化系统的快速生产。这些文化系统可能更天然生物结构的代表，和描述的技术可以适应各种应用。

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 ...

fabrication-micropatterned-hydrogels-for-neural-culture-systems-using

Research

JoVE Journal

Bioengineering

18.6K 次观看.  Tulane University. 简单的技术是使用常规的细胞培养基质的动态遮罩投影光刻数字微镜器件的微型神经文化系统的快速生产。这些文化系统可能更天然生物结构的代表，和描述的技术可以适应各种应用。

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

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

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.

的微型双元应变计测量加工和植入<em&gt;体内</em&gt;胃肠收缩鼠害。

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

基因转染向着文化微模式球体板细胞基因修饰的细胞移植

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

微京尼平沉积技术的微模式化血管肌肉薄膜的扩展文化

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

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 ...

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 ...