Name: microfluidic genipin deposition technique for extended culture of micropatterned vascular muscular thin films
Uploaded: 2015-06-26T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Watch this Scientific Journal Video about Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films at JoVE.com

We acknowledge financial support from the American Heart Association Scientist Development Grant, 13SDG14670062 (PWA) and the University of Minnesota Doctoral Dissertation Fellowship (ESH). We also acknowledge the microfabrication resources of the Minnesota Nano Center (MNC) and the image processing resources of the University Imaging Centers (UIC), both at the University of Minnesota. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRS program.

注意:此协议的目标是建立和利用血管肌肉薄膜（vMTF）与图1所示的PDMS衬底 ​​的血管平滑肌细胞（VSMC）的延长培养期间，以评估收缩的结构。为了延长VSMC活力，我们利用交联剂京尼平化合物。衬底这些vMTFs被设计来分析组织收缩所发展Grosberg 等人 8其他vMTF方法5也可以使用，以微妙的变化对所呈现的衬底制造协议。 1.衬底制造<ol><li>盖玻片清洁<ol><li>将直径25毫米的玻璃盖玻片到盖玻片染色架。放置架成一个大烧杯或容器（ 如一个空100 - 1000微升枪头容器）。 </li><li>加70％的乙醇的容器完全浸没盖玻片。超声处理了至少30分钟。&lt;/ LI&gt; <li>除去从乙醇溶液盖玻片机架。由悬挂架在无菌培养罩（以防止在盖玻片上粒子积聚）为1- 2小时使盖玻片风干。 注:盖玻片必须以下步骤之前完全干燥。 </li></ol></li><li>聚（N-异丙基丙烯酰胺）上盖玻片（PIPAAm）剥离隔离<ol><li>使用胶带，胶带掀起了清洗盖玻片的侧面，留下暴露的带材中心的盖玻片（ 图1A）。修改基于应用和/或微流体设计该露出条带的宽度。 </li><li>标记带条上使用实验室标记以供以后参考（ 图1A）的盖玻片的边缘。 </li><li>周围的盖玻片的周边切割以从盘（ 图1A，红色虚线）释放。 </li></ol></li><li>聚（N-异丙基丙烯酰胺）（PIPAAm）涂层<ol><li>用分析天平，称量1克的PI的聚丙烯酰胺粉末。没有使用的未聚合的N-异丙基丙烯酰胺，这是一种致癌物。 </li><li>转移PIPAAm到50ml离心管中。加入10毫升1-丁醇中的化学罩内，得到10％w / v的溶液。注意:1-丁醇闪点为37℃。存储在易燃柜所得溶液，并避免加热。 </li><li>允许PIPAAm溶解10分钟。如果粉末依然可见，用涡旋混合器，直到所有的粉末溶解混合的解决方案。 注意:下面的步骤需要使用旋涂器的。对于每个盖玻片: </li><li>放置在旋转涂布机夹头用钳子盖玻片录音。 </li><li>转移150微升PIPAAm溶液到盖玻片放置沿着露出的玻璃微滴在盖玻片的中心。确保暴露的区域的完全覆盖。 </li><li>采用旋涂PIPAAm以下几招: <ol><li>坡道10秒〜3000转。停留5秒。 </li><li>坡道10秒〜6.000转。停留60秒。 </li><li>坡道10秒〜3000转。停留5秒。 </li></ol></li><li>将盖玻片成有盖培养皿与PIPAAm朝上。风干至少15分钟。 </li><li>小心地从所有的盖玻片取出胶带，留下完整的盖玻片PIPAAm涂层的中心带一薄层暴露。 </li></ol></li><li> PDMS涂层<ol><li>混合和脱气15克PDMS在10:1群:交联剂的比例。添加7 - 8滴超声0.2微米微珠荧光混合之前。罩杯的PDMS与铝箔的不使用时，以防止灰尘和其它微粒污染的PDMS。 注意:下面的步骤需要使用旋涂器的。对于每个盖玻片: </li><li>放置在旋转涂布机夹头镊子PIPAAm涂层的盖玻片。 </li><li>转移的PDMS上的盖玻片，覆盖至少三分之一的盖玻片面积。 </li><li>旋涂用下列RECIPE: <ol><li>斜5秒至500rpm。停留5秒。 </li><li>斜5秒到1000转。停留5秒。 </li><li>坡道10秒〜3000转。停留10秒。 </li><li>坡道10秒〜4000转。停留60秒。 </li><li>坡道10秒〜2000转。停留15秒。 </li><li>坡道10秒〜1000转。停留10秒。 </li><li>斜5秒至500rpm。停留5秒。 </li></ol></li><li>将盖玻片成有盖培养皿中的PDMS朝上。记录时盖玻片旋涂的时间。跟踪与每个盖玻片整个实验过程中，供以后使用在确定的PDMS基片厚度相关联的时间。 </li><li>把这个包含盖玻片在90℃的烘箱中至少1.5小时的培养皿，以确保适当的PDMS固化。如果烤箱不可用，让盖玻片固化至少48小时，在室温。 </li><li>从烤箱中取出盖玻片，并将它们存储在一个黑暗的抽屉里，直到准备使用。 </li><li>抛开每四个盖玻片˚F或基片厚度的后测量，如旋涂时间的函数，用轮廓。 </li></ol></li></ol> 2.微流控图案为组织工程<ol><li>微流体设备的制造<ol><li>组织微流控光罩设计<ol><li>使用任何适当的计算机辅助设计程序设计模式的微流体。动脉薄片人脐带动脉平滑肌细胞组成，使用10微米的通道用10微米壁的交替图案。 </li><li>使用二进制信道如果可能的话17分支，但其它支设计可以被使用。减少信道为每个分支迭代的宽度和长度，直到获得所期望的组织图案间距（墙壁和通道， 图1B）。 </li><li>设计该装置具有表面处理解决方案布局和应用真空的单一出口的单一入口。 </li><li> FABricate含有微流体设计（多个）的光掩模，如前所述18。 </li></ol></li><li>光刻晶圆制造 注:在合适的洁净室或类似设施进行光刻。为了使硅片的模式组织微流体装置（约20 - 25微米通道高度）软平版印刷制作使用光刻: <ol><li>清洁的丙酮，甲醇和异丙醇的硅晶片为每1分钟。干燥用氮气枪的晶片。 </li><li>预烘烤的热板上的晶片5分钟，在115℃，以除去过量的水分。 </li><li>旋涂用下面的食谱，以产生与SU-8 3025光刻胶的晶片提供20 - 25微米的高度: <ol><li>斜5秒至500rpm。停留5秒。 </li><li>斜坡15秒到4000转。停留15秒。 </li></ol></li><li>软烘烤的热板上的晶片在95℃下进行15分钟。 </li><li>加载光掩模，并揭露牛逼他晶片16秒，使用真空接触程序上的联系人掩模对准。 </li><li>硬烤的热板上的晶片在95℃下进行4分钟。 </li><li>开发晶圆6分钟的开发商。然后，洗净晶片两次，持续2秒的新鲜显影剂和漂洗，用异丙醇晶片。 </li><li> 3滴十三三氯硅烷的空盘真空干燥器内 - 通过将2 Silanate的图形晶片O / N。使用培养皿，以使晶片的底部和顶部被暴露支撑晶片向上。 注意:Tridecafluro - 三氯氢硅是一种易燃，腐蚀性液体。做好个人防护设备及局部排气是必要的使用。 </li></ol></li><li>组织微流控设备制造<ol><li>放置硅烷化，图案的晶片的特征的一面朝上在培养皿。 </li><li>混合和脱气100克PDMS的一个10:1群:交联剂的比例。倒入PDMS入菜，完全并均匀地覆盖晶片。</li><li>将培养皿在真空干燥器，直到所有气泡被从所述未固化的PDMS除去，约30分钟。固化在培养皿中的PDMS在90℃至少1.5小时。时间和温度可以根据所决定通过制造准则，以获得完全治愈进行调整。 </li><li>一旦PDMS固化后，切出的晶片周围的PDMS用刀片，并仔细释放从盘与PDMS覆盖晶片。去除多余的PDMS晶片下方，慢慢地剥离PDMS远离晶片的顶部。 </li><li>将PDMS磁盘功能端在一个干净的菜，使用后从光储存晶片而去。 </li><li>切去多余的PDMS从周围使用刀片的模式。切设备分成矩形形状（ 图1C），以简化从在后面的步骤中基片的剥离装置。精确削减是没有必要的，只要存在用于入口，出口，和组织图案区域（ 图1C）充足的空间。 </li><li>冲床使用1mm的外科活检穿孔入口孔和出口孔（ 图1C）。 </li></ol></li></ol></li><li>微流体装置沉积 注意:在这个协议中，微流体输送用于沉积图案化的京尼平，关键交联剂长期组织培养，以及纤连蛋白。之前青霉素/链霉素灭菌（2.2.3）的步骤并不需要发生在无菌条件下，但限制了污染和灰尘收集鼓励整个协议。盖玻片消毒用青霉素/链霉素（2.2.3）后发生的所有步骤应该利用无菌技术。注意:该协议的这部分必须前一天细胞接种启动。 <ol><li>基板和微流体器件的制备<ol><li>超声处理在70％乙醇的微流体装置至少30分钟。 </li><li>使用加压空气或氮气干燥超声微流体装置，并将其放置在培养皿中的无线个通道具有面朝上，以防止不必要的磨损的特点。 </li><li>放置最多10 vMTF衬底盖玻片在UVO清洁器（上盘盖除去，从而表面被官能化）为8分钟。 </li><li>除去UVO处理盖玻片，上下放置的微流体装置特征侧到每个滑一次一个（取向应该类似于图1C）。用力按下设备，以确保密封的PDMS涂层盖玻片。 </li></ol></li><li>京尼平与纤维连接蛋白的沉积<ol><li>通过加入1毫升无菌DDH 2 O的以冻干京尼平的5毫克容器准备有5毫克/毫升京尼平溶液中。混合使用一个旋涡混合器将溶液。搁置在RT至少30分钟。 注意:该粉末是难以溶解在RT，所以重复混合至少一分钟，常常需要。 </li><li>很快，放置一滴70％的乙醇在每个器件的器件吸入口。注:ETHanol应灯芯通过设备。 </li><li>经过5 - 10分钟，小心地吸去多余的乙醇在入口，立即用1X磷酸缓冲盐水（PBS）在入口替换它。从这点出发，一定不允许进口变得完全干燥，以避免空气进入到设备。 </li><li>放置在真空吸气器尖端在每个装置的出口。通过设备绘制PBS洗涤，漂洗乙醇而去。离开少量的1X PBS中的入口。注:如果出现进近干，加入更多的1X PBS。 </li><li>吸过量的1X PBS，使得只有少量的施加之前的京尼平溶液保持在入口。 </li><li>放置60微升在每个入口（ 图1D）的5mg / ml的京尼平溶液中。通过将真空抽吸尖端处的出口（ 图1D）绘制通过设备的京尼平溶液中。一定不要通过绘制所有的解决方案，留下了少量溶液在入口处。 </li><li>地方滴（约硬币大小）的1X PBS中的两个入口和出口到孵化过程中保持湿润。移动包含设备的潮湿烘箱或恒温箱（无菌环境是没有必要）设定为37℃的盘，并孵育4小时。菜不需要被覆盖。 </li><li>在培养过程中，悬浮纤连蛋白的50微克/ ml的浓度在无菌DDH 2 O在冰上至少30分钟前，应用到微流体装置。 </li><li>京尼平的孵育后，吸出所有剩余的1X PBS在装置插座。继续施加真空抽吸器在每个设备出口，通过剩余的1X PBS拉在入口。 </li><li>放置100微升的50μg/ ml的纤连蛋白溶液在每个入口，加入到剩余的1X PBS中的入口的最小量（ 图1D）。 </li><li>绘制通过使用真空吸气器尖端的设备进行纤连蛋白溶液在出口（ <s仲&gt;图1D）。一定要通过不是绘制所有的解决方案。注:纤维连接蛋白下降，将看起来比1X PBS和京尼平不同，由于表面张力的差异。 </li><li>移动包含设备的烘箱或恒温箱设置为37℃的露天菜，并培育24小时。注意:纤连蛋白步骤不需要入口和出口用1X PBS中润湿。纤维连接蛋白在入口其余池会干出。这是预期的。 </li></ol></li><li>消毒和准备细胞种植<ol><li>制备青霉素/链霉素的图案vMTF盖玻片的灭菌的溶液中。加入5 ml青霉素/链霉素（10000单位/毫升10,000微克/毫升），500毫升无菌1X PBS的。 </li><li>将包含在无菌生物安全罩装置的菜。 </li><li>小心地慢慢剥离装置在一个角落里，一边轻快地抓在另一只手的盖玻片删除盖玻片的设备。注意:此步骤需要实践，以减少盖玻片损坏的去除处理。一个替代方案是用注射器注入的1X PBS在入口和/或出口在装置释放，以帮助。 </li><li>将在无菌六井菜盖玻片。加至少5毫升青霉素/链霉素溶液，以每孔中。放置在无菌保育箱的菜，在37℃下进行至少30分钟。 </li><li>灭菌后，吸出青霉素/链霉素溶液，和种子与培养的人脐动脉血管平滑肌cells19（ 图1D）的盖玻片。播种浓度血管平滑肌细胞是〜 个每平方厘米80,000个细胞。为了减少所需的各样品的细胞数，使用一个减速器来减少播种面积。一个减速器的一个例子是连接到用无菌真空润滑脂前播种盖玻片15毫升锥形管的切割顶部。 </li><li>孵育在无菌恒温箱中播种盖玻片，在37℃和5％CO 2的</s的ub&gt;并允许接种的细胞附着并形成对准动脉薄片模仿O / N（ 图2A-B）。 </li></ol></li><li>长期vMTF组织培养<ol><li>播种后一天，除去细胞培养基和减速器。冲洗1X PBS组织。加入4毫升血清无细胞培养基以诱导收缩表型的平滑肌细胞20。 </li><li>根据需要长期培养重复的1X PBS冲洗，加入新鲜的无血清培养基每隔一天。 </li></ol></li></ol></li></ol><img alt="图1" src="/files/ftp_upload/52971/52971fig1.jpg" /> 图1.微流控蛋白输送设备。 （一）胶带封闭盖玻片PIPAAm涂层。红色虚线圆圈:切割路径释放盖玻片（二）组织微掩模图形的代表AutoCAD图形。插图:详细的二进制分支来altern阿婷10微米的微流体装置×10微米的组织图案。（℃）放置于具有入口和出口的盖玻片衬底指示。微流体蛋白构图和交付（D）的示意图。左到右:微流体通道的扫描电子显微镜图像（比例尺为50μm）;详细图解法蛋白沉积;免疫组化染色纤维连接蛋白（比例尺:50微米）;细胞种植血管平滑肌细胞，编造组织（E）示意图。 1日插图:详细分层构建。 第二插图:详细PDMS基板的微流体沉积后京尼平的修改。 ©I​​OP出版。再现和/或修改的许可。保留所有权利。19 <a href="https://www.jove.com/files/ftp_upload/52971/52971fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 与vMTF收缩试验3.组织功能分析注意:这里介绍的MTF收缩试验中Grosberg 等人开发的技术是仿照8。 <ol><li> vMTF收缩试验<ol><li>发生在100毫米的菜的组织样本。添加无菌1X蒂罗德solution8在pH7.4加热到37℃，以覆盖样品。 </li><li>用剃刀刀片，使垂直于PIPAAm边缘几个平行的切口。使能产生更广泛的组织切片，这将是vMTFs（与宽度〜2毫米）的薄带（ 图3A，侧切口）交替的方式削减。使清洁的削减，放置在与样品接触刀片和牢牢拖动到一侧。 </li><li>旋转盘90°，使两个直线，在该组织的中间平行切口，平行于PIPAAm的条带（ 图3A中 ，末端切口）。移走和处置的组织，这些切口之间的松散条s，将薄带在vMTFs之间（切在之前的步骤），以防止邻近的薄膜从制造接触。 </li><li>使样品休息在RT 10分钟，或直到所有的PIPAAm溶解。注意:如果PIPAAm保留在将来​​的步骤，样品可以被返回到所述切割盘以溶解残余PIPAAm。 vMTF的下侧的小心刮擦可以清除PIPAAm帮助，根据需要。 </li><li>将一小点真空脂在干净的35mm平皿。加入5毫升新鲜，无菌1X台氏液，在37℃。与转切膜从100毫米的菜到35毫米的菜，然后按盖玻片到真空润滑脂以防止盖玻片移动。 </li><li>放置在一个温度控制的平台上的立体显微镜阶段的菜。 </li><li>发送捕获的时间推移和在所需的时间间隔的荧光图像（ 例如 ，30秒）在整个治疗试验。 </li><li>串联治疗vMTFs用50nM内皮素-1 20分钟（引起的收缩）和100μM的HA-1077为30分钟（组织松弛）。添加各处理浓缩溶液的实验皿含有在指定的时间点加入5ml无菌1X台氏液，得到在5 ml，此所需的治疗浓度。使隔时图像采集之间的时间间隔内处理补充，以避免捕捉吸管中的图像。 </li></ol></li><li> vMTF收缩分析<ol><li>使用抛开1.4.8盖玻片，测量PDMS基板厚度与profilometer21。创建一个与厚度旋转时间曲线每套盖玻片。使用该曲线来估计vMTF厚度用于在收缩试验中使用每个盖玻片。 </li><li>在实验过程中测量vMTF凸起长度对每个时间点，并计算曲率使用先前报道的方法8的相关联的半径（ 图3B）。 </li><li>计算vMTF应力每次PO诠释以前的使用方法vMTF 5。 注意:使用来自3.2.1计算的估计vMTF厚度。使用共焦图像测量血管平滑肌细胞的厚度，如之前报道9。获得PDMS的杨氏模量从公司的数据表。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这里，我们提出了一个协议，建立在先前开发vMTF技术，允许延长试验时间比较典型的慢性血管疾病的途径1,23,24。要做到这一点，我们微图案京尼平，这已被证明可以提供的PDMS基板11的长期功能化，使用微流体沉积技术以产生工程改造动脉薄片具有改善血管组织的生存力为在MTF收缩实验。麦凯恩等人 。开发工程心脏组织的扩展文化替代的微成形的明胶凝胶基质几个星期在...

血管疾病，如脑血管痉挛1,2，高血压3，和动脉粥样硬化4，发展缓慢，在性质上通常是慢性的，并且通过血管平滑肌细胞（VSMCs）涉及功能失调力代。我们的目标是研究用体外方法与实验条件下比在体内模型更精确的控制，这些缓慢进展的血管功能障碍。我们以前曾开发血管肌薄膜（vMTFs），用于测量的体外功能收缩工程心血管组织5，但这种方法已被限制于相对短期研究。在这里，我们提出了扩大我们以前vMTF技术长期测量底物改性技术。 而内皮也是很关键的整体血管功能，设计动脉薄片提供了一个有用的模型系统，用于评估变化血管在疾病进展期间的收缩。工程师的官能血管疾病组织模型，二者的结构和动脉瓣的功能，该船只的基本收缩单元，必须概括以高的保真度。动脉薄片是同心，周向排列的弹性由6张分离收缩血管平滑肌细胞的表。细胞外基质（ECM）蛋白的微接触印刷到聚二甲基硅氧烷（PDMS）衬底先前已用于提供指导线索为组织机构，以模仿对齐心血管组织5,7-10。然而，组织使用微接触印刷在培养3-4天会失去诚信，限制了其应用在慢性研究构图。这个协议提供了通过用一个新的微流体沉积技术代替以往的微接触印刷技术的解决这个问题。 现地等改性PDMS基板与京尼平以及found延长细胞的存活长达一个月中培养11。在这里，我们使用类似的方法来延长图案的血管平滑肌细胞的培养上PDMS。京尼平，栀子果实的自然水解衍生物，是一种理想的候选衬底修改由于其相对低的毒性相比类似的交联剂和其越来越多地使用作为在组织修复12,13和ECM变形例14中的字段的生物材料， 15。在这个协议中，纤连蛋白被用作细胞指导线索，如在前面的微接触印刷法;然而，京尼平沉积到之前​​纤连图案的PDMS衬底。因此，当细胞降解的图案矩阵，从附着的VSMC新合成的细胞外基质可以结合到京尼平涂覆PDMS衬底。 这个协议利用两步京尼平和ECM沉积的微流体输送装置。微流体装置模拟microco的设计ntact印刷图案在以前的研究中16用于改造动脉片状结晶。因此，我们预计该协议产生动脉薄片模仿，成功地概括了高度一致的体内结构和动脉薄片的收缩功能。我们还评估组织收缩，以确认京尼平是用于长期体外血管疾病模型合适的基材变形化合物。

<table><tbody><tr><td>Coverslip staining rack</td><td>Electron Microscopy Sciences</td><td>&lt;a href=&quot;http://www.emsdiasum.com/&quot;&gt;www.emsdiasum.com/&lt;/a&gt;</td><td>72239-04</td></tr><tr><td>Microscope cover glass - 25 mm</td><td>Fisher Scientific, Inc.</td><td>&lt;a href=&quot;http://www.fishersci.com/&quot;&gt;www.fishersci.com&lt;/a&gt;</td><td>12-545-102</td></tr><tr><td>Poly(N-iso-propylacrylamide) (PIPAAm)</td><td>Polysciences, Inc.</td><td>&lt;a href=&quot;http://www.polysciences.com/&quot;&gt;www.polysciences.com/&lt;/a&gt;</td><td>#21458</td></tr><tr><td>1-butanol</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>360465</td></tr><tr><td>Spincoater</td><td>Specialty Coating Systems, Inc.</td><td>&lt;a href=&quot;http://www.scscoatings.com/&quot;&gt;www.scscoatings.com&lt;/a&gt;</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Ellsworth Adhesives (Dow Corning)</td><td>&lt;a href=&quot;http://www.ellsworth.com/&quot;&gt;www.ellsworth.com&lt;/a&gt;</td><td>184 SIL ELAST KIT 0.5KG</td></tr><tr><td>Fluorescent microbeads</td><td>Polysciences, Inc.</td><td>&lt;a href=&quot;http://www.polysciences.com/&quot;&gt;www.polysciences.com/&lt;/a&gt;</td><td>17151</td></tr><tr><td>Silicon wafers</td><td>Wafer World, Inc.</td><td>&lt;a href=&quot;http://www.waferworld.com/&quot;&gt;www.waferworld.com&lt;/a&gt;</td><td>2398</td></tr><tr><td>Photoresist&amp;nbsp;</td><td>MicroChem Corp.</td><td>&lt;a href=&quot;http://www.microchem.com/&quot;&gt;www.microchem.com&lt;/a&gt;</td><td></td></tr><tr><td>Contact mask aligner</td><td>Suss MicroTec</td><td>&lt;a href=&quot;http://www.suss.com/&quot;&gt;www.suss.com&lt;/a&gt;</td><td></td></tr><tr><td>Developer</td><td>MicroChem Corp.</td><td>&lt;a href=&quot;http://www.microchem.com/&quot;&gt;www.microchem.com&lt;/a&gt;</td><td></td></tr><tr><td>Tridecafluro-trichlorosilane</td><td>UCT Specialties, Inc.</td><td>&lt;a href=&quot;http://www.unitedchem.com/&quot;&gt;www.unitedchem.com&lt;/a&gt;</td><td>T2492</td></tr><tr><td>Surgical biopsy punch</td><td>Integra LifeSciences Corp.</td><td>&lt;a href=&quot;http://www.miltex.com/&quot;&gt;www.miltex.com&lt;/a&gt;</td><td>33-31AA-P/25</td></tr><tr><td>Genipin</td><td>Cayman Chemical</td><td>&lt;a href=&quot;http://www.caymanchem.com/&quot;&gt;www.caymanchem.com&lt;/a&gt;</td><td>10010622</td></tr><tr><td>1X phosphate buffered saline</td><td>Mediatech, Inc.</td><td>&lt;a href=&quot;http://www.cellgro.com/&quot;&gt;www.cellgro.com&lt;/a&gt;</td><td>21-031-CV</td></tr><tr><td>Fibronectin</td><td>Corning, Inc.</td><td>&lt;a href=&quot;http://www.corning.com/&quot;&gt;www.corning.com&lt;/a&gt;</td><td>356008</td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies, Inc.</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/&quot;&gt;www.lifetechnologies.com&lt;/a&gt;</td><td>15140-122</td></tr><tr><td>Umbillical artery smooth muscle cells</td><td>Lonza</td><td>&lt;a href=&quot;http://www.lonza.com/&quot;&gt;www.lonza.com&lt;/a&gt;</td><td>CC-2579</td></tr><tr><td>Tyrode&#039;s solution components</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>various</td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>&lt;a href=&quot;http://www.zeiss.com/&quot;&gt;www.zeiss.com&lt;/a&gt;</td><td>4350020000000000</td></tr><tr><td>Temperature-controlled platform</td><td>Warner Instruments</td><td>&lt;a href=&quot;http://www.warneronline.com/&quot;&gt;www.warneronline.com&lt;/a&gt;</td><td>641659; 640352; 641922</td></tr><tr><td>Endothelin-1</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>E7764-50UG</td></tr><tr><td>HA-1077</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;www.sigmaaldrich.com&lt;/a&gt;</td><td>H139-10MG</td></tr></tbody></table>

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.

这项工作的主要目的是扩大微图案化血管平滑肌细胞的生存能力疏水PDMS衬底。这是通过将微流体递送系统以沉积图案化的京尼平与纤连蛋白上的PDMS（图1）来完成的。使用微流体递送ECM蛋白的沉积，得到沟道图案与京尼平与纤连蛋白（图1D）的线之间的裸露PDMS高保真传输。贴壁细胞（图1E）形式汇合单层模仿动脉薄片的体内结构（ 图2），?...

Watch this Scientific Journal Video about Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films at JoVE.com

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

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 ...

microfluidic-genipin-deposition-technique-for-extended-culture

Research

JoVE Journal

Bioengineering

7.6K 次观看.  University of Minnesota. 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.

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

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 ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

发展多深的圆形横截面微内皮化的一个芯片的程序

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

影像引导下，血管源性微流控网络的基于激光的研制

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

模仿神经血管化初始事件的微流体模型

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...