我们承认，从自然科学和工程研究理事会，加拿大和加拿大健康研究机构（CHRPJ 323533-06），安大略研究生奖学金计划到CM，和加拿大研究主席在微型和纳米工程系统的财政支持黏， Mechanobiology到CAS。

<html> A.设备的描述和操作设备都采用多层软光刻技术1，在聚二甲基硅氧烷（PDMS），能够同时产生一系列机械在单个细胞的微阵列跨文化地点条件。在这个协议中，首先描述的步骤来制作气动驱动microposts的阵列，步骤来修改设备，使机械动态二维（2D）和三维（3D）的文化范式文化。概述了微加工方法增加吞吐量比现有的宏观系统，适用于各种机械条件的影响，是最好的筛选。 设备的工作原理是基于垂直驱动的microposts阵列。 Microposts制造一个自由悬浮隔膜，并提出和下方的驱动隔膜（图1）申请的正面和负面的压力降低。数组的一个主要特点是一个单一的压力源，通过改变驱动隔膜的大小，可以用来获得一系列跨阵列的垂直位移。这个原则是用来迅速屏幕上的跨越单一设备的机械刺激条件下，大量的细胞反应。 谢弗等人。一个类似的宏观设计的基础上，我们的设计，包括第二次暂停和润滑的较后的细胞培养膜，使细胞的经验作为文化电影滑倒在提出的加载后的2D基板变形。另外，photopatterning在装载员额细胞载货凝胶的数组允许压刺激细胞的三维培养。设立这些系统的详细说明如下。 B.制造的气动微驱动器阵列编造气动微驱动器阵列，需要在PDMS的多层结构的严格对齐。这是具有挑战性，由于收缩引起的对齐登记错误。为了解决这个问题，我们使用一个制造过程称为“夹心模具制造”，有效地消除这种问题4。 材料的制备<ol><li>单或多级SU - 8各级大师制作在洁净室使用的标准程序。对于这些设备，船长200-400微米厚，根据不同的应用。船长hardbaked在80 ° C时3天前使用。 </li><li>清洁玻片和SU - 8的主人是在真空室中，以防止硅烷固化硅橡胶材料的附着力。在通风柜，60μL硅烷代理（tridecafluoro - 1 ,1,2,2 - tetrahydrooctyl）- 1 -三氯硅烷（美国化学技术，布里斯托尔，PA）是吸管成在会议厅内的培养皿。裸露的玻璃和SU - 8的主人放置在会议厅内，并在真空条件下过夜。 </li><li>两个泡沫垫（可在工艺用品商店）和开销喷墨透明胶片切割尺寸稍大比夹心模具制造所使用的SU - 8的主。 </li></ol>夹心模具制造<ol><li>每一层的多层设备需要使用此方法成型的硅橡胶层。夹心模具制造过程的示意图如图1。 </li><li> PDMS的单体和交联剂彻底混合在标准的比例为10:1。 3毫升固化的硅橡胶混合物沉积到以前捏造的SU - 8的主，并在一个真空室脱气。 </li><li>泡沫垫和SU - 8的主，被放置在一个有机玻璃底座。 </li><li>透明度是仔细降低到未固化硅橡胶，一定要避免捕获任何气泡。喷墨透明胶片有一个粗糙和光滑的一面，重要的是，光滑的一面接触到未固化的硅橡胶。 </li><li>甲硅烷载玻片放置在顶部的透明度，第二个泡沫垫和一个有机玻璃顶板下方。三明治，然后使用C钳钳制在一起。 </li><li> PDMS夹心固化烘箱中在80 ° C至少4个小时。 </li><li>三明治是从烤箱中删除，并让其冷却后再取出钳。 </li><li>钳夹心拆解，被丢弃的泡沫垫。从SU - 8的主透明度仔细剥离，保留图案的PDMS薄膜。 </li><li>这些图案的PDMS层和其处理的透明度层可以存储在一个无尘的环境中中，直到他们准备使用。我们已经成功地使用硅橡胶层达两个月之久，没有明显的有害影响。 </li></ol>构建多层设备T“&gt;的微驱动器阵列的制造原理是在图2A，和编造的设备执行机构的运作是在图2B和C所示： <ol><li>最下面的图案的PDMS层准备夹心模具制造。对于这些实验中，使用了直径从600到1200μm不等的圆形图案。 </li><li>硅橡胶层，然后转移到一个干净的玻片上。电晕放电单元是用于与氧等离子体处理PDMS和玻璃表面，并放置在相互接触的两个表面。栈是一个烤盘上，然后放置在80 ° C至完成粘接工艺。然后被剥离的透明度和丢弃。在这个过程中，图案的PDMS薄膜是永远不会释放从刚性基板，防止个别层收缩。 </li><li>根据夹心成型步骤，薄的硅橡胶之间的SU - 8的功能和透明度，没有被挤压出的电影的成功，可能需要被删除。这可以使用立体25G针手工完成。 </li><li>第二个夹心层成型是形成一个负副本模具所需的隔膜和岗位结构。这是因为制造大面积的SU - 8的主人是具有挑战性的，但使用替代制造工艺可以消除这种额外的步骤。在这个演示中，使用圆形帖子500微米直径。负副本模具硅烷，暂时使用双面胶带贴于载玻片。未固化的PDMS的沉积，脱气，并在1000 rpm到这个45秒的“三明治”模具层旋涂，产生驱动60微米厚的隔膜。旋涂硅橡胶层部分固化在80 ° C的〜20分钟，或直至的PDMS是粘性的，但触摸时不变形，永久。 </li><li>部分固化的硅橡胶层，然后等离子体处理，倒在放在一个特制的对准的玻片上，双面胶带被删除。对准由一个真空吸盘安装一个显微（锡斯基尤;团Viego，CA，USA）。 PDMS的第一层，然后等离子体处理，并置于一个旋转舞台上（纽马克，资助传球，或美国），下方的真空吸盘，两层之间的对齐方式是使用一个Navitar的12倍变焦的机器视觉系统（NAVITAR观察;罗切斯特，纽约，美国）。 </li><li>使用机械臂层对齐，然后小心地接触到。三明治，然后从对准中删除，并固化为30分钟，在80 ° C的烤箱中。从设备，然后将去皮的透明度和硅烷副本的PDMS模具丢弃。该设备，然后在80 ° C至少有四个小时完全固化。 </li><li>根据正在开展的机械性刺激实验的类型，就可以将更多的PDMS层粘合此基础设备通过重复的概述过程。在C和D节提供各类机械性刺激实验的详情</li></ol>连接器<ol><li>一旦设备完成后，一针可以用来清除在连接点的PDMS层。 </li><li> Nanoport（厄普丘奇科学;奥克港，WA，USA），如商业连接器，然后可以用于连接外部油管。为了避免商业连接器的高度要求，从而为设备容器使用标准的培养皿中，我们制造我们自己的如下。 </li><li>大约35克的PDMS演员和治愈以及全方位托盘（NUNC;罗切斯特，纽约，美国）。该设备是去皮，切成小垫片部分，从1 / 8“孔被删除，用一记重拳。 </li><li>一个18G的钝针是用来核心一段从一侧的垫片。一个较小的21G针是用来清除前撤回18G针的核心。 </li><li>大约是硅橡胶15克投治愈以及全方位托盘盖，去皮，切成同样大小的部分。本节用透明胶带清洁，等离子保税垫片顶部，和单位等离子体设备，一拳方下来保税。 </li><li>第二个18G钝针，然后分开的彩色塑料鲁尔接头，插入芯孔和密封到位的PDMS几滴。造成连接器是相当有效的，经济的和死空间体积足够大，以防止气泡在液体灌装阶段进入渠道。 </li></ol> C.机械活性的二维培养基质阵列驱动microposts可用于创建各种应变型材在暂停的高分子膜，细胞培养。提高到一个润滑膜后会导致滑和变形后各地的电影。 equibiaxial应变分布使用一个循环加载后的结果，但在不同的岗位形状设计是多才多艺的，可用于创建各种应变场。要修改为二维培养实验（原理图如图3）执行机构数组： <ol><li>债券PDMS的第三层的设备，如在图3a所示。 PDMS的第三层由两层结构。较低的圆匹配下的驱动microposts的驱动腔的大小。上部圆的直径是100微米，比直径装载职位更大。一个200微米宽的微连接这些功能的连接区，将用于润滑加载的职位和文化电影。 </li><li>制造和债券连接器的设备，如前面所述。 </li><li>旋涂10-15微米厚层到一个透明光滑的一面（旋转参数：4000 RPM，120秒）的PDMS。在80 ° C，至少4个小时的治疗。 </li><li> Appy一个低级别的真空（Barnant航空青年团抽吸泵）执行器，降低阵列的职位。 </li><li>等离子体处理和债券的旋涂PDMS薄膜驱动微装置。广场上的电磁炉在80 ° C 10分钟。很短的时间将保持亲水性的职位和文化电影。 </li><li>与去离子水，溶液中90％的甘油，填充润滑通道。除了润滑，这一提法无限期地保持的PDMS的亲水性，降低摩擦系数。将继续和文化之间的后电影被困的气泡。炉具装置放置于40 ° C，并不断注入润滑剂通道，造成小的压力增加。几分钟后，气泡会漫过的PDMS，和所有表面进行润滑。 </li><li>删除负面压力，释放出的职位。 </li><li>使用手术刀，切薄的PDMS薄膜各地。小心剥离的透明度远离设备的支持。 </li><li>设备周围的文化领域的手工切割硅橡胶垫片等离子债券。 </li><li> 70％的乙醇中浸泡5分钟，然后下杀菌的紫外线灯在45分钟的生物安全柜消毒设备。 </li><li>等离子体处理表面用100微克/毫升的胶原蛋白（或替代的ECM蛋白）和孵育过夜，4 ° C。 </li><li>清洗设备，用磷酸盐缓冲液（PBS）和种子细胞在10-15000细胞/平方厘米以下标准的细胞培养协议。 </li></ol>别处7已发表 ​​微装置的运作，并在这个平台上进行的生物实验的细致刻画。 D.机械活性三维凝胶设备进行修改，也可以用来使photopatterned压缩。细胞载货，立体水凝胶在高通量的方式。在这个例子中，我们创建了直径为350微米的聚乙烯乙二醇（PEG）凝胶气瓶，封装间质干细胞C3H10T1 / 2鼠标，并适用于从5至25％，跨阵列的压缩应变。该协议可以使用更先进的水凝胶聚合化学品。要使用三维mechanobiology（原理图如图4）的实验平台： 设备的修改： <ol><li>大衣1微米的聚对二甲苯- C的厚厚的一层，以减少透气性，并提高在PEG凝胶体系的聚合时间的职位。这将减少自由基聚合反应的细胞毒作用。透明胶带饮片是用来屏蔽后周围的地区，并涂在PDS 2010年2 LabCoter系统设备（特种涂料系统，印第安纳波利斯，在美国）。 </li><li>镀膜设备是从涂料体系，透明胶带剥离，留下的聚对二甲苯- C形层以上的职位。 </li><li>玻璃盖玻片methacrylated浸泡在2％V / V解决方案3 8 - （三甲氧基硅基）丙基甲基丙烯酸甲酯在95％乙醇2分钟。 </li><li>盖玻片是100％的乙醇冲洗，烘烤，在100℃，表面上留下甲基丙烯酸甲酯组。 </li><li>投一个200微米厚的硅橡胶垫片电影是在培养皿​​中，并切成小部分。这些四个小部分methacrylated盖玻片边缘周围保税血浆。 </li><li>垫片等离子保税PDMS区的设备，覆盖- C的聚对二甲苯涂层职位。 </li><li>在70％乙醇洗涤，暴露在45分钟的生物安全柜的杀菌紫外灯灭菌设备。 </li></ol> 原位聚合： 载货细胞的PEG凝胶，然后光刻图案到8,9如下设备： <ol><li>准备10％W / V PEGDA（3.4kDa，Laysan生物，阿拉伯，AL，美国）和10％W / V聚乙二醇（8kDa，Sigma - Aldrich公司）在unsupplmented细胞培​​养基中的解决方案。 </li><li> 1 - 乙烯基- 2 - pyrolidinone准备的100毫克/毫升Irgacure 2959（汽巴精化;塔里敦，纽约，美国）的光引发剂原液。 </li><li>彻底混合准备的两个解决方案，获得0.4％W / V的光引发剂的浓度的解决方案。混合物用0.45μm注射器过滤器过滤，消毒和去除颗粒。 </li><li> Trypsinize细胞培养和两倍所需的细胞浓度为终点，准备在文化传媒一个细胞悬液。 </li><li>以1:1的比例混合细胞悬液和过滤后的水凝胶的易制毒化学/光引发剂溶液。 </li><li>注入微装置的使用很长的25G针解决方案。小心避免被困在设备中的气泡。 </li><li>使印刷的透明度屏蔽设备表面上的对准标记。设置一个紫外照明系统，在设备的表面提供了17.5毫瓦/平方厘米的紫外线剂量。通过195号的面具照亮凝胶此时已为我们的系统进行了优化，并可能需要调整。 </li><li>远离unpolymerized凝胶前驱体溶液，用PBS清洗，并取代与文化传媒的PBS。驱动加载的职位，以压缩的凝胶结构。 </li></ol>在这个平台上进行的微装置的操作和生物实验的详细特征已出版在别处10。 E.周边设备修改培养皿中，用于设备的驱动细胞培养过程中保持无菌。一个迟钝和剥离的18G针epoxied到在培养皿中的侧钻了一个洞。套管（亚当斯Intramedic PE190粘土; VWR International的阿灵顿高地，IL，美国）是按装到这些接口连接到控制电磁阀和压力源。 到设备的压力是由外部的微型泵。对于二维的实验中，从30-55千帕的正压值产生的使用电压控制偏心隔膜泵（SP 500 EC - LC 4.5VDC，施瓦泽精密，德国）。一个基于脉宽调制的电压调节器是用来改变施加电压控制输出压力。电磁阀和阀组（S10MM - 30 - 12 - 3和MSV10 - 1; Pneumadyne，普利茅斯，美国明尼苏达州）用于创建一个循环的压力波形。该阀驱动12 V信号由一个方波函数发生器控制。 F.器件表征和使用成像设备一个常见的​​问题是成像设备上的细胞或颗粒光学分辨率差，由于支持加载后的PDMS薄膜下方的水滴凝结。这凝结来自培养箱内的温度和湿度的差异，并固定后，或在显微镜舞台上。要解决这个问题： <ol><li>连接到开放的压力进气口水库，并用去离子水填充。我们用一个开放的注射器鲁尔锁连接器为方便。 </li><li>放置在一个真空室的设备和填充水库，并抽空。 </li><li>等待5分钟，使大气压力室。重复两次或三次。由于空气是流离失所设备的渠道，它是取代水从水库，消除了液滴的凝析油和光学显微镜提供了明确的路径。 </li><li>使用明场或荧光显微镜的影像文化领域。 </li></ol>活细胞成像，这个过程可以完成播种前对设备的细胞。然而，渠道的设计必须足够大，以防止单位之间在设备上的任何压力的粘性损失。 由珠跟踪应变特性在二维培养系统： <ol><li>稀释至所需浓度甲醇的直径为1微米的荧光珠（邦斯实验室;费舍尔，IN）。 1:150稀释液从原来的浓度（珠缓冲液中1％的固体）被发现适合我们的需要。 </li><li>等离子体处理设备的表面，和存款珠悬浮5-10微升。 </li><li>等待甲醇蒸发。重复珠浓度太低。 </li><li>图像处于静止状态的细胞培养领域，并根据负载。 </li><li>开源自动化粒子跟踪算法，然后可以使用在ImageJ（NIH）的跟踪珠的位移。 </li><li>位移的大小，方向和位置参数，然后可以使用一个数学模型来描述设备的表面上的应变场。 </li></ol>类似的程序可以遵循的特点在三维系统的菌株。混合成的H荧光珠的数量ydrogel易制毒化学解决方案。在PEG水凝胶系统的情况下，孔径比直径为1微米的珠小。珠子都被封装在水凝胶，并可以使用激光共聚焦显微镜成像系统。珠的位移，然后可以进行跟踪，分析，并安装到变形模型。 G.代表性的结果代表器件的制备，表征和操作的结果在图5和6。 <img src="/files/ftp_upload/2224/2224fig1.jpg" alt="图1" /> 图1。夹心模具制造过程中。 （一）架空透明度仔细降低到未固化的PDMS的SU - 8的主。 （二）放置在一个泡沫，玻璃和金属的堆栈，夹层，并在炉下压缩治愈。 （三）堆栈是拆卸和透明度剥离，保留图案的PDMS的3层。复制物理研究所的许可。 <img src="/files/ftp_upload/2224/2224fig2.jpg" alt="图2" /> 图2垂直驱动micropost示意图（一）休息和（B）当驱动。 （三）多层制造过程的示意图，需要做出一个 microposts 3数组。改编物理研究所的许可。 <img src="/files/ftp_upload/2224/2224fig3.jpg" alt="图3" /> 图3。进程修改微驱动器阵列的一个变形的二维衬底 7培养细胞进行实验。转载由英国皇家化学学会的<a href="http://dx.doi.org/10.1039/B914460A">许可</a> http://dx.doi.org/10.1039/B914460A 。 <img src="/files/ftp_upload/2224/2224fig4.jpg" alt="图4" /> 图4修改微驱动器阵列，立体photopatterned水凝胶中培养的细胞进行实验的过程构造10。复制的ELSEVIER许可。 <img src="/files/ftp_upload/2224/2224fig5.jpg" alt="图5" /> 图5（A）为2D mechanostimulatory文化的样本设备。红色染料用于标记的工作压力传递渠道，和蓝色染料，用来标记的润滑通道。 （二）应变特性的实验样品图像。红点代表的珠子未变形的位置，而绿点代表变形的位置。转载由英国皇家化学学会的<a href="http://dx.doi.org/10.1039/B914460A">许可</a> http://dx.doi.org/10.1039/B914460A 。 <img src="/files/ftp_upload/2224/2224fig6.jpg" alt="图6" /> 图6（A）3D压缩实验样品装置。绿色染料用于标记驱动的压力渠道。 （二）上往下看micropost阵列（三）水凝胶上兴建后上方图案。 （D，E）侧重建水凝胶中的荧光珠的图像构造（四）休息和（E）55千帕的驱动压力下，展示在一个三维培养系统10的应变表征过程。复制的ELSEVIER许可。

<ol>
	<li>Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolithic+microfabricated+valves+and+pumps+by+multilayer+soft+lithography.">Monolithic microfabricated valves and pumps by multilayer soft lithography.</a> Science. 288, 113-116 (2000).</li><li>Schaffer, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Device+for+the+application+of+a+dynamic+biaxially+uniform+and+isotropic+strain+to+a+flexible+cell+culture+membrane.">Device for the application of a dynamic biaxially uniform and isotropic strain to a flexible cell culture membrane.</a> J Orthop Res. 12, 709-719 (1994).</li><li>Jo, B., Lerberghe, L. V. a. n., Motsegood, K., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+micro-channel+fabrication+in+polydimethylsiloxane+(PDMS)+elastomer.">Three-dimensional micro-channel fabrication in polydimethylsiloxane (PDMS) elastomer.</a> Journal of Microelectromechanical Systems. 9, 76-81 (2000).</li><li>Moraes, C., Sun, Y., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solving+the+shrinkage-induced+PDMS+alignment+registration+issue+in+multilayer+soft+lithography.">Solving the shrinkage-induced PDMS alignment registration issue in multilayer soft lithography.</a> J. Micromech. Microeng. 19, 065015-065015 (2009).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab on a chip. 6, 1548-1549 (2006).</li><li>Bongaerts, J., Fourtouni, K., Stokes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft-tribology:+Lubrication+in+compliant+PDMS-PDMS+contact.">Soft-tribology: Lubrication in compliant PDMS-PDMS contact.</a> Tribology International. 40, 1531-1542 (2007).</li><li>Moraes, C., Chen, J. H., Sun, Y., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+arrays+for+high-throughput+screening+of+cellular+response+to+cyclic+substrate+deformation.">Microfabricated arrays for high-throughput screening of cellular response to cyclic substrate deformation.</a> Lab on a chip. 10, 227-234 (2010).</li><li>Liu, V. A., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+photopatterning+of+hydrogels+containing+living+cells.">Three-dimensional photopatterning of hydrogels containing living cells.</a> Biomedical Microdevices. 4, 257-266 (2002).</li><li>Hahn, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolithographic+patterning+of+polyethylene+glycol+hydrogels.">Photolithographic patterning of polyethylene glycol hydrogels.</a> Biomaterials. 27, 2519-2524 (2006).</li><li>Moraes, C., Wang, G., Sun, Y., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfabricated+platform+for+high-throughput+unconfined+compression+of+micropatterned+biomaterial+arrays.">A microfabricated platform for high-throughput unconfined compression of micropatterned biomaterial arrays.</a> Biomaterials. 31, 577-584 (2010).</li><li>Wipff, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+covalent+attachment+of+adhesion+molecules+to+silicone+membranes+for+cell+stretching+applications.">The covalent attachment of adhesion molecules to silicone membranes for cell stretching applications.</a> Biomaterials. 30, 1781-1789 (2009).</li><li>Moraes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+polyurethane+culture+substrates+into+poly(dimethylsiloxane)+microdevices.">Integrating polyurethane culture substrates into poly(dimethylsiloxane) microdevices.</a> Biomaterials. 30, 5241-5250 (2009).</li><li>Ifkovits, J. L., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+photopolymerizable+and+degradable+biomaterials+for+tissue+engineering+applications.">Review: photopolymerizable and degradable biomaterials for tissue engineering applications.</a> Tissue engineering. 13, 2369-2385 (2007).</li></ol>

虽然概念上很简单，设备制造并采取了一定的实验技能和实践。设备中的多层次的对齐，特别是在2D细胞培养的情况下，可以是具有挑战性的的，尤其是在一个大面积的数组。实事求是地讲，我们可以可靠地实现了100％对齐的成功率，使用50微米的公差，在间距之间多层次的相邻功能的设备。我们还成功地展示了低至15微米的公差对齐，但对齐所需时间大大提高，并实现在一个较低的成功率。对准?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sylgard 184 PDMS Monomer and Crosslinker Kit</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SU-8 masters</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Silanization agent: (tridecafluoro-1, 1, 2, 2-tetrahydrooctyl)-1-trichlorosilane</td>
<td>&nbsp;</td>
<td>United Chemical Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Foam pads</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Craft supply stores, 1-2 mm thick</td>
</tr>
<tr>
<td>Overhead inkjet transparencies</td>
<td>&nbsp;</td>
<td>Grand &amp; Toy</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plexiglass plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>C-clamp</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>hardware store</td>
</tr>
<tr>
<td>Micromanipulator system</td>
<td>&nbsp;</td>
<td>Siskiyou</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Custom-made vaccum mount</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vision system, Navitar 12x zoom</td>
<td>&nbsp;</td>
<td>Navitar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Connecting tubes</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>Clay Adam Intramedic PE190</td>
<td>&nbsp;</td>
<tr>
<td>Blunt 18G needles</td>
<td>&nbsp;</td>
<td>Smallparts.com / Amazon Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Eccentric diaphragm micropump</td>
<td>&nbsp;</td>
<td>Schwarzer Precision</td>
<td>SP 500 EC-LC4.5V DC</td>
<td>&nbsp;</td>
<tr>
<td>Solenoid valves</td>
<td>&nbsp;</td>
<td>Pneumadyne</td>
<td>S10MM-30-12-3</td>
<td>&nbsp;</td>
<tr>
<td>Solenoid manifold</td>
<td>&nbsp;</td>
<td>Pneumadyne</td>
<td>MSV10-1</td>
<td>&nbsp;</td>
<tr>
<td>Function generator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3-(trimethoxysilyl) propyl methacrylate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene glycol diacrylate 3.4 kDa</td>
<td>&nbsp;</td>
<td>Laysan Bio</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene glycol 8 kDa</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Irgacure 2959</td>
<td>&nbsp;</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-vinyl-2-pyrolidinone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Standard cell culture reagents</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

microfabricated platforms for mechanically dynamic cell culture

在体外细胞组织工程，药物发现或基本的细胞生物学研究的mechanobiological刺激组合系统探测能力由目前的生物反应器技术，它不能同时适用于各种培养细胞的机械性刺激是有限的。为了解决这个问题，我们已经制定了一系列旨在为机械刺激的影响画面在高通量格式的微型平台。在这个协议中，我们展示了微驱动器阵列的垂直流离失所的技术为基础，进一步展示如何可以修改此基础技术进行高通量的机械动态细胞培养两种维和职位的制造三个立体养殖的范例。

Watch this Scientific Journal Video about Microfabricated Platforms for Mechanically Dynamic Cell Culture at JoVE.com

Microfabricated Platforms for Mechanically Dynamic Cell Culture

在这个协议中，我们展示的微驱动器阵列的垂直流离失所该技术是基于职位的制造，以及如何可以修改此基础技术，进行二维和三维培养高吞吐量的机械动态细胞培养范例。

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 ...

microfabricated-platforms-for-mechanically-dynamic-cell-culture

Research

JoVE Journal

Bioengineering

13.6K 次观看.  University of Toronto. 在这个协议中，我们展示的微驱动器阵列的垂直流离失所该技术是基于职位的制造，以及如何可以修改此基础技术，进行二维和三维培养高吞吐量的机械动态细胞培养范例。

视频: Microfabricated Platforms for Mechanically Dynamic Cell Culture

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

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

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 ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

环境控制机械自适应聚合物纳米复合材料的微拉伸测试<em&gt;体外</em&gt;表征

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

研究微加工通道细胞迁移的

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

微流控控制环境的红细胞聚集动态调查

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...