作者承认口腔医学院的在匹兹堡，健康奖K01 AR062598的国家机构，并授予P30 DE030740的大学的财政支持。作者还感谢金章博士为ICUE1质粒，韦恩Rasband开发的ImageJ和迈克尔Cammer为ImageJ的宏。

The authors have no conflicts to declare.

这项工作表明FRET成像如何可用于分析细胞信号在三维水凝胶。虽然现有的研究已经表明与水凝胶38接种在水凝胶基质35-37，细胞外相互作用的细胞的FRET成像和截留在水凝胶39-41的分子，这是第一次出版物描述嵌入细胞FRET成像基于细胞的分析3D水凝胶。从2D转换FRET成像3D时，工作解决了一些执行问题。首先，需要高数值孔径目​​标FRET成像收集到足够的发射信号，但它们限制景深。这里的允许细胞通过离心来增加细胞数目的焦平面玻璃附近以补偿此稍向定居。第二，三维水凝胶支架可变得复杂分析成像期间横跨视场漂移。水凝胶在这里被结合到COVerslip使得它们保持固定在整个实验。因为水凝胶可能会阻碍细胞的可视化三，3D FRET适当的水凝胶组合物的选择是至关重要的。 PC-凝胶和TR-凝胶这里透明的水凝胶被使用。然而，收集用离心分离使细胞在盖玻片附近的焦平面可以用于图像的细胞在水凝胶具有较高的水凝胶的衍射仪选择取决于微环境条件必须模拟，其可在评论中找到对凝胶42 。第四，一个备用计算此处使用的单链ICUE1探针的FRET比率（ 即 ，E QE = QE / CAEM），以尽量减少分析所需的图像的信道数，从而减少光漂白。每个细胞的平均FRET比率被计算为平均每像素的比率的小区内，以尽量减少实际的FRET比率的低估。最后，一​​个线性比例分析和装置计算单链二元探针的激活的级分进行说明。 有使用广角镜进行成功的FRET实验的几个关键方面。对于硬件，相机必须足够敏感来解决小信号变化，而使新科学的CMOS摄像机和电子倍增CCD比传统的CCD更适合。到最好的分析的FRET比率的空间分布，相机必须至少具有物镜（奈奎斯特准则）的点扩散函数的空间分辨率的两倍。这使得双视图系统不太适合的任务。更关键的是，选择用于给定FRET对适当的曝光和图像采集速率以便最小化的荧光团的光漂白。降低采集速率被推荐为实验的持续时间增加。使用快速滤光轮的提高采集速率和视场进行分析，同时避免了数ING与双视图和炮塔滤镜立方体图像配准的问题。不均匀的照明领域的复杂化，从样品自发荧光和摄像头的系统噪声产生的背景荧光校正。一些水凝胶，特别是那些含有胶原蛋白是高度自体荧光43,44。该FRET比率被低估没有背景校正。在这里，我们采用依赖于它可以经常与落射荧光照明在图像上（ 图3B，步骤7.2）的中心被配置在相对 ​​平坦的照明场的简单的背景校正方法。因此，这种方法限制了感兴趣的那些该照明领域内的细胞。这里所描述的背景校正不考虑在读取二进制探针波长蜂窝自发荧光。在这样的情况下，未标记的细胞（无探针转染）应在相同的条件和背景减去下进行成像。 A M可以使用标记和未标记的细胞的九和选定为背景的ROI的未标记细胞。这为在整个图像领域细胞分析替代方法包括使用遮光膜的，多背景的投资回报，或相同的样品不含细胞和协议可应要求提供。 具体到3D FRET成像，探测响应是向水凝胶渗透性分析物（ 图6）高度敏感。因此相同的水凝胶区域横跨实验处理相比，优选在几何对称点如邻近如这里使用的支架中心。可替代地，微流体腔室/生物反应器可用于迅速而均匀地灌注与分析物溶液45的水凝胶。音柱信号可能不会被检测到（信噪比过低）时，水凝胶的自发荧光过高;应使用的较薄的水凝胶或不同探针（不同的荧光团）。在光交联水凝胶相对于3D FRET成像，建议使用最小辐射曝光和探针的荧光团的激发光谱外的波长。 LAP可以与可见光源在405nm被用来进一步减少潜在的细胞损伤31,46。但是，建议使用365纳米的照射LAP因为这减少了探测器漂白。 365nm处在于在CFP施主激发光谱的尾端，而405nm处在于在峰值激发。可替代地，探针的表达可以被允许一天恢复。二进制探针的使用最小化灵敏度的问题，以在表达水平，并在供体和受体荧光团的分子扩散的差异。非二进制探针可以被使用，但具有SE代替QE成像和附加校正激发和发射光谱的光谱渗出至（见下文）。此外，低商业可用性和设计的FRET探针的复杂性可能是一个障碍。对于成功的实验的最关键因素仍然是荧光信号的适当的修正和分析。 FRET的比率的替代计算被用于除了光漂白的最小化几个其他原因。对于二进制单链探针时，常见的比例是根据捐助者的激励（致敏的排放，SE），以供体发射下捐助激励（淬火排放，QE）， 即受体发射，FRET比= SE / QE 25,47,48。 SE / QE是非线性相对于FRET效率21,22,47变化。即，该比例具有与探针（EI）的固有的FRET效率的指数的非线性关系（Donius，AE，Taboas，JM 未发表的作品。（2015）），与荣小于大约15％的比例最线性的。 SE / QE还将低估激活探针（FAP， 即，探针结合分析物的级分）的分数。 Therefo再次，SE / QE的分析需要繁琐的平衡剂量反应研究，曲线拟合。幸运的是，SE或QE到受体发射下受体激发的比例（AEM）是相对于Ei和FAP， 即直链的，所述的FRET比率SE / AEM和QE / AEM。 SE / AEM通常用于二进制探针，只需要结束点校准（在无探针的活性和全探针活性），但基底细胞信号使这个困难。为了消除对端点校准的需要，SE能对供体和受体的激发和发射光谱的光谱重叠（光谱泄放通）和用于将合并的探针的荧光团和显微镜的量子产率和光谱灵敏度（QS）进行校正（CSE）成像系统。基于CSE校正后的SE的FRET比率限定的图像， 即，E SE = CSE / AEM的每个像素内的探针的观察FRET效率。商业和免费软件可供SE纠正这些影响和读者可参考陈和2006 Periasamy的工作的方法50的详细说明。但是，计算êSE要求每个时间点（SE，QE，AEM）三个图像通道捕获。因此，在此工作中，我们也使用一个替代的FRET比E QE = 1 - QE / CAEM，其中只需要两个图像通道的捕获（QE和AEM）。从这些渠道计算ËQE只需要修正为AEM QS（CAEM = AEM x质量得分）。 QS = DEM的/ AEM利用从一个校准样品，其中，数字高程模型是根据供体激发的供体发射与受体漂白两个图像容易地估计。 FAP的在任何时刻可以用E SE或E QE比较探针的荣来计算。 最终，FRET比选择（E SE = CSE / AEM 与 ËQE = QE /航空气象学委员会）应基于考虑探头类型，并与最佳的信号通道。既是pproaches包括如上所述以考虑随时间的自体荧光的改变每帧的图像的背景噪声的校正。他们不承担任何AEM激发光重叠到民主党激发光谱，这往往是由于探头设计和显微镜的过滤器配置的情况下。单链探针可以使用和选择是基于最好的探测信号（亮度）到相机噪声，并就SE和QE通道背景信号。对于ICUE1探头和这里使用的实验条件（细胞和水凝胶型），SE比QE更强的信号。双链探头需要采用E SE由于供体和受体荧光团非等摩尔分布。对于在结合分析物如ICUE1在FRET降低，FRET效率可以"倒"呈现在结合分析物的积极信号的变化，因为我们在这里做的，有E SE = 1探测器- CSE / AEM或E QE = QE / （AEM x质量得分）。 日分析ËFAP是对FRET比率适当修正，并荣的估计敏感。它可以与用于QS上的相同的校准样品和常规的端点来估计FRET效率测定为荣= 1-（QE / DEM）（QE图像后跟受体漂白和DEM的图像）28，但必须小心，以尽量减少捐助意外漂白。我们描述那里调整QS的民主党基于AEM估计被取代，修改后的版本，即 ，EI = 1 - （QE /（AEM x质量得分））。交替，可以使用荣= CSE / AEM。在活细胞荣估算，要求所有探头被驱动到全FRET。这是在实践中难以为探针，如ICUE1即在结合分析物中的FRET减少来实现。使用细胞裂解物和纯化的分析物荣的体外基于计算是最好的，但这种工作的范围之外。在这里，我们建议使用2&#39;，5&#39;-二脱氧腺苷，以减少在细胞内cAMP。然而，一个相对的FAP可be。使用从信令的基线水平得出的EI估计计算。由于这些原因，研究最经常报告基于端点校准，其中加入激动剂之前，信令基线是下界和信号并称饱和信令的第二激动剂后的FRET比率（如这里完成）或相对FAP在上界。福斯克林常被用作对照激动剂饱和的cAMP信号。交替，可以使用cAMP类似物如8- Bromoadenosine 3&#39;，5&#39;-环一磷酸。在的研究，以确定在试验性治疗中的信号传导途径的潜在变化的完成使用阳性对照建议。 每个细胞的平均信号传导应答的计算需要考虑的几个因素。首先，将平均FRET比率应计算的平均值的比率在每个像素的小区内， 即 ，（Σ（QE/cAem））/（像素数），而不是岭平均QE和AEM在细胞， 即 （ΣQE）/（ΣcAem）的蒂奥。尽管后者不需要小心掩蔽作为背景噪音低，它严重低估了真实平均FRET比率。然而，像素比值需要浮点运算和单元面积来定义的ROI和避免包含从细胞外的背景噪声产生的寄生比的仔细二进制掩蔽。整个小区区域必须量化以避免对细胞形状偏压的结果。掩蔽可能需要重新定义随时间根据在细胞形状和迁移的变化。或者，如果排除标准定义为从QE并且大大低于细胞水平和接近背景水平AEM信号中去除的像素的比率，可以使用的ROI比电池大。所描述的分析方法的细节在此工作的FRET分析中使用无需专门软件/插件的基本步骤。显微镜制造商和其他供应商销售附加模块S代表他们的显微镜软件，它支持自动比例和FRET分析。同样，公共领域的ImageJ软件具有可用于颗粒和FRET分析几个插件，如RiFRET。第二，基于像素的平均的FRET比率低估，因为2D图像使用三维微孔结构的真正的平均比率。二维信号表示沿z轴的平均值。在活细胞中的FRET比率的三维空间分布的计算是不可能的，而不高速三维成像（ 例如 ，使用旋转盘共焦显微镜）。这是二维和三维培养物中的问题，但在3D较大效果更蜂窝结构中存在的平面外。这对定位于细胞结构，例如细胞膜EPAC1探针PM-ICUE探针的极大关注。见Spiering等。 2013年的建议，以修正对比率计算，24电池厚度的影响。 AEM的分布的分析可以用来如果检测探头调节细胞和成像平面的区域聚集。这也将有助于解释FRET比率的空间分布，并消除虚假的结果， 例如 ，识别探针饱和度（ 即，低的AEM区域将具有低探针浓度和可以表现出探针饱和和高的FRET比率）。第三，不同的FRET基准水平可以指示在转染效率的差，探测实验组之间的表达，或基底信令。 "相关性分析"（步骤10.1.1）的帮助，如果存在删除漂在FRET比率随着时间的推移。它促进的样品之间的反应的相对大小的比较，但阻碍比较响应为参考样品（对照区是一个平线）的时间过程。 "绝对分析"（步骤10.1.2）便于在样品反应速度的比较，但因为在每行由一个dissim缩放妨碍反应的幅度的比较ILAR不变。研究经常使用在基线上的线性回归以校正在FRET比率随时间漂移。 所描述的3D FRET方法是制造和执行小区载货三维水凝胶，其可以被应用于其它探针和成像方式的时间推移成像有用的和实际的方法。除了单链二元探针其它FRET系统将需要基于强度的信号的更复杂的校正，如上所述。另外，FRET（FLIM-FRET）的荧光寿命成像可用于通过探头供体发射寿命的变化来计算FRET效率。不像基于强度的FRET，FLIM-FRET是不敏感的背景噪声，光谱渗出通过，量子效率，和检测器的光谱灵敏度2。然而，FLIM系统是昂贵的，复杂的，和不常见的，并与单指数衰变的荧光团和无FLIM的径流49工作最好。所描述的方法也可以与莫用于再先进的显微镜平台（ 例如 ，FRET的TIRF，荧光各向异性，谱相关FRET）。应用这种方法对三维成像高速共聚焦和多光子显微镜将促进信令响应的亚细胞分布的分析和增加信令分析的准确度。这种3D FRET方法将使模拟3D微环境细胞的高级细胞生物学研究。因此，它可以容易地应用到药理学和再生医学的需求，包括研究细胞间信号传导和筛选在改造的水凝胶基microtissues药物反应。

细胞信号既复杂又衍生了众多领域，包括药理学，组织工程和再生医学。有用的研究性工具需要进一步我们的细胞生物学的理解，并发展组织再生的最佳材料。荧光共振能量转移（FRET）成像是一个重要的工具，使受体激活，分子构象，并在活细胞中的超分子相互作用（ 例如 ，蛋白质/ DNA复合）的分析。 FRET是一种荧光团1之间的非辐射能量转移的。它有一个效率是成反比的供体荧光团和受体荧光团之间的距离的第六功率。因此，它可以提供有关的空间距离在纳米尺度两个不同分子之间或跨越一个分子的区域的信息（通常为1 - 10纳米）2。供体和受体荧光团可以是不同的Molecule类型（杂-FRET），其中该供体具有更高的能量发射（较短波长），或者相同类型的（同型FRET）。 FRET成像可以在固定的或活细胞中进行，但在一般的固定细胞用于分子间的相互作用的成像在高空间分辨率时高速共聚焦系统不可用。活细胞用于研究的相互作用和过程被抑制或通过定影改变，如实时细胞内信号响应3。 雇用活细胞研究FRET显像使用二维（2D）细胞培养物中，因为简单和低的背景（无从平面外的细胞的发射）的一部分。然而，2D培养物也相比于生理微环境和三维（3D）培养4,5-改变众多细胞过程。例如，细胞和细胞外基质相互作用天然细胞被彻底改变在二维培养导致EASILY观察细胞形态和极性6的变化。膜微（ 例如 ，小窝和脂筏）和受体信号强烈的文化环境，因为它们结合细胞骨架7,8和细胞形态和细胞骨架结构在很大程度上受到空间的养殖环境调控9上调，部分。机械传导不仅由3D矩阵的影响，但由更复杂的二级载荷条件在3D环境主义及其相比2D培养物10。最后，三维矩阵的渗透率小于培养基，一般具有降低的扩散和增加的信号传导分子相比2D培养细胞的结合。与3D培养人能够创建并观察一个微环境更类似于生理相对于粘合剂，化学和机械的线索。细胞与细胞的相互作用可促进和粘接性能能够控制瓦特第i个的结构中，组合物，和3D材料11-13的体系结构（图案形成）。该材料的机械性能同样可以通过组成和结构14,15量身定做。培养细胞在水凝胶，因此允许FRET上，否则将去分化的原代细胞或使用常规技术在表型变化， 例如研究中，从关节软骨细胞。因此，需要一种方法，以使在现场的3D培养物的FRET测定。 水凝胶的支架是理想的基于三维的FRET的研究，因为它们可以被制成光学透明，并适合控制微环境，并提供蜂窝线索。从天然聚合物制成的水凝胶已经在细胞培养中使用了几十年，包括明胶和纤维蛋白16。在细胞微环境更大的控制可以与这些聚合物的化学改性，并与使用合成聚合物17,18来实现。 HYDRogel机械刚度和渗透性可以通过改变它们的聚合物组合物进行控制和网眼尺寸（聚合物和交联密度）19,20。此外，水凝胶可以通过生长因子和细胞的配体的结合进行的生物活性。由于这些可能性，水凝胶生物传感，药物输送和组织工程应用的发展研究21一 ​​个非常活跃的领域。水凝胶的三维打印也正在开发微型组织制造和-organs 15。因此，在水凝胶的细胞FRET成像不仅作为近似生理细胞行为的手段是有用的，但也可作为工具来研究和优化工程化组织的生长。 二进制比率的FRET探针是在研究细胞信号传导非常有用的，可以在水凝胶培养物被利用。对于荧光公布的部分名单，FRET探头，看到细胞迁移联合会网站<sup&gt; 22。最常见的探针类型包含由一个识别元件（多个）（分析物敏感区域）关联或连​​接的两个不同的荧光团。这些区域经历构象变化，联系或解离在检测分析物（ 例如 ，离子或分子，该区域的酶裂解的结合），该改变荧光团，因此FRET幅度（或高或低）之间的距离23。一个不太常见的但却很有用的探头类型依赖于两个荧光基团的光谱重叠，从而改变FRET幅度分析物敏感的变化。 "单链"比例式传感器利用荧光团对上的单分子相连。 "双连锁"探测器利用在不同的分子荧光团对，但他们的表现可以在相同的表达盒24下耦合。不同于单一的荧光表达研究（ 如 ，荧光融合蛋白），研究与比率FRET探针不需要双重转染，以控制转染效率或细胞生存力。最低门槛（浓度不敏感）23,25以上时，表示比率FRET探针是相对不敏感的转染效率。它们是不敏感的激发源的波动和其它细胞内环境因素（ 例如 ，pH值，离子），只要这两个荧光团也同样敏感。此外，他们的反应是不受转录延迟，不像荧光和生物发光启动子-报道构建26,27。 成比例的FRET探针轻松，频繁地在传统的宽视场落射荧光显微镜系统中采用。宽视场显微镜优于由于超过系统成本，荧光漂白，并以足够的时间分辨率信号改变捕获的关注线扫描共聚焦系统为活细胞成像。另外，单电池肛门ysis在一个人口众多的细胞是可能的机动阶段和图像采集了多视野。广角镜需要异质FRET探针信号的强度基础分析。虽然强度分析不需要像其他的FRET方法复杂的硬件，更采集后工作是需要以校正荧光行为和显微镜/成像系统配置28的影响。荧光团的拍摄发光强度的激发能量的函数，荧光量子产率，并且将合并的过滤器和相机/检测器的光谱灵敏度和线性度2。这些可以是不同的供体和受体，并且需要进行定量分析的校准。此外，该受体发射的成像由荧光团的光谱重叠的影响（受体的激发供体激发光和渗出通过供体发射的到受体发射光谱）2。4单链"探针内部归因为它们的荧光团等摩尔在纳米尺度，而荧光团"双链"探针可以在不同的化学计量存在整个电池25,28如果荧光团。"单链"探针是相似亮度（消光系数和量子产率的功能），非线性检测器的灵敏度的效果是对于背景荧光和耦合量子收率/检测器的灵敏度，需要23小并且仅校正的。因此，"单链"比例FRET探针是最容易在广角镜24实现。 本文介绍一种简单的技术来执行使用传统的宽视场啶显微镜在三维水凝胶文化的活细胞成像FRET。它可以通过已经开展二维FRET实验，以及有意间实验室的实验室很容易地通过在microtissues细胞信号。在这里，我们证明用环磷酸腺苷（cAMP）的光交联内活软骨细胞水平（PC-凝胶，聚乙二醇二甲基丙烯酸酯）和温敏（TR-凝胶，基质胶）水凝胶的FRET成像基于测量的方法。进行比例FRET探针是基于EPAC1（ICUE1）来检测响应佛司可林，对腺苷酸环化酶的化学激动剂其催化ATP向cAMP的转化cAMP水平利用。的cAMP是主要的第二信使介导的G蛋白偶联受体信号之一，并激活各种因素，包括由腺苷（EPACs）直接激活下游交换蛋白。荧光团移动分开时cAMP结合，导致在FRET的减少的EPAC域。这里所描述的是水凝胶材料的在三维水凝胶的细胞的制备中，细胞转染与ICUE1探针和嵌入。三维FRET实验程序和必要的图像分析以评估每个细胞活性探头解释。我们还讨论了在2D和3D用广角镜FRET分析的内在局限性。这里提出的技术是在现有的二维方法的改进，因为它使在一个更生理上下文分析和需要更少的图像校正和校准。极大效用在于，可用于许多透明材料，同时细胞和转染的偏好可以维持这一事实。

<table><tbody><tr><td>Polydimethylsiloxane (PDMS) [Sylgard 184 Elastomer Kit]</td><td>Fisher Scientific (Ellsworth Adhesives)</td><td>NC0162601</td><td>Reagents</td></tr><tr><td>Polyethylene glycol dimethacrylate (PEGDM)</td><td>Prepared according to Lin-Gibson with PEG from Sigma Aldrich</td><td>95904</td><td>Reagents</td></tr><tr><td>3-(Trimethoxysilyl)propyl methacrylate</td><td>Sigma Aldrich</td><td>M6514</td><td>Reagents</td></tr><tr><td>3-glycidoxypropyltrimethoxysilane</td><td>Sigma Aldrich</td><td>440167</td><td>Reagents</td></tr><tr><td>Dimethyl Phenylphosphonite</td><td>Sigma Aldrich</td><td>149470-5g</td><td>Reagents</td></tr><tr><td>2,4,6-trimethylbenzoyl Chloride</td><td>Fisher Scientific</td><td>AC244280100</td><td>Reagents</td></tr><tr><td>2-Butanone</td><td>Fisher Scientific</td><td>AC149670010</td><td>Reagents</td></tr><tr><td>Lithium Bromide</td><td>Fisher Scientific</td><td>AC199871000</td><td>Reagents</td></tr><tr><td>Isopropanol</td><td>Sigma Aldrich</td><td>190764</td><td>Reagents</td></tr><tr><td>Sigmacote (siliconizing reagent)</td><td>Sigma Aldrich</td><td>SL2</td><td>Reagents</td></tr><tr><td>Toluene&amp;nbsp;</td><td>Fisher Scientific</td><td>AC36441-0010</td><td>Reagents</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Hyclone</td><td>SH3025601</td><td>Reagents</td></tr><tr><td>Opti-MEM Reduced Serum Medium, no phenol red (phenol- and serum-free medium)</td><td>Life Technologies</td><td>11058-021</td><td>Reagents</td></tr><tr><td>FuGENE HD Transfection Reagent (transfection reagent)</td><td>Promega</td><td>E2311</td><td>Reagents</td></tr><tr><td>Cellstripper (cell dissociation solution)</td><td>Corning</td><td>25-056-CI</td><td>Reagents</td></tr><tr><td>Growth Factor Reduced Matrigel, Phenol Free</td><td>Corning</td><td>356231</td><td>Reagents</td></tr><tr><td>Forskolin</td><td>Sigma Aldrich</td><td>F6886</td><td>Reagents</td></tr><tr><td>Pipette Tips (10,200,1,000 &amp;mu;l)&amp;nbsp;</td><td>Fisher Scientific</td><td>02-707-474, 02-707-478, 02-707-480</td><td>Disposable lab equipment</td></tr><tr><td>Powder Free Examination Gloves</td><td>Fisher Scientific</td><td>19-149-863B</td><td>Disposable lab equipment</td></tr><tr><td>Aluminum foil</td><td>Fisher Scientific</td><td>01-213-100</td><td>Disposable lab equipment</td></tr><tr><td>Biopsy punch (3 mm)</td><td>Miltex</td><td>3332</td><td>Disposable lab equipment</td></tr><tr><td>Glass coverslips</td><td>Fisher Scientific</td><td>12-544C</td><td>Disposable lab equipment</td></tr><tr><td>Precoated glass coverslips (epoxysilane)</td><td>Arrayit</td><td>CCSL</td><td>Disposable lab equipment</td></tr><tr><td>Glass slide</td><td>Fisher Scientific</td><td>12550D</td><td>Disposable lab equipment</td></tr><tr><td>Sterile vials (15 ml, 50 ml conical)</td><td>Fisher Scientific</td><td>14-959-70C, 14-959-49A</td><td>Disposable lab equipment</td></tr><tr><td>Cell culture dish (6-well)</td><td>Falcon</td><td>351146</td><td>Disposable lab equipment</td></tr><tr><td>Epifluorescent Microscope with motorized stage</td><td>Nikon</td><td>MEA53100 (Eclipse TiE Inverted)</td><td>Large/non-disposable lab equipment</td></tr><tr><td>Adjustable Specimen Holder for XY Stage</td><td>Nikon</td><td>77011393</td><td>Non-disposable lab equipment</td></tr><tr><td>60X (1.3 NA) objective (CFI Plan Apochromat 60XH Lamda)</td><td>Nikon</td><td>MRD01605</td><td>Non-disposable lab equipment</td></tr><tr><td>CFP/YFP light source and excitation / emission filter holders (Lambda light source &amp;amp; wheel, Lambda emission filter system)</td><td>Nikon (Sutter Instrument reseller)</td><td>77016231, 77016088</td><td>Non-disposable lab equipment</td></tr><tr><td>CYF/YFP filter set</td><td>Nikon (Chroma Technology Corp)</td><td>77014928</td><td>Non-disposable lab equipment</td></tr><tr><td>Camera (ORCA Flash 4.0 v1)</td><td>Nikon (Hamamatsu reseller)</td><td>77054076</td><td>Non-disposable lab-equipment</td></tr><tr><td>NIS-Elements 40.20.02 microscope software)</td><td>Nikon</td><td>MQS31100&amp;nbsp;</td><td>Non-disposable lab equipment</td></tr><tr><td>Prism (spreadsheet and graphing software)</td><td>GraphPad Software, Inc</td><td>Version 6</td><td>Non-disposable lab equipment</td></tr><tr><td>OmniCure S1000 UV Spot Curing Lamp System with 365nm filter or other light source (&amp;lambda; = 365 or 405)</td><td>OmniCure</td><td>S1000</td><td>Large/non-disposable lab equipment</td></tr><tr><td>Pipette Aid&amp;nbsp;</td><td>Drummond Scientific Co.&amp;nbsp;</td><td>DP-100</td><td>Non-disposable lab equipment</td></tr><tr><td>Hemacytometer</td><td>Hausser Scientific</td><td>Reichert Bright-Line 1475</td><td>Non-disposable lab equipment</td></tr><tr><td>Vacuum desiccator chamber/degasser&amp;nbsp;</td><td>Any</td><td></td><td>Large/non-disposable lab equipment</td></tr><tr><td>Tissue Culture Hood&amp;nbsp;</td><td>Any</td><td></td><td>Large/non-disposable lab equipment</td></tr><tr><td>Chemical Fume Hood&amp;nbsp;</td><td>Any</td><td></td><td>Large/non-disposable lab equipment</td></tr><tr><td>Oven</td><td>Any</td><td></td><td>Large/non-disposable lab equipment</td></tr><tr><td>Spatula</td><td>Any</td><td></td><td>Non-disposable lab equipment</td></tr><tr><td>Beaker</td><td>Any</td><td></td><td>Non-disposable lab equipment</td></tr><tr><td>Incubator&amp;nbsp;</td><td>Any</td><td></td><td>Large/non-disposable lab equipment</td></tr></tbody></table>

fret imaging in three-dimensional hydrogels

荧光共振能量转移（FRET）的成像是在实时检查细胞生物学的有力工具。利用FRET研究通常采用的两维（2D）培养，其不模仿三维（3D）细胞微环境。使用3D凝胶环境内的细胞的常规广角荧光显微镜进行骤冷发射FRET摄像用方法。这里描述了比例FRET探针产生在探头激活范围线性比例的分析方法。细胞内环磷酸腺苷（cAMP）水平的测定证明在根据福斯克林刺激软骨细胞用探针为EPAC1活化（ICUE1）和检测cAMP的信号依赖于水凝胶材料的类型，在此光致水凝胶（PC-凝胶差异的能力，聚乙二醇二甲基）和一个温敏水凝胶（TR-凝胶）。与2D FRET方法相比，这种方法需要很少的额外工作。已经利用FRET成像2D实验室可以很容易地采用这种方法在3D微环境进行细胞学研究。它可以进一步工程化三维microtissues应用于高通量药物筛选。此外，它与其他形式的FRET成像，诸如各向异性测量和荧光寿命成像（FLIM），并与使用共聚焦调制照明先进显微术平台，脉冲，或兼容。

如上所述制备所有水凝胶前体，细胞和铸模。小区载货前体溶液流延在PDMS模具，然后凝胶化之前离心/光交联固定化细胞的盖玻片附近并促进FRET成像分析（ 图1）。附带盖玻片水凝胶置于准备PDMS成像井显微成像（ 图2）内。光学配置和图像采集参数然后设置为如图3A所示。为CFP-YFP荧光团对所有四个FRET相关图像通道的捕获证实（参见"光学CONF"。在图3中），因为需要对非二进制探针。然而，在这项工作只有两个图像都需要对单链二元ICUE1探头，QE ="CFP CFP"和AEM ="YFP YFP"（激发发射）。多视场（XY位置多个）被选择，其中几个细胞的均匀的照明区域（ 图3B）内居住。时间的推移收购完成后，将探头信号导出数据。感兴趣区为信道信号的背景校正和细胞分析采用阈值AEM图像从实验（ 图4）的开始被指定。从细胞池因为这些表达中选出足够的细胞（不是太暗淡），但不能太高（太亮）探针（ 图4B）。在每个采集每个象素的QE和SE的FRET比率使用背景相减图像通道的浮点除法计算。每个细胞的平均FRET比率（即每ROI）计算归一化到之前刺激基线信号（ 即，来自所有时间点中减去基线回归），并用误差棒为一体的标准误差作图平均（SEM）。无论是QE和基于CSE FRET比德TECT下福斯克林刺激（100纳米， 图5）软骨细胞的cAMP增加的活性。在QE FRET比是比CSE FRET比背景校正更敏感，因为QE信号由于嵌入水凝胶内的cAMP软骨细胞活性的低基础水平较低。这两种水凝胶类型的（TR-凝胶和PC-凝胶）显示在FRET比率随时间增加（ 图6），这表明在加入毛喉素的增加cAMP的信号传输响应。正如QE FRET比表示，在PC-凝胶水凝胶软骨细胞显示cAMP的变化较大（如图所示）和cAMP（E QE更高的基础水平= 0.19 PC-凝胶与 ËQE = 0.09 TR-凝胶，在基线减去图中未示出）。 <img alt="图1" src="/files/ftp_upload/54135/54135fig1.jpg" /> 图1: 在光交水凝胶（PC-凝胶）细胞分布 </STRONG&gt; 答:细胞载货水凝胶前体离心在盖玻片附近焦平面最大化细胞的数目。 B:这增加了视场的细胞数，并从平面外的细胞减少背景信号。 （比例尺= 200微米） <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54135/54135fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54135/54135fig2.jpg" /> 图2:水凝胶在PDMS菜 水凝胶放在盖玻片基地显微成像和分析物刺激一个高大的PDMS良好（10倍量水凝胶）内。在PC-凝胶水凝胶是透明和结合到盖玻片，使它们保持在适当位置在整个测定法。将PDMS孔可以增大以包含比使用除了在更宽的活检穿孔，水凝胶体积的10倍多平台hicker PDMS表。 （比例尺= 2mm）的<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54135/54135fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54135/54135fig3.jpg" /> 图3: 采集配置FRET成像 答:本次收购在多个位置配置了图像采集每7秒。对于QE FRET比例计算，只需要两个图像通道的下此处使用的ICUE1探头（二进制单链探针），QE ="CFP CFP"和AEM ="YFP YFP"（激发发射）"光CONF。"在显微镜软件的λ标签栏。 A:视场（"XY"位置）被选中，其中几个细胞均匀的照明区域内居住。图像下面的图显示沿着蓝色的光照强度箭头线遍历通过FOV的中心。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54135/54135fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54135/54135fig4.jpg" /> 图4:FRET成像分析投资回报率的选择 答:利息率（ROI）游离细胞的一个区域选择纠正每个通道的背景信号。无细胞的水凝胶的图像可以替代地用于背景减除如果细胞密度或迁移是高，或者如果当细胞不均匀的照明场中位于阴影掩模不被使用。 B:使用图像阈值与AEM通道的二进制掩蔽被选定单元格的投资回报。选择比细胞大的投资回报率将每个细胞的平均比例FRET计算产生不利影响，由于夹杂背景ñ产生的胞外比率瓦兹。不建议使用每个小区的每个信道的平均蜂窝FRET比率的计算，因为这低估的比率。 （比例尺= 50微米）， <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54135/54135fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54135/54135fig5.jpg" /> 图5:FRET比率的温敏凝胶（TR-凝胶）的比较 无论QE和基于SE FRET检测的比率下毛喉素刺激软骨细胞的cAMP增加活动。毛喉素是一种腺苷酸环化酶激动剂诱导cAMP的产生。当ICUE1探针结合的cAMP FRET减小，并且因此QE FRET比（E QE = SQE /（SAEM x质量得分））增加。相反，SE FRET比率下降，因此绘制为E SE = 1 - CSE / SAEM。在水凝胶嵌入式软骨细胞，在QE˚FRET比率是背景校正比SE FRET比率由于QE信号为低更为敏感，由于低的基础cAMP的活性。误差棒= SEM，N = 25。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54135/54135fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54135/54135fig6.jpg" /> 图 6: 不同的水凝胶FRET率的比较 在这两种类型的水凝胶的软骨细胞回应毛喉素刺激这表现在增加QE FRET比例随时间增加的cAMP活动。水凝胶型的影响通过多个作用，包括在渗透性福斯克林和基底细胞cAMP活性的差异的细胞反应（E QE = 0.19在PC的凝胶与 ÊQE = 0.09在TR-凝胶，未示出）。误差棒= SEM，正为TR-凝胶= 25，正为PC-凝胶= 15。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54135/54135fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about FRET Imaging in Three-dimensional Hydrogels at JoVE.com

FRET Imaging in Three-dimensional Hydrogels

荧光共振能量转移（FRET）成像是用于实时细胞生物学的研究的强有力的工具。这里提出了在使用常规荧光显微镜生理三维（3D）水凝胶的微环境FRET显像细胞的方法。被描述为比例FRET探针产生在激活范围线性比例的分析。

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

fret-imaging-in-three-dimensional-hydrogels

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.0K 次观看.  University of Pittsburgh. 荧光共振能量转移（FRET）成像是用于实时细胞生物学的研究的强有力的工具。这里提出了在使用常规荧光显微镜生理三维（3D）水凝胶的微环境FRET显像细胞的方法。被描述为比例FRET探针产生在激活范围线性比例的分析。 

视频: FRET Imaging in Three-dimensional Hydrogels

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

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

观察和量化纤维细胞介导的纤维素凝胶压实

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

一种优化的O9-1/水凝胶系统，用于研究神经嵴细胞中的机械信号

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

发育生物学

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

基于聚丙烯酰胺的多井格式实验研究细胞外基质硬度对黏附细胞细菌感染的影响

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

免疫与感染

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

无参考牵引力显微镜平台的制造和实施

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

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

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

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

使用水凝胶图案化技术控制细胞粘附，用于牵引力显微镜的应用

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

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

微图案牵引显微镜的图案生成

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

水凝胶阵列可提高 3D 肿瘤模型中基质成分和治疗药物筛选效果的通量

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...