我们要感谢华盛顿大学的集体知识，这个协议可能在整个毛雷尔组。这项工作的经费是由国家精神卫生研究所（1R01MH085495）。

1。制备的图案硕士（图1） <ol><li>中心硅片上的自旋涂布在两个周期自旋方案的表1中的最初步骤，并用丙酮冲洗晶圆。丙酮蒸发过程中的自旋留下一个清洁，干燥的晶圆计划的第二步。 </li><li>应用约1毫升AZ9245光阻/（直径）晶圆和自旋大衣，使用表1中所述的条件。 </li><li>软烤的光致抗蚀剂涂层的晶圆在110 ° C为2米，用高均匀烤盘。 </li><li> Photopattern基材，使用一个直写光刻系统或光刻系统和适当的口罩。可以从商业来源购买口罩。此外，紫外线吸收剂，油墨贴光学平面，印刷的透明胶片，可用于生产与大的特点模式。 </li><li>在1:2 400K开发商开发图形晶片：1米45的小号轻轻摇动半导体级去离子水。彻底冲洗半导体级去离子水和干燥的氮（N 2）气体的流。具有紫外线过滤器使用显微镜可以检查发展格局。如果模式没有完全开发，晶圆可以返回到有更多的时间发展的解决方案。 </li></ol>注：为了达到最佳效果，photopatterning应在洁净室环境。 2。制备PDMS的邮票（图2） <ol><li>准备10:1的重量树脂：固化剂混合Sylgard 182（PDMS）和完全覆盖主一次性培养皿中的混合物（photopatterned晶圆）。 </li><li>德气体的PDMS覆盖在真空dessicator中的主，直到无气泡是可见的，允许邮票治愈在65 ° C烘箱中为1.5小时。固化之前，重要的是确保主盘底部是在脱气的一步，因为它可能上升到表面。 </li><li>削减的PDMS邮票主，修剪到合适的大小。储存在有盖容器（功能的一面朝上），以防止灰尘和杂物它图章。 </li></ol> 3。图案的金基底的制备（图3） <ol><li>没有准备25毫米。 1金属沉积与氧等离子体处理10米的圆形盖玻片18.2MΩ的去离子水其次是用乙醇的两倍，每一步之间的 N 2气体流与干燥，冲洗两次。 </li><li>使用钛存款50多口袋里的电子束沉积系统，其次是150金。不泄的蒸发器之间的钛层和金层沉积。另外，镀金盖玻片可以从各种不同的供应商购买，但是，它是重要的金属层是由电子束蒸发和不热蒸发的准备。如果从外部购买，黄金基板可能由等离子体氧化或“食人鱼”（7:3浓H 2 SO 4：30％的H 2 O 2）11，在使用前进行清洗。 </li></ol>注：“食人鱼”的解决方案是在有机化合物的存在爆炸性。 <ol start="3"><li>准备冲压解决方案，在无水乙醇10毫米hexadecanethiol，和回填的解决方案，1毫米乙二醇终止无水乙醇巯基。 </li><li>邮票用乙醇冲洗，并彻底干燥与 N 2气体。应用冲压解决方案，直到完全包覆的PDMS邮票滴加。与N 2气体彻底干燥的邮票。适当的PDMS邮票的特点的基础上，继续5A或5B。 </li><li><ol class="lalpha"><li>轻轻按下的金基底上的邮票，并允许单层，形成15条</li><li>放入培养皿含有18.2MΩ去离子水菜金基底，确保基材被淹没。轻轻按下的金基底上的邮票，并允许单层，形成15条</li></ol></li><li>用乙醇冲洗两次加盖基板，与N 2气体干燥后，每冲洗，并放置在培养皿基板。 </li><li>回填解决方案覆盖的基板和菜用封口膜密封，以防止水分蒸发。 </li><li> 12-14小时，允许单层的背景，在黑暗中形成</li><li>回填解决方案中删除的图案盖玻片，并用乙醇冲洗两次，每冲洗后干燥与 N 2气体。 </li></ol> 4。应用蛋白质和细胞图案的基材<ol><li>广场图案中的一个小培养皿或细胞培养室和盖盖玻片，用500μl1毫升贝科磷酸盐缓冲液（DPBS）。 DPBS必须完全覆盖在蛋白质孵化基材，以确保蛋白质覆盖。 </li><li>添加蛋白浓缩液的DPBS和管道的混合解决方案婷若干倍。孵育与基体蛋白混合物，在37 ° C为1小时层粘连蛋白和纤维连接蛋白终浓度一般为12μg/ mL和20微克/ ml，分别。蛋白质可与胺反应的荧光染料标记，以便容易格局可视化。然而，标记的蛋白质混合1:1蛋白标记的未标记的蛋白质可干扰生物活性。 </li><li>孵育后，用DPBS（4 - 5倍）彻底冲洗基板去除未结合的蛋白质，照顾，不会干的基板，或把它通过空气 - 水界面的。经过前三个漂洗，加入约500μL完整的细胞生长介质，以保持湿基材。 </li><li>更换的增长，用于冲洗与新媒体的基板媒体。 </li><li>分离和计数细胞电镀基板上。无论是分离的主细胞，如海马神经元，或永生细胞株，如CHO - K1细胞，可以利用。 </li><li>板基板上分离的细胞。通常使用30至200个细胞/毫米2。 </li></ol> 5。代表性的成果： <img src="/files/ftp_upload/3164/3164fig1.jpg" alt="图1"/> 图1。一个图案主光刻准备的一般原理图。在这个过程中，用丙酮清洗硅片，涂光致抗蚀剂，接触到的利益格局，和开发模式。 <img src="/files/ftp_upload/3164/3164fig2.jpg" alt="图2"/> 图2。PDMS的邮票准备示意图。在这个过程中，图案的主人是覆盖Sylgard（10:1树脂：固化剂），毒气在一个真空干燥器烘箱中固化，在60 ° C，并切割成一定的尺寸。 <img src="/files/ftp_upload/3164/3164fig3.jpg" alt="图3"/> 图3。基板图案的一般原理图。在这个过程中，玻璃基板上涂有钛（50A）和黄金（150A），利用电子束蒸发器，微接触印刷hexadecanethiol使用的PDMS邮票图案，与乙二醇终止的烷烃硫醇回填，并涂有荧光标记的蛋白质。 <img src="/files/ftp_upload/3164/3164fig4.jpg" alt="图4"/> 图4主图案（A）和PDMS的邮票（二）使用描述的方法制备。比例尺是100微米。 <img src="/files/ftp_upload/3164/3164fig5.jpg" alt="图5"/> 图5。图案地空导弹AlexaFluor 647标记纤维连接蛋白（A）和CHO - K1细胞禁闭（二）可视化。比例尺是100微米。 <img src="/files/ftp_upload/3164/3164fig6.jpg" alt="图6"/> 图6。图案层粘连蛋白与E18小鼠海马神经元在4天的体外种子。 AlexaFluor 350标记的抗层粘连蛋白抗体用于模式的可视化（一）和E18小鼠海马神经元MitoTracker红580（二）染色。比例尺是100微米。 <img src="/files/ftp_upload/3164/3164fig7.jpg" alt="图7"/> 图7。AlexaFluor 647 -共轭纤维连接蛋白吸附可视图案基材的准备潜在的隐患。 （一）不足，不平衡的蛋白质吸附的混合信息。 （二）应用的压力不平衡，在冲压导致部分彭定康转移过程中。 （三）在冲压过程中过度的压力可能会导致邮票崩溃。 （四）暴露到空气中的水界面图案的表面，在漂洗，可以在背景中蛋白质的吸附结果。比例尺是100微米。 <img src="/files/ftp_upload/3164/3164fig8.jpg" alt="图8"/> 图8。埋弧焊图案可以产生小的特点，很难在空气中的传统的微接触印刷打印模式。图像（a）和（b）显示不同地区的相同的图案，印有相同的PDMS邮票（一）在空气中或去离子水（二）。 10μm的广泛支持线环绕的模式（添加，以帮助防止邮票崩溃）（一），然而，（二）的小圆点所示的功能都没有见过。这表明，在空气中的印刷作品以及较大的特点，但在水中的印刷可能会需要较小的特点，模式。比例尺是20微米。 <table border="1"><tbody><tr><td> 周期 </td><td> 加速率（转/秒） </td><td> 最终速度（RPM） </td><td> 时间（s） </td></tr><tr><td> 1 </td><td> 500 </td><td> 1000 </td><td> 5 </td></tr><tr><td> 2 </td><td> 3800 </td><td> 3800 </td><td> 30 </td></tr></tbody></table> 表1。双周期自旋方案，用于创建一个4.5微米厚涂在硅片上的AZ9245。 要准备PDMS的邮票的图案衬底，在光致抗蚀剂的主形成首先是捏造（图1，图4A）。法师是逆邮票和创建使用无论是直写光刻系统或光刻。当积极的光致抗蚀剂，如AZ9245，主生产使用，抵制涂层的晶圆被暴露在光线下具有相同的图案将出现在最后的基板。虽然它并不总是可行的的，它已经报道（特征尺寸抵制厚度）的PDMS邮票的主人是理想的长宽比为 1:2 13。我们发现，1:40的长宽比是可能的，根据性质的格局。这里描述的给予与标称厚度为4.5微米的光致抗蚀剂AZ9245的条件下，涂硅晶片。我们发现，这AZ9245厚度可以被用来生产PDMS的主人，从100微米到2微米的功能不等。 PDMS的邮票是从Sylgard 182（或Sylgard 184）使用光致抗蚀剂（图2）捏造主投。光致抗蚀剂的主人，可多次使用相同的邮票，以创建多个副本。硬化后的PDMS，邮票从主机使用刀片和由此产生的邮票可在显微镜下可视化邮票功能的一面朝下放置在一个玻璃盖玻片（图4B） 适当的冲压结果在一个锐利，清晰的蛋白质的模式，应用荧光标记的蛋白（图3和图5），可以通过可视化。另外，免疫组化可用于可视化后，细胞固定的蛋白质模式（图6）。细胞生长局限于为永生细胞株和原代细胞（图5和6）的蛋白质模式。 虽然这种技术很容易掌握，几种常见的可能会出现问题。没有足够的应用程序在DPBS浓缩蛋白质溶液混合的蛋白质可导致不平衡蛋白质模式（图7a）。冲压不当可导致局部图案转移或邮票崩溃（图7B - C）。此外，露出图案基板吸附蛋白含在空气中能破坏导致抵抗力下降的背景（图7D）单层。非常小的特征（&lt;5微米）和高宽比组成的图案往往需要淹没微接触印刷的使用。在此过程中（3.5B）水是用来作为屏障，以防止外的格局基板上沉积（图8）hexadecanethiol 14。

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没有利益冲突的声明。

一个用于PDMS的邮票创造主的平版印刷生产中可能出现的一些问题。曝光不足，在若隐若现的模式和曝光过度放大或缺少的功能抵抗涂​​层的晶圆结果抵抗涂层的晶圆结果。在一般情况下，与大特征尺寸（&gt; 10微米）的主人是相对容易的图案和开发，而较小的特点的主人可以要求广泛photopatterning和发展参数优化（超越光阻制造商推荐的参数）。最困难的主人，生产大型和小型的功能结合起来。 <...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>硅片</td><td>晶圆回收服务</td><td></td><td> 2英寸</td></tr><tr><td>旋转涂布机/热板</td><td>布鲁尔科技</td><td> CEE 200CB旋转烘烤系统</td><td></td></tr><tr><td> AZ9245光刻胶</td><td>梅斯化工公司</td><td> 105880034-1160 </td><td></td></tr><tr><td>直写光刻系统</td><td>迈特SRL </td><td> LW325 LaserWriter系统</td><td></td></tr><tr><td>掩模对准器</td><td> HTG </td><td> 3小时</td><td></td></tr><tr><td> AZ 400K开发</td><td>梅斯化工公司</td><td> 105880018-1160 </td><td></td></tr><tr><td> Sylgard 182有机硅弹性体套件</td><td>道康宁</td><td></td><td></td></tr><tr><td>没有25毫米。 1圆形盖玻片</td><td> VWR </td><td> 16004-310 </td><td></td></tr><tr><td>等离子体氧化</td><td>迪纳</td><td>飞秒</td><td></td></tr><tr><td>钛片Kamis </td><td>注册成立</td><td></td><td> 99.95％的纯</td></tr><tr><td>黄金颗粒</td><td> Kamis团</td><td></td><td> 99.999％的纯</td></tr><tr><td>电子束蒸发</td><td>库尔特J. Lesker </td><td> 75物理气相沉积薄膜沉积系统</td><td>电子束配件</td></tr><tr><td> Hexadecanethiol </td><td>阿法埃莎</td><td> A11362 </td><td></td></tr><tr><td> 1，mercaptoundec - 11 - YL）TETRA（乙二醇） </td><td>西格玛爱秩序</td><td> 674508 </td><td></td></tr><tr><td>乙醇</td><td> Pharmco - aaper </td><td> 111000200 </td><td> 200证明，绝对</td></tr><tr><td>封口膜</td><td> VWR </td><td> 52858-000 </td><td></td></tr><tr><td> DPBS </td><td> VWR </td><td> 4500-434 </td><td>如果没有钙和镁</td></tr><tr><td>鼠标层粘连我</td><td> VWR </td><td> 95036-762 </td><td></td></tr><tr><td>人类血浆纤维连接蛋白</td><td> Invitrogen公司</td><td> 33016-015 </td><td></td></tr><tr><td> AlexaFluor ® 647羧酸琥珀酰亚胺酯</td><td> Invitrogen公司</td><td> A - 20006 </td><td></td></tr><tr><td> MitoTracker红580 </td><td> Invitrogen公司</td><td> M22425 </td><td></td></tr><tr><td> AlexaFluor ® 350羧酸琥珀酰亚胺酯</td><td> Invitrogen公司</td><td> A - 10168 </td><td></td></tr><tr><td>抗层粘连蛋白抗体</td><td> Fisher Scientific则</td><td> AB2034MI </td><td></td></tr></tbody></table>

creating two-dimensional patterned substrates for protein and cell confinement

微接触印刷提供了一个快速，为创建良好定义的图案衬底的高度重复性的方法1虽然微接触印刷，可直接打印大量的分子，包括蛋白质，DNA的 2，3和硅烷，形成自我金长链烷烃硫醇组装单层膜（SAMS）提供了一个简单的方法来限制​​蛋白质和细胞含有粘合剂和耐地区的具体模式。这禁闭可用于控制细胞的形态，是用于研究的蛋白质和细胞生物学的各种问题。在这里，我们描述了细胞研究创造良好定义的蛋白质模式的一般方法，5这个过程包括三个步骤：一个图案主生产采用光刻，创造一个PDMS邮票，和微接触印刷金涂基材。图案后，这些细胞培养基质围蛋白质和/或细胞（原代细胞或细胞株）的格局。 对图案的蛋白质/细胞粘附地区和非粘性地区的精确控制允许使用的自组装单层化学，不能使用直接的蛋白质冲压实现。 Hexadecanethiol，微接触印刷步骤所用的长链烷烃硫醇，产生一个疏水表面容易吸附溶液中的蛋白质。乙二醇终止的巯基，用于回填基板的非打印区域，创建一个单层抗蛋白质吸附，因此，细胞的生长。6这些巯基的单体生产的高度结构化膜，精确地界定地区的基板，可以支持蛋白质吸附和细胞的生长。因此，这些承印物是有用的各种广泛的应用，从7间行为研究创造微电子8。 虽然已用于细胞培养的研究，包括从本集团的工作，创建模式，直接在玻璃基板上使用trichlorosilanes的其他类型的单层化学，9图案形成单分子膜对黄金的烷烃硫醇直截了当的准备。此外，单层准备使用的单体商业，​​稳定，不需要储存或处理惰性气氛下。烷烃硫醇准备的图案基板也可以被回收和重复使用多次，维持细胞禁闭10。

Watch this Scientific Journal Video about Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement at JoVE.com

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

金长链烷烃硫醇形成的自组装单层膜（SAMS）提供了定义良好的蛋白质模式的形成和细胞禁闭基板。使用一个邮票回填与聚二甲基硅氧烷（PDMS）微接触印刷hexadecanethiol乙二醇终止烷烃硫醇单体生产蛋白质和细胞吸附加盖hexadecanethiol地区唯一的模式。

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

creating-two-dimensional-patterned-substrates-for-protein-cell

Research

JoVE Journal

Bioengineering

12.5K 次观看.  Washington University in St. Louis. 金长链烷烃硫醇形成的自组装单层膜（SAMS）提供了定义良好的蛋白质模式的形成和细胞禁闭基板。使用一个邮票回填与聚二甲基硅氧烷（PDMS）微接触印刷hexadecanethiol乙二醇终止烷烃硫醇单体生产蛋白质和细胞吸附加盖hexadecanethiol地区唯一的模式。

视频: Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

创建瞬态细胞膜毛孔，使用标准的喷墨打印机

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

科研

生物工程

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

在光刻技术定义的聚对二甲苯-C细胞图案：二氧化硅<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

订货单细胞和单胚胎的三维约束：高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

合并一个液滴后荧光恢复来衡量磷脂单层的二维扩散

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

扩大奈米基材使用缝合技术的细胞行为Nanotopographical调制

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

移动式单分子FRET探头的自动化二维时空分析

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...