笔者想承认博士托马斯·博兰细胞印刷使用HP DeskJet打印机的想法。从NSF，RII-EPS 0903795和NIH K25中HL0922280项目提供资金。

1。转换的HP DeskJet 500 应当指出，这种技术应与许多商用喷墨打印机。然而，旧的打印机往往更好的工作，因为他们使用的墨盒，直径较大的喷嘴，不堵塞一样容易。此外，旧的打印机往往使用机械的进纸传感器，很容易绕过。打印机与光学传感器可以被欺骗，但使用打印机在每个周期远缘的小片纸，但比机械系统的​​“猫腻”多一点困难。目前市面上的打印机工作最好有低的分辨率（DPI）。 更高分辨率的打印机往往更容易堵塞。惠普Deskjet 500的分辨率为300 dpi。有许多市售的打印机（HP Deskjet系列及其他），有一个600 dpi的分辨率。这种类型的打印机可以使用只是一个小喷嘴增加堵塞可缓解（第3）仔细清洗的问题。 <ol><li>解锁从底座打印机的几个塑料夹，慢慢抬起顶部关闭删除打印机顶部的塑料外壳。 </li><li>松开按钮/显示打印机顶部的光面板，离开它连接到打印机的主板。 </li><li>清洁打印机内部，尤其是地区的地方在于墨盒和印刷发生。 </li><li>找到电缆供电纸机制，并从主板上拔下。 <ol><li>在HP DeskJet 500，这些被发现略低于朝着左前方的纸盒。 </li></ol></li><li>找到纸张检测机制。绕过字符串或线环作为手动拉手追究机制。 <ol><li>纸检测机制中的HP DeskJet 500，上方和后方的印刷/纸机制发现一个灰色的塑料杆。 </li></ol></li><li>创建一个阶段，在进纸机制，以便将美联储从纸张和沉积前把所需的打印墨盒打印头正下方水平的幻灯片。 <ol><li>对于我们的实验中，泡沫航运持有15 mL离心管，用几个显微镜在印刷区域的地方录音，幻灯片，使幻灯片的最后一级所需的高度。 </li></ol></li><li>要保持无菌技术，打印机可以被放置在一个标准的生物危害柜或桌面层流罩。 </li><li>重要的是要注意，修改市售喷墨打印机通常会打印机制造商的保修失效。 </li></ol> 2。转换为股票的惠普墨盒（HP 26黑色墨盒） <ol><li>从包装中取出墨盒，留下暂时覆盖打印机的接触和打印头的保护胶带。 </li><li>稳住身体用夹具或台钳（只需牢牢抓住通常工作以及手工墨盒），留下任何障碍绿色明确墨盒（黑色部分）。 </li><li>用钳子或活动扳手，把握绿色墨盒的顶部和扭动，来回几次，直到它打破了自由。 <ol><li>这是不应该花太多的力量，但不再需要的顶端，所以它是好的，如果它打破取出时。 </li></ol></li><li>用螺丝刀撬开现在暴露的透明塑料片。 <ol><li>再次，这不应该花太多的力量，但它不会需要，因此它是好的，如果它在搬迁过程中打破。 </li></ol></li><li>清空任何其余水库。 </li><li>取下塑料覆盖打印机的接触和打印头的保护胶带。 </li><li>水库的水彻底冲洗。 <ol><li>使用吸管或注射器推水通过渠道。 </li><li>水从打印头中可能会泄漏这个过程中，这是可以接受的，并不会造成任何破坏墨盒的功能。 </li></ol></li><li>当水运行明确，使墨盒晾干。 </li></ol> 3。清洁墨盒<ol><li>为了保持一个干净的印刷环境，使用前后应清洗墨盒。 <ol><li>这也有助于避免结晶盐和其他生物材料的墨盒可能会导致堵塞。 </li></ol></li><li>完全淹没在去离子水的烧杯墨盒，超声15分钟之前和之后，印刷。 </li><li>超声波处理后，取出墨盒从水中，摇出多余的水分。 </li><li> 70％的乙醇喷入墨盒，以创造更多的无菌环境。 <ol><li>确保乙醇加入bioink印刷解决方案之前，干燥。 </li></ol></li></ol> 4。细胞悬液 - “生物墨“ <ol><li>培养细胞，直到准备到通过。 <ol><li> 3T3成纤维细胞：约1.3x10 4细胞/厘米2 Dulbeccos改性老鹰培养基（DMEM培养基），10％胎牛血清（FBS），对T-75瓶种子细胞。两天孵化器培养细胞在37°C间和5％的CO 2。 </li></ol></li><li>使G-肌动蛋白的荧光浓度原液在磷酸盐缓冲液（PBS）的50微克/毫升。 </li><li>传代细胞。 <ol><li> 3T3成纤维细胞：从烧瓶中取出的媒体，用PBS冲洗两次。覆盖细胞用5毫升0.25％胰蛋白酶+ EDTA和孵育5分钟。 </li><li>新鲜培养基中加入5毫升到15毫升锥形离心管中，并在5分钟1000转离心吸取细胞悬液。抽吸介质花。 </li></ol></li><li>荧光G-肌动蛋白原液创建bioink PBS重悬细胞。 <ol><li> bioink应该有一个G-肌动蛋白的终浓度10微克/毫升和细胞合作ncentration的1×10 5细胞/毫升。这个浓度已经得到了优化，以限制每一滴水和打印头堵塞的细胞数量。 </li><li>请注意，250μl的bioink的打印模式如图2所示的三个盖玻片。 </li></ol></li></ol> 5。 Bioprinting <ol><li>电源和让打印机热身。 </li><li>地方所需的印刷面（在这里，我们使用22毫米×22毫米盖玻片）在步骤1.6准备阶段的中心。 </li><li>创建一个印刷图案文件。 <ol><li>打开Microsoft Word（或任何其他的绘图软件），并绘制所需的图案。 </li><li>挑选一个细胞中的预制文件印刷所需的打印模式。 </li></ol></li><li>加载所需的细胞悬液的制备墨盒。 <ol><li>移液器悬挂墨盒舱底部的小圆形。使用约100-120微升的溶液。打印的墨滴大小，大约是130皮升8。 </li></ol></li><li>惠普Deskjet 500打印机打印文件。 <ol><li>为了达到最佳效果，打印小图案（5）多次，通过改变在文字处理程序所需的副本数量。 </li></ol></li><li>打印机将再次热身，然后墨盒会移动到“准备位置”。 </li><li>当墨盒移动到准备位置（稍远低于左侧的墨水滴，并在那里停留），拉进纸机制电线，并应开始打印。 <ol><li>对于多个副本的印刷，纸机制将每个周期或页打印后公布。墨盒，然后将返回到“准备就绪”的位置，进纸机制电线应该再次被提出。 </li></ol></li><li>打印机将打印到盖玻片上的印刷阶段的中心放置在步骤5.2（ 见图2）。 </li></ol> 6。代表结果&lt;/ P&gt; 整个转换过程中的一个标准的，现成的惠普Deskjet 500的有代表性的成果，标准HP 26系列墨盒，将创建一个打印机与如前所述印刷多种类型的细胞分析解决方案的能力。转换完成后的打印机， 如图3所示，放置在显微镜的盖玻片上印刷阶段。打印机可以在许多领域的分析是有用的，包括但不限于：单细胞力学，组织工程，基因转染，缩微传感器，和直接的细胞疗法1-10，16-22。 在这个例子中的HP Deskjet 500打印机和惠普26系列墨盒被为bioprinting修改。使用该打印机设置了一个G-肌动蛋白单体溶液中的成纤维细胞悬液组成bioink，细胞被印到玻璃显微镜的盖玻片。 图1说明了代表水库<html>印刷的成纤维细胞的ULTS，这表明成立荧光的肌动蛋白单体。结果在控制的无菌环境中的细胞被印制成定制模式。 在这个例子中使用的模式是建立在Microsoft Word中（ 图2）。这种模式建立了一个横跨大多数的显微镜幻灯片的打印解决方案的不断线。 图4显示了直线的bioink和细胞相互存放。应当指出后，立即打印，有一个背景荧光的增加，细胞被暂停，因为bioink解决方案，包含免费过量荧光的肌动蛋白单体。此背景荧光显着减小后增加细胞（ 图1），洗去多余的基板单体的生长介质。 <html> ad/3681/3681fig1.jpg“/&gt; 图1。3T3成纤维细胞3小时后，使用修改后的喷墨打印机打印的代表图像。细胞内部，显示纳入荧光标记的肌动蛋白单体。标尺代表50微米。 <img alt="图2。" src="/files/ftp_upload/3681/3681fig2.jpg" /> 图2。设计用于打印bioink的。 <img alt="图3。" src="/files/ftp_upload/3681/3681fig3.jpg" /> 图3。与泡沫塑料阶段转换后的打印机。在中间的橙色线绕过纸杆传感器。 <img alt="图4。" src="/files/ftp_upload/3681/3681fig4.jpg" /> 图4。代表的形象印在本地化印刷区3T3成纤维细胞。 5分钟后打印在20倍放大倍率的显微镜图像。 / files/ftp_upload/3681/3681fig5.jpg“/&gt; 图5。3小时后，呈现出成纤维细胞具有内在荧光的肌动蛋白单体的印刷所采取的荧光图像（40倍放大倍率）。比例尺代表50微米。 <img alt="图6。" src="/files/ftp_upload/3681/3681fig6.jpg" /> 图6。印刷15分钟后，采取了细胞的荧光显微镜（20倍放大倍率）。图像显示两个G-肌动蛋白（绿色）和细胞核（蓝色）。从左至右依次为：G-肌动蛋白的Alexa Fluor 488，细胞核用DAPI，两者的叠加。 <img alt="图7。" src="/files/ftp_upload/3681/3681fig7.jpg" /> 图7显示在印刷后3小时的在线模式两成纤维细胞的荧光显微镜。左边的图像显示荧光标记的细胞内的肌动蛋白单体。右边的图片是一个荧光通道与背景图像的叠加显示，虽然幻灯片（左下角），可能会有一些碎片，它不会发出荧光。标尺代表50微米大小，如果不表示最右边是3个小时的培养与荧光标记单体的非印刷细胞的对照组。此控件是为了表明，单体无法穿透无膜通透性细胞膜细胞印刷。

<ol>
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我们什么都没有透露。

也不是特别困难的过程转换为bioprinting一个标准的喷墨桌面打印机。最具挑战性的一步是确定如何绕过进纸机制，这是取决于打印机的品牌和模型。然而，这是相对简单时，进纸传感器是机械此处所述。对于饲料用光学传感器的型号，其他技术可能需要采用欺骗，以为它是使用纸的打印机，例如，可以通过打印机的运行，而它在显微镜幻灯片打印的小纸条。旁路送纸机制是不同型号的打印机在应用这些程序可能是最困难的一步。 在建设阶段举行印刷盖玻片，它是重要的，以确保正确的路线和高度。该阶段应允许被放置在中间的印刷面积的盖玻片。此外，它应该PL王牌盖玻片，在适当的高度，使墨盒间隙过而不破坏它的幻灯片。该阶段的确切高度将取决于打印机型号。 为了确保印刷细胞没有得到冲走被放置在一个孵化器，幻灯片后立即打印约30分钟，使细胞附着前增加了额外的细胞生长介质。因为细胞被印在一个解决方案，包括大量的PBS没有干细胞仍然可行。细胞成像使用荧光显微镜，以可视化的标签G-肌动蛋白在细胞内的单体（ 图1，5-7）的分布。 3T3成纤维细胞打印时， 图5显示了一个有代表性的结果，与肌动蛋白导致细胞发出荧光，但展示增加亮度线。荧光标记的G-肌动蛋白细胞骨架成立细胞骨架动力学和细胞力学的研究非常有用。12-15这种技术的局限性在于它是只适用有直径小于约10纳米的分子和蛋白质。 为了确保细胞实际上被转换打印机处理的DAPI（1:5000）在PBS添加到bioink纯PBS量的一半的地方。荧光染色的DAPI结合氨基酸丰富的DNA位于细胞核中的部分。因此的DAPI是一个有用的污渍印刷细胞荧光成像原子核。 图6显示了双方的Alexa Fluor 488（肌动蛋白）和荧光的DAPI（核），两者的叠加图像显示的单元格的形象代表。 图7还说明了如何细胞会发出荧光，一旦G-肌动蛋白单体已被纳入非生物材料已进入样品存放不会因为的absenc的荧光E G-肌动蛋白单体。 一个重要的考虑因素bioprinting，是被用于bioink的水介质。结果发现，使用标准血清细胞的生长介质产生不一致的结果。这很可能是由于血清蛋白的打印头喷嘴堵塞。使用的PBS提高了印刷图案的一致性和沉积的细胞数量。用PBS的一个缺点是，不应悬挂在长时间离开细胞。然而，在这些例子中的成纤维细胞能够容忍没有改变细胞活力至少一个小时的bioink条件。这是与前几个结果，报告说，通过热喷墨机制印制的细 ​​胞已被证明具有较高的可行性率一致。2-8 此修改打印机设置可用于细胞印刷以外的应用程序。16-22矩阵的蛋白质，如胶原蛋白或fibronectin，可以很容易地印上使用这种技术，它可以是有用的细胞图案的衬底。例如，型胶原，我把线条图案印刷，会导致排列的胶原蛋白，可用于在体外细胞培养的研究17。基板除了基质蛋白，其他的分子，包括生长因子，可以可靠地本地化基板上细胞研究和潜在的治疗应用18。 在上述步骤中所述的设计的一个主要限制是，这台打印机是不能够在多个维度打印。这就限制了使用，如脚手架印刷图案的应用潜力。为了让3D印刷，需要使用一个专门的阶段。该阶段需要有增量调整高度层层沉积bioink。 bioprinting作为组织工程的效率和成本效益的方法的承诺基因转染，缩微和芯片制造1-12，16-22这种类型的设备，未来的应用有很多，包括：支架细胞微环境控制和图案，纳入细胞的细胞质中的大分子，细胞沉积的创作上。结构不发生自然或有效的。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>的HP DeskJet 500 </td><td>惠普</td><td> C2106A </td><td>停止从制造商。购买翻新DEC的交易。 </td></tr><tr><td>惠普26黑色墨盒</td><td>惠普</td><td> 51626A </td><td></td></tr><tr><td>从兔肌肉，肌动蛋白的Alexa Fluor 488共轭，200微克</td><td> Invitrogen公司</td><td> A12373 </td><td></td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td> MP的Biomedicals </td><td> ICN1860454 </td><td></td></tr><tr><td> dulbeccos改性老鹰培养基（DMEM培养基） </td><td> Thermo Scientific的</td><td> SH3002201 </td><td></td></tr><tr><td>胎牛血清</td><td&gt; Sigma-Aldrich公司</td><td> F4135 </td><td></td></tr><tr><td>两性霉素B </td><td> Sigma-Aldrich公司</td><td> A2942 </td><td>在0.5％用于细胞培养基</td></tr><tr><td>青霉素链霉素</td><td> Sigma-Aldrich公司</td><td> P4333 </td><td>在0.5％用于细胞培养基</td></tr><tr><td>单向流洁净工作台</td><td> Envirco公司</td><td>甚低频797 </td><td>为保持无菌的打印机可选住房</td></tr></tbody></table>

creating transient cell membrane pores using a standard inkjet printer

，热喷墨打印bioprinting具有广泛的应用和意义，包括组织工程，细胞直接应用疗法和生物传感器的微细1-10最近也被用于基因转染。8,9热喷墨印刷过程中被证明暂时中断的细胞膜，在不影响细胞活力。在膜的瞬态毛孔可以用来引进的分子，这本来是太大，通过细胞膜，进入细胞的细胞质中，8,9,11 这里展示的应用是利用荧光标记的G-肌动蛋白单体掺入到细胞的热喷墨打印。使用热喷墨打印注入细胞分子的优点是，该技术是相对良性的细胞。，8，12印刷后的细胞活力，已被证明是类似的，以标准单元解放军婷方法1,8。此外，喷墨印刷可以在数分钟内处理数以千计的细胞，这是比手动显微注射快。印刷所创建的毛孔已被证明约两小时内关闭。然而，有一个创建印刷技术，这就限制了小的蛋白质和/或颗粒细胞注射技术（10纳米）的孔隙大小的限制。8,9,11 修改了一个标准的HP DeskJet 500打印机，使细胞印刷。3，5，8打印机盖被删除，送纸机制是绕过使用机械杠杆。一个阶段是创建允许直接打印头下放置载玻片和盖玻片。打开墨盒，墨水被拆除和清理之前使用细胞。印花图案是使用标准的绘图软件，然后通过一个简单的打印命令的打印机控制。 3T3纤维爆炸分别增长汇合，胰酶消化，然后进入磷酸盐水溶性荧光标记的G-肌动蛋白单体悬浮缓冲盐水。细胞悬液滴加入墨盒，印到玻璃显微镜盖玻片的细胞系。使用荧光显微镜的活细胞成像和肌动蛋白被发现在整个细胞质中。进入细胞内的肌动蛋白荧光团允许短时间的细胞骨架的动态影像，并为广泛的应用范围非常有用。13-15

Watch this Scientific Journal Video about Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer at JoVE.com

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

用于成bioprinter转换的HP DeskJet 500打印机的方法的描述。打印机是能够处理活细胞，这将导致膜的瞬态毛孔。这些孔隙可以利用纳入印刷的细胞，包括荧光灯的G-肌动蛋白，小分子。

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 ...

creating-transient-cell-membrane-pores-using-a-standard-inkjet-printer

Research

JoVE Journal

Bioengineering

38.8K 次观看.  Clemson University. 用于成bioprinter转换的HP DeskJet 500打印机的方法的描述。打印机是能够处理活细胞，这将导致膜的瞬态毛孔。这些孔隙可以利用纳入印刷的细胞，包括荧光灯的G-肌动蛋白，小分子。

视频: Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

开发自定义的中国仓鼠卵巢宿主细胞蛋白检测使用声膜微粒子技术

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

科研

生物工程

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

增加从单细胞的数量标准实验室逆转录反应的mRNA的cDNA产量，使用声Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

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 ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

通过创建一个的合成Curli的操纵子工程粘附的细菌

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

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 ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

定量的光学显微镜：测量细胞生物物理特性与标准光学显微镜

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

烟草中瞬时表达的蛋白作为一个案例研究：复杂系统采用的实验方法设计的表征

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Bioprinted蜂窝构造的可行性使用三分配器笛卡尔打印机

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.