Name: sandwich-like microenvironments to harness cell/material interactions
Uploaded: 2015-08-04T14:00:00.000+00:00
Duration: 6 min 50 s
Description: Watch this Scientific Journal Video about Sandwich-like Microenvironments to Harness Cell/Material Interactions at JoVE.com

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1.生产背基质<ol><li>生产PLLA的通过溶剂浇铸平坦膜构成。 <ol><li>在通风橱中操作，在室温下（RT）下，直到完全溶解（3小时左右）搅拌，以制备2％溶液（重量/体积）的PLLA的氯仿（100毫升氯仿2克PLLA）。 </li><li>放在玻璃培养皿的垫圈。 注意:使用不锈钢垫圈10.5毫米（比腹侧样品直径稍小）的内径，使得所述垫圈被放置在腹侧衬底的顶部。其他材料，如聚四氟乙烯（PTFE）或玻璃垫圈可以使用。 </li><li>加入200μl在洗衣机的PLLA溶液，并让蒸发慢慢为在室温30分钟。为了使溶剂缓慢蒸发，关闭培养皿用铝箔与几个洞。 </li><li>加热在120℃下将样品5分钟以蒸发溶剂的痕迹。 </li><li>让样品做凉WN在RT。 </li><li>一旦样品已经冷却下来（30分钟左右），盖上18.2兆欧·厘米水的培养皿孵育在室温8分钟。 注意:如果样品没有完全冷却下来，他们可能会采取比上年增长18.2MΩ·cm的水，并采用橡胶态。另外，培养在18.2MΩ·cm的水为小于8分钟，可能会导致不正确的脱离，用于从所述垫圈在脱离超过8分钟。 </li><li>用刀片撬垫圈剥离其关闭的培养皿。 </li><li>从用镊子垫圈的边缘除去的任何缺陷，并让样品风干。 </li><li>存储在真空干燥器（80毫巴），直至试验当天的样品，因为PLLA是降解由水解。 </li></ol></li><li>生产聚乳酸纤维通过静电纺丝。 <ol><li>在通风橱中操作，制备了8％的溶液（重量/体积）的PLLA在六氟异丙醇（100毫升六氟异丙醇8克PLLA）由stirr荷兰国际集团在室温至完全溶解（3小时左右）。 </li><li>放置的聚四氟乙烯（PTFE）垫圈上的铝薄片或在滚筒上的静电，以获得随机或排列的纤维分别。 注意:使用聚四氟乙烯（PTFE）垫圈10.5毫米（比腹侧样品直径稍小）的内径，使得所述垫圈被放置在腹侧衬底的顶部。其他材料，如不锈钢或玻璃垫圈可以使用。所述滚筒应以角速度的160.7秒-1（半径= 7厘米），以获得排列的纤维旋转。较慢的速度可能不会触发光纤对准和更快的速度会导致断丝。 </li><li>装载注射器与聚合物溶液，并放置于注射器泵。 </li><li>静电纺丝用0.9毫升/小时的流速为30千伏的电压和为12cm的集电极的距离的PLLA溶液。 </li><li>停止静电纺丝过程中船尾呃20-30分，一旦一个良好的纤维密度达到（因为细胞有同时与若干纤维进行交互）。 </li><li>加热在120℃下将样品5分钟以蒸发溶剂的痕迹。 </li><li>存储在真空干燥器（80毫巴），直至试验当天的样品，因为PLLA是降解由水解。 </li></ol></li></ol> 2.三明治文化<ol><li>组装夹层状培养一旦细胞被粘附到腹侧2D衬底。 <ol><li>在培养罩工作，以确保在无菌条件并通过紫外线照射消毒的所有材料（玻璃盖玻片上，背侧基片，镊子等 ）30分钟。 </li><li>涂层的腹侧和背侧基板用纤连蛋白，以便引导特异性细胞蛋白的粘附。要做到这一点，孵育的样品中的纤连蛋白的20微克/毫升溶解在Dulbecco氏磷酸盐缓冲盐水（DPBS）含Ca 2+和Mg 2+（200微升/秒充足）1小时。 注意:由于在制造过程中，背侧基片具有一个指定顶侧和底侧，因为溶剂浇铸膜和电纺丝纤维附着到垫圈的侧面之一。因此，这一边是一面临的细胞（ 图3）。 <ol><li>蛋白质涂布后，用DPBS两次洗样品，以在过量除去蛋白质。然后孵育在DPBS中的样品，直到其以防止样品从得到干燥，因为这会导致蛋白质变性用途。含Ca 2+和Mg 2+，因为二价阳离子使用DPBS调节蛋白质折叠。 </li></ol></li><li>在玻璃上的种子细胞的盖玻片13。 注意:作为夹层状培养基于2D基板，细胞接种将同样作为一个2D样本来执行。例如，C2C12成肌细胞接种17,500细胞厘米-1分化实验和NIH3T3成纤维细胞接种在7000ÇELLS厘米-1孤立的文化研究细胞的形态和附着力。 </li><li>放置在温箱中的样品在37℃，5％的CO 2，并允许细胞粘附于腹侧基底3小时。 </li><li>将腹侧播种基板在12多孔板的一个新井（其中垫圈适合）。 注:在组装新井夹心样的文化是一种方式来摆脱那些坚持的底部播种后好细胞，因为这些会消耗养分和分泌，影响细胞内的培养生长因子和浪费三明治类的系统。这也是蜂窝萃取以裂解仅夹层状环境中培养的细胞是必须的。 </li><li>小心并用一对镊子的帮助下，覆盖在细胞上的背衬底。笔芯与介质（1毫升），37℃，5％的CO 2。 注意:该垫圈的重量将防止背子浮动STRATE。 </li><li>一个星期换两次培养基作为标准的2D文化。别动背底，因为这可能会导致细胞损伤。 </li></ol></li><li>刚过细胞种植组装三明治般的文化。 <ol><li>在培养罩工作，以确保在无菌条件并通过紫外线照射消毒的所有材料（玻璃盖玻片上，背侧基片，镊子等 ）30分钟。 </li><li>涂层的腹侧和背侧基板用纤连蛋白，以便引导特异性细胞蛋白的粘附。要做到这一点，孵育的样品中的纤连蛋白的20微克/毫升溶解在含有Ca 2+和Mg 2+（200微升/样品）进行1小时的DPBS。 <ol><li>蛋白质涂布后，用DPBS两次洗样品，以在过量除去蛋白质。然后孵育在DPBS中的样品，直到其以防止样品从得到干燥，因为这会导致蛋白质变性用途。含有钙和镁的使用DPBS <s达&gt; 2+，因为二价阳离子调节蛋白质折叠。 </li></ol></li><li>在玻璃盖玻片在12多穴板的孔种子细胞（其中垫圈适合）。要做到这一点，使用高浓缩细胞悬浮液，以避免铺设背底后的细胞损失。 </li><li>小心并用一对镊子的帮助下，覆盖在细胞上的背衬底。笔芯与介质（1毫升），37℃，5％的CO 2。 注意:该垫圈的重量将防止背衬底浮起。 </li><li>一个星期换两次培养基作为标准的2D文化。 注意:不要将背底，因为这可能会导致细胞损伤。 </li></ol></li></ol> 3.分析注意:三明治状培养物基于二维基板，所以可以通过已制定了标准的2D培养程序可以共同进行分析。例如，由于聚乳酸是透明和大公LS被约束为在xy平面内移动，显微镜是作为二维衬底。细胞迁移可因此分析作为2D培养物，而不在z轴作为用于3D培养，从而简化了实验和图像分析追踪单元的需要。要通过划痕试验研究伤口愈合实验遵守此协议: <ol><li>研究细胞迁移（伤口愈合测定法）的三明治般的文化中。 <ol><li>在培养罩工作，以确保在无菌条件并通过紫外线照射消毒的所有材料（玻璃盖玻片上，背侧基片，镊子等 ）30分钟。 </li><li>涂层的腹侧和背侧基板用纤连蛋白，以便引导特异性细胞蛋白的粘附。要做到这一点，孵育的样品中的纤连蛋白的20微克/毫升溶解在Dulbecco氏磷酸盐缓冲盐水（DPBS）含Ca 2+和Mg 2+ 1小时。 <ol><li>蛋白质涂布后，用DPBS洗两次的样品为了在过量除去蛋白质。然后孵育在DPBS中的样品，直到它们的使用，以防止样品从因为这获得干燥可能会导致蛋白质变性。含Ca 2+和Mg 2+，因为二价阳离子使用DPBS调节蛋白质折叠。 </li></ol></li><li>种子细胞在玻璃盖玻片以高密度13。 </li><li>放置在温箱中的样品在37℃，5％的CO 2，并允许细胞达到汇合。 </li><li>使用移液管头划伤细胞单层以获得由间隙隔开的2细胞群。 </li><li>放置在一个新井合适的腹侧播种底物时间推移采集并且其中背基板适合（ 即 12多孔板）。 </li><li>小心并用一对镊子的帮助下，覆盖在细胞上的背侧基板，然后用培养基填充（1毫升12多孔板）。 注意:该垫圈的重量将防止吨他背底浮动。 </li><li>将样品中的时间推移显微镜（设定为37℃和5％的CO 2），并采取间隙闭合，每20分钟的图像。 </li><li>使用特定软件的差距封闭分析如ImageJ的插件MiToBo。14 </li></ol></li></ol>注:蛋白质和核酸提取作为2D基板同样进行。只有一个额外的步骤，由在拆卸夹层状培养直接添加裂解缓冲液的细胞以提高提取效率。例如，对于mRNA提取: <ol start="2"><li>从三明治般的文化基因提取<ol><li>根据制造商的说明从商业试剂盒制备的所有缓冲区。 </li><li>就拿三明治般的文化从孵化器。 </li><li>从样品中取出培养基。 </li><li>含有钙DPBS洗一次样本2+和Mg 2+（1毫升），并删除所有的溶液。 </li><li>用一对镊子的帮助下，取下背基板与裂解缓冲液（350微升）添加到腹侧基底。再次叠加腹侧基底上的背衬底以便也溶解粘附于背基片上的​​细胞。 </li><li>除去背侧基板以分离出裂解溶液。 </li><li>按照制造商的说明，以获得表达。 </li></ol></li></ol>注:蛋白质的免疫检测可以也执行与二维衬底。因为夹层状培养物会妨碍抗体和缓冲液的正确扩散，孵育时间应该增加。此外，夹层可以在开始染色方案之前被拆卸，但在后一种情况下，一些细胞将保持附着于背基片和一些向腹侧基底。 <ol start="3"><li>内形成夹芯状培养Immunodetect蛋白<ol&gt; <li>用DPBS洗涤一次样品并用在PBS中的4％甲醛为20分钟，在4℃下固定。 注意:背衬底可以在固定过程，从而避免扩散问题被除去。 </li><li>冲洗用DPBS（1毫升）中，然后用0.5％的Triton X-100中的DPBS（1毫升）通透5分钟，室温。 </li><li>阻断非特异性结合位点与在DPBS中1％白蛋白（1毫升）中在室温30分钟。 </li><li>孵育与初级抗体（2微克/毫升1毫升）的阻断溶液中3小时，在室温。 </li><li> 10分钟用DPBS / 0.5％吐温20（1毫升）洗3次。 </li><li>孵育2小时用二级抗体（2微克/毫升）和1微克/毫升的DAPI在封闭溶液（1毫升）中于RT。 </li><li>在DPBS 10分钟/ 0.5％吐温20（1毫升）洗3次。 </li><li>在DPBS洗。 注意:样品可以被安装在一个具有的Vectashield滑动并用指甲油密封时背衬底之前移除免疫检测。 </li><li>在荧光显微观察范围作为标准的2D衬底。 </li></ol></li></ol>

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The authors have nothing to disclose.

如今，3D培养是用于制药和生物技术工业中的重要课题，以及研究在细胞生物学，包括癌症和干细胞。因此几个3D​​培养系统已经开发出来。不幸的是，三维系统之间的差别通常导致不同的细胞行为，阻碍细胞命运的理解。此外，实验程序通常不那么简单，因为二维培养系统。因此开发新的培养系统寻求克服一些上述缺点的是非常重要的。 夹芯状培养已显示强烈影响关键的?...

传统上，细胞培养物已进行了对两维（2D）基板，但大多数的体内细胞微环境有一个三维（3D）的性质。这种不自然的2D环境触发一个平坦的世界，改变细胞的行为作为自适应性的一种方式，这直接影响细胞的命运1,2。因此，获得的二维细胞培养结果并不总是可再现的体内 。这鼓励了新的有关文化系统寻求提供更多的生理条件一样获得进一步深入了解任何尺寸依赖性生物学机制3,4发展。 之一的二维培养和3D 体内环境之间的主要区别是锚定到细胞外基质（ECM）的细胞受体的分布:而在2D基板细胞附着腹侧，大部分细胞在体内的由ECM和被完全包围因此CELL粘附是通过受体的三维分布。这会触发不同的细胞粘附信号传导途径，从而调节重要的过程，如细胞生长，细胞分化和基因表达。在过去的十年中，许多不同的3D培养系统已经建立5-8，虽然他们的变异性和复杂性，阻碍它们在通常的细胞培养过程标准化。此外3D系统通常不容易处理和二维衬底目前的实验过程不能用于3D培养容易建立。此外，文学比较罕见的3D文化具有同等条件的2D或其他3D系统，阻碍了在这些模型中细胞行为的正确理解。 一旦具有附着一个2D衬底，背侧受体的激发在细胞上 - 由叠加新材料（夹芯状培养物）的膜 - 可以触发细胞的反应一样的3D环境。该REA子后面，这是同时激活两个背侧和腹侧受体粘附及夹心环境内传播（ 图1）9,10。其结果是，细胞经历相对于2D培养11,12重要的变化。因此，细胞的命运，是因为夹层文化的组装过程中确定，因为背的刺激触发的变化的关键细胞途径。因此，在细胞命运是高度由当夹层状培养组装11的时间来确定。 由于该系统的性质，形成夹芯状培养是一个简单的和通用的工具，它允许在小区/材料相互作用，诸如化学，地形，刚度和蛋白质涂层不同的参数同时在腹侧和背侧两侧的研究。这提供了一个更高的通用性相比其他3D系统（ 图2），由于独立背宽诉和腹侧组合ariety的表面状况。此外，不同的细胞系和不同的时间来组装夹芯状培养可以研究，增加的可能性的宽光谱。 夹心状培养物的标准协议是以下详述使用任一聚L-乳酸（PLLA）电纺丝纤维或薄膜作为背侧基板，玻璃盖玻片作为腹侧基底和纤连蛋白作为蛋白质涂层。三明治般的培养物刚过细胞种植或3小时二维培养后组装。然而，请注意，其他材料的系统和蛋白质可用于;同样的夹层状培养物可以在不同的时间点进行组装。

<table><tbody><tr><td>Ploy(lactic acid)</td><td>NatureWorks</td><td>4042D</td><td>Reagent</td></tr><tr><td>Cover glasses (12 mm&amp;Oslash;)</td><td>Marienfeld</td><td>631-0666</td><td>Equipment</td></tr><tr><td>Chloroform</td><td>Scharlab</td><td>CL0200</td><td>Reagent</td></tr><tr><td>1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP)</td><td>Sigma</td><td>105228</td><td>Reagent</td></tr><tr><td>Syringe (1 ml)</td><td>Henke Sass Wolf</td><td>4010-200V0</td><td>Equipment</td></tr><tr><td>Syringe pump</td><td>New Era Pump Systems</td><td>NE1000</td><td>Equipment</td></tr><tr><td>High Voltage DC Power Supply</td><td>Glassman High Voltage</td><td>Series FC</td><td>Equipment</td></tr><tr><td>Incubator</td><td>Hucoa-Herl&amp;ouml;s</td><td>3111</td><td>Equipment</td></tr><tr><td>Laminar flow&amp;nbsp; hood</td><td>Telstar</td><td>AV30/70</td><td>Equipment</td></tr><tr><td>Human Fibronectin</td><td>Sigma</td><td>F2006</td><td>Reagent</td></tr><tr><td>RNeasy Micro Kit</td><td>Qiagen</td><td>74004</td><td>Reagent</td></tr><tr><td>Inverted microscope</td><td>Leica Microsystems</td><td>DMI 6000</td><td>Equipment</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Reagent</td></tr><tr><td>Albumin</td><td>Sigma-Aldrich</td><td>A7409</td><td>Reagent</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P2287</td><td>Reagent</td></tr></tbody></table>

sandwich-like microenvironments to harness cell/material interactions

细胞培养物已被传统上进行二维的（2D）基板，其中细胞粘附使用腹侧受体的生物材料的表面上。然而， 在体内 ，大部分细胞完全由细胞外基质（ECM）所包围，从而导致受体的三维（3D）分布。这可能会触发外信号传导途径，从而在细胞行为​​中的差异。 本文表明刺激已经附着到2D衬底通过叠置一种新材料（形成夹芯状培养物）的膜单元的背侧受体触发相对于标准的2D培养物的重要变化。此外，腹侧和背侧受体的同时激发转移细胞行为更接近，在3D环境中。另外，由于该系统的性质，形成夹芯状培养是一种多用途的工具，它允许在小区/材料INTE不同参数的研究ractions， 例如 ，地形，刚度和不同的蛋白质涂层同时在腹侧和背侧两侧。最后，由于三明治般的文化是基于二维基质，已经开发了几个分析程序的标准的2D文化都可以正常使用，需要克服的3D系统更复杂的程序。

背受体的夹层状培养中的刺激触发细胞形态变化，细胞粘附和细胞内信号通路（ 例如粘着斑激酶，FAK）10-12。作为一个例子，成纤维细胞的夹层状系统内培养过表达的α5整亚基相比2D，作为观察到的其他3D培养15,16。 细胞命运是高度依赖于当背受体受到刺激的时间和由背相互作用的性质，类似于其他3D系统发生诸如水凝胶。例如，水凝胶，其中蛋...

Watch this Scientific Journal Video about Sandwich-like Microenvironments to Harness Cell/Material Interactions at JoVE.com

Sandwich-like Microenvironments to Harness Cell/Material Interactions

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

sandwich-like-microenvironments-to-harness-cellmaterial-interactions

Research

JoVE Journal

Bioengineering

7.5K 次观看.  Universitat Politècnica de València. The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

视频: Sandwich-like Microenvironments to Harness Cell/Material Interactions

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

癌症研究

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

操纵细胞与细胞间相互作用的硅微芯片

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

生物学

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

制造和使用微环境芯片（MEArrays）

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.