The support from ERC through HealInSynergy (306990) and the FPU program AP2009-3626 are acknowledged.

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ploy(lactic acid)</td>
			<td>NatureWorks</td>
			<td>4042D</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Cover glasses (12 mm&#216;)</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 (1mL)</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&#246;s</td>
			<td>3111</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Laminar flow&#160; 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 align="right">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

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

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

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

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

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

细胞物质相互作用研究微细的纳米多孔金模式

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

膜脊柱：一个生化工具来确定膜蛋白的蛋白质 - 蛋白质相互作用<em&gt;在体内</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

The Use of a &amp;#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

基于β-酶的电导生物传感器检测技术在分子间相互作用中的应用

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip：蛋白质 - 磷酸肌醇相互作用的无标记研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

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 ...