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

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?...

<ol>
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Effect on cell differentiation.</a> Biotechnol Bioeng. 110 (11), 3048-3058 (2013).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Moratal, D., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-matrix+sandwich-like+microenvironments+to+manipulate+cell+fate.">Fibronectin-matrix sandwich-like microenvironments to manipulate cell fate.</a> Biomater. Sci. 2 (3), 381-389 (2014).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Rico, P., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+and+ventral+stimuli+in+cell-material+interactions:+effect+on+cell+morphology.">Dorsal and ventral stimuli in cell-material interactions: effect on cell morphology.</a> Biointerphases. 7 (1-4), 39 (2012).</li><li>Ballester-Beltr&#225;n, J., Lebourg, M., Rico, P., Salmer&#243;n-S&#225;nchez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+within+confined+sandwich-like+nanoenvironments.">Cell migration within confined sandwich-like nanoenvironments.</a> Nanomedicine. , (2015).</li><li>Gla&#223;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Migration+Analysis:+Segmenting+Scratch+Assay+Images+with+Level+Sets+and+Support+Vector+Machines.">Cell Migration Analysis: Segmenting Scratch Assay Images with Level Sets and Support Vector Machines.</a> Pattern Recogn. 45 (9), 3154-3165 (2012).</li><li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+cell-matrix+adhesions+to+the+third+dimension.">Taking cell-matrix adhesions to the third dimension.</a> Science. 294 (5547), 1708-1712 (2001).</li><li>Huebsch, N., Arany, P. R., Mao, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+traction-mediated+manipulation+of+the+cell/matrix+interface+to+control+stem-cell+fate.">Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate.</a> Nat. Mater. 9 (6), 518-526 (2010).</li><li>Petrie, R. J., Gavara, N., Chadwick, R. S., Yamada, K. 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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. ...

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