作者要感谢提供技术援助与ACC基材的准备凯利A.伯克。的文章发表在生物材料，戴维斯KA， 等 ，动态细胞行为的形状记忆聚合物衬底材料 ，DOI:10.1016/j.biomaterials.2010.12.006，版权爱思唯尔（2011年）的基础上。基于这种材料是由美国国家科学基金会的支持下批准号DMR - 0907578工作。

1。温紫外光固化的NOA63 <ol><li>开发一个自定义的固化室商会使用载玻片（75毫米x 25毫米x 1毫米），1毫米厚的聚四氟乙烯垫片，及铝合金板（75毫米× 25毫米× 3毫米），如图1所示。使用小型长尾夹一起举行。 </li><li>通过在聚四氟乙烯垫片孔使用18号针头注入到腔NOA63。 NOA63可以轻轻加热，以减轻注射。 </li><li>广场上设置在125 ° C，并允许以一个统一的温度加热5分钟热板腔。 </li><li>预固化NOA63紫外灯室（λ 最大 = 365 nm处;见表）灯泡的玻璃表面从6.5厘米为20分钟。 </li><li>从室中取出NOA63温暖而使用刀片。 </li><li>后固化，紫外光下为3小时40在125米上的热点板块° C。NOA63 </li><li>储存在-20 ° C干燥的NOA63 </li></ol> 2。形状记忆表征NOA63 <ol><li>准备一个哑铃试样由热压固化NOA63电影与一个定制的冲压（见表），其尺寸如图2所示。 </li><li>装入一个动态机械分析仪（DMA;见表）试样的拉伸夹具。仪器设置"武力控制"的模式，那么程序的测试程序如下: <ol><li>平衡在95.00 ° C </li><li>温为10.00分</li><li>斜坡力量0.300 N /分钟2.500 ñ </li><li>温为5.00分</li><li>斜坡2.00 ° C /分钟到20.00 ° C </li><li>温为10.00分</li><li>斜坡力量0.300 N /分钟0.015 ñ </li><li>温为5.00分</li><li>斜坡2.00 ° C /分钟至95.00 ° C </li><li>重复步骤2-9两个更次</li></ol></li></ol> 3。活跃​​的细胞培养基质的制备<ol><li>个别样品可以从等温固化的SMP电影准备。剪切带所需的样本大小的剃刀SMP的电影。放置在SMP的一个热点板块，设置，以减少在温度高于 Tg的高模量和缓解切割。 </li><li>临时的形状可以是固定的，在许多不同的方式。在这里，我们使用加热/冷却压板一个台式液压机，压花一个临时的地形。设置在压板的温度高于 Tg的温度。 </li><li>是压花固化环氧乙烯基记录。这将产生一个平行沟槽的临时形状。在这里，压花高35至40微米的三角形峰，宽60微米，间距80微米。压花可以从其他材料和不同的地形，但必须在压花温度比NOA63围追堵截。放置在SMP样本正面朝下放在压花和地方在报刊上的样品和压花。 </li><li>应用A〜100千帕的预紧力，使加热板及样本之间的联系和按住〜5分钟，使样品达到温度均匀。 </li><li> 1-6兆帕的样品，并保持1分钟。 4.7兆帕斯卡的压力，导致不完整的复制压花地形。产生的临时地形圆润的山峰，高150微米，宽25至35微米。如果施加更大的压力是在SMP将骨折。应用较小的应力有较小幅度的拓朴。 </li><li>降低温度低于Tg的。在这里，我们使用水冷却能力按压板。 </li><li>当温度低于Tg的是，除去施加的力。 </li><li>可以储存在-20 ° C干燥样品当存储在这些条件下，我们所观察到小于1微米的下降在振幅恢复后两个月样本唱片压花浮雕。 </li></ol> 4。主动细胞培养实验<ol><li>生物安全柜（BSC）的紫外灯是用来消毒的样品。排列样品面朝下在无菌无盖菜和BSC紫外灯开启6小时</li><li>面对新的无菌菜翻转样品。 BSC紫外灯开启6小时</li><li>样品现在需要一个相对稳定的状态，与细胞的电镀前平衡。放置在一个96孔板的样品，加150μL完全培养基。 </li><li>置于5％的CO 2板，直至达到所需的部分恢复，在30℃培养箱。在这里，我们用30小时生产足够大的幅度保持一致细胞样本。 </li><li>样品现在可以镀细胞。放置在一个新的96孔板的样本。 </li><li>加入150μL细胞溶液样品。在这里，我们使用20,000细胞/ ml C3H/10T1/2小鼠胚胎成纤维细胞，以实现分离的细胞（一般不与其他细胞接触）。 </li><li>为了让细胞附着和传播上的临时地形，在30℃孵化器为9.5小时</li><li>检测细胞morpholOGY之前的过渡，取出的样品和执行样品的染色和荧光成像。这种材料具有自体荧光的紫外线和可见光范围内的大部分。的Alexa Fluor 647，如频谱远红端的荧光基团的建议，以减少背景。 </li><li>要触发的样本，以恢复，将37 ° C培养箱板，并继续为19 H的文化，使材料恢复，并允许细胞，以适应新的地形形态。 </li><li>取出样品，并运用适当的染色（鬼笔环肽对丝状肌动蛋白成像）和成像过程。 </li></ol> 5。代表性的成果: 固化NOA63是一种透明的玻璃状固体，具有优异的形状记忆如在图3所示的属性。在这种情况下，材料被治愈，在上述议定书第1显示了一个统一的Tg 51.1 ° C（从E"下降的发病确定）。据观察，从单程形状记忆周期（加热，变形，冷却，恢复， 图3），很大比例的应变是固定的卸货后，在20 ° C，对应一个固定的比例 15 89.3（R F ） ％（三个周期的平均值; R R，下同） 。固定应变恢复比例为84.4％（R R）在一个相对较小的温度范围内，在加热过程中恢复。此外，形状记忆性能表明没有恶化到三个周期，在所有的曲线都遵循相互几乎一模一样。 NOA63使用这个协议，因为它很容易从制造商和供应容易治愈的无溶剂型光引发剂，预聚物。然而，其组成是由供应商没有透露。它被发现，让高细胞附着和生存能力。最后，转变温度可以调节，允许两种细胞之间的兼容温度恢复有意义的幅度。也可用于其他聚合物系统转变温度是兼容细胞培养和促进细胞的粘附和活力，如果他们与本议定书​​。 临时地形（槽）的幅度降低随着时间的推移在30 ° C在30℃30 h，幅度已经降低〜50％（ 图4，时间为0）16。它可以减少另外10％，在未来的9.5小时当复苏引发的温度提高到37 ° C，幅度降低初始幅度为0.5％，9.5小时内所用的压花和压花压力4.9兆帕，这相当于一个13微米沟槽近平坦的表面功能性的改变。 通过使用活跃的细胞培养基质控制细胞行为的一个例子是在细胞骨架的组织变化。临时沟槽基板前恢复被触发后，肌动蛋白微丝排列沿方向的沟槽（ 图5a）16。回收温度升高后，微丝有重组和随机取向。对照样品，静态沟槽或静态平坦的表面不重组后的温度上升（ 图5B，C）。 <img src="/files/ftp_upload/2903/2903fig1.jpg" alt="图1"/> 图1:NOA63固化室示意图。截面视图（左）和自上而下，没有玻璃盖（右）。 <img src="/files/ftp_upload/2903/2903fig2.jpg" alt="图2"/> 图2:用于散装形状记忆特性的哑铃几何宽 : 地下狭窄段的长度:长度量具，禾:宽度整体而言，劳:总长度，D:交手，R之间的距离狭窄段，L宽度:半径的圆角，和RO:外径。 <img src="/files/ftp_upload/2903/2903fig3.jpg" alt="图3"/> 图3:散装单向单一NOA63治愈的形状记忆，重复3次（星号表示实验发病） 。在标有星号的点，聚合物被加热和变形，然后通过施加压力界定其临时形状。这株是保持不变，温度下降到修复下面的聚合物的 Tg临时形状。温度上升，作为临时应变降低材料恢复其永久的形状。 <img src="/files/ftp_upload/2903/2903fig4.jpg" alt="图4"/> 图4:SMP的恢复可以触发细胞培养兼容的温度下。幅度为25.6 ± 0.8微米目前以下压花RECovered ± 1.5微米后30小时平衡在30至12.6℃（时间为0，黑色圆圈）。后的样品被转移到37℃培养箱（9.5小时），振幅3.5小时内恢复到1.1 ± 0.2微米振幅恢复到〜0.3 ± 9.5 h和0.1微米范围内检测到没有超过最后的9.5小时减少（红色三角形）。误差棒代表一个标准差（N = 4-6）。痕迹接触有代表性的样品进行轮廓扫描。 <img src="/files/ftp_upload/2903/2903fig5.jpg" alt="图5"/> 图5:细胞肌动蛋白细胞骨架重新排列下面的地形过渡的鬼笔环肽染色细胞上压印基板，共聚焦显微镜图像显示与沟槽的方向过渡和温度升高之前（白色箭头）一致的微丝。过渡期后，微丝重新排列是随机取向，B，平面控制基板上的单元格显示随机取向的微丝前后温度升高，C，沟槽控制基板上的单元格显示微丝沟方向一致，前后温度升高。比例尺为100μm。痕迹， 如图4。

<ol>
	<li>Liu, C., Qin, H., Mather, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+progress+in+shape-memory+polymers.">Review of progress in shape-memory polymers.</a> J. Mater. Chem. 17, 1543-1543 (2007).</li><li>Mather, P. T., Luo, X. F., Rousseau, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+Memory+Polymer+Research.">Shape Memory Polymer Research.</a> Annu. Rev. Mater. Res. 39, 445-445 (2009).</li><li>Lendlein, A., Kelch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+Memory+Polymers.">Shape Memory Polymers.</a> Angew. Chem. Int. Edit. 41, 2034-2034 (2002).</li><li>Ratna, D., Karger-Kocsis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+shape+memory+polymers+and+composites:+a+review.">Recent advances in shape memory polymers and composites: a review.</a> J. Mater. Sci. 43, 254-254 (2008).</li><li>Rousseau, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+of+shape+memory+polymers:+A+review+of+the+progress+toward+overcoming+SMP's+limitations.">Challenges of shape memory polymers: A review of the progress toward overcoming SMP's limitations.</a> Polym. Eng. Sci. 48, 2075-2075 (2008).</li><li>Pelham, R. J., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility.">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proc. Natl. Acad. Sci. U. S. A. 94, 13661-13661 (1997).</li><li>Addae-Mensah, K. A., Kassebaum, N. J., Bowers, M. J., Reiserer, R. S., Rosenthal, S. J., Moore, P. E., Wikswo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flexible,+quantum+dot-labeled+cantilever+post+array+for+studying+cellular+microforces.">A flexible, quantum dot-labeled cantilever post array for studying cellular microforces.</a> Sensor Actuat. a-Phys. 136, 385-385 (2007).</li><li>du Roure, O., Saez, A., Buguin, A., Austin, R. H., Chavrier, P., Siberzan, P., Ladoux, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+mapping+in+epithelial+cell+migration.">Force mapping in epithelial cell migration.</a> Proc. Natl. Acad. Sci. U. S. A. 102, 2390-2390 (2005).</li><li>Lam, T., Clem, W. C., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+on-demand+cell+alignment+using+reconfigurable+microtopography.">Reversible on-demand cell alignment using reconfigurable microtopography.</a> Biomaterials. 29, 1705-1705 (2008).</li><li>Stevens, M. M., George, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+and+engineering+the+cell+surface+interface.">Exploring and engineering the cell surface interface.</a> Science. 310, 1135-1135 (2005).</li><li>Tan, L., Tien, J., Pirone, D. M., Gray, D. S., Bhadriraju, K., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+lying+on+a+bed+of+microneedles:+an+approach+to+isolate+mechanical.">Cells lying on a bed of microneedles: an approach to isolate mechanical.</a> 100, 1484-1484 (2003).</li><li>Teixeira, A. I., Nealey, P. F., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+human+keratocytes+to+micro-+and+nanostructured+substrates.">Responses of human keratocytes to micro- and nanostructured substrates.</a> J. Biomed. Mater. Res. A. 71A, 369-369 (2004).</li><li>Yang, M., Sniadecki, N., Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+Considerations+of+Micro-+to+Nanoscale+Elastomeric+Post+Arrays+to+Study+Cellular+Traction+Forces.">Geometric Considerations of Micro- to Nanoscale Elastomeric Post Arrays to Study Cellular Traction Forces.</a> Adv. Mater. 19, 3119-3119 (2007).</li><li>Zhao, Y., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Mechanics+Study+in+Cardiac+Myocytes+Using+PDMS+Pillars+Array.">Cellular Mechanics Study in Cardiac Myocytes Using PDMS Pillars Array.</a> Sensor Actuat. a-Phys. 125, 398-398 (2006).</li><li>DiOrio, A. M., Luo, X., Lee, K. M., Mather, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functionally+Graded+Shape+Memory+Polymer.">A Functionally Graded Shape Memory Polymer.</a> Soft Matter. 7, 68-68 (2011).</li><li>Davis, K. A., Burke, K. A., Mather, P. T., Henderson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+cell+behavior+on+shape+memory+polymer+substrates.">Dynamic cell behavior on shape memory polymer substrates.</a> Biomaterials. 32, 2285-2285 (2011).</li></ol>

的NOA63 的 Tg可以很容易地通过固化温度控制。我们用这个生成SMP基板，可以在单元格中的兼容范围引发。 NOA63是由水，降低干燥的 Tg塑化，所以我们增加了固化在125 ° C至30日和37 ° C之间移动的Tg范围湿干的 Tg 激活的细胞培养基质表明，能够控制细胞的行为。微丝重组的结果，强调控制和化验动态基板上的细胞行为的潜力。硫化模具和压花形状的基础?...

<table border="1"><tbody><tr><td>试剂或仪器名称</td><td>公司</td><td>目录编号</td><td>评论（可选） </td></tr><tr><td> NOA63 </td><td>诺兰产品公司</td><td> NOA63 </td><td>批号111 </td></tr><tr><td>狗骨头打孔</td><td> TestResource公司Shakopee监狱，MN </td><td></td><td>按比例缩小的IV型狗骨头（ASTM D638 - 03） </td></tr><tr><td>台式液压出版社</td><td>卡弗</td><td> 3851 </td><td></td></tr><tr><td> C3H10T1 / 2小鼠胚胎成纤维</td><td> ATCC </td><td> CCL - 226 </td><td></td></tr><tr><td>生物安全柜</td><td>赛默飞世尔</td><td> 1357 </td><td></td></tr><tr><td>紫外灯</td><td> Spectroline </td><td> SB - 100PC </td><td></td></tr><tr><td>动态力学分析仪（DMA） </td><td> TA仪器公司</td><td> Q800 </td><td></td></tr><tr><td>倒置荧光显微镜</td><td>徕卡</td><td>徕卡的DMI 4000B </td><td></td></tr><tr><td>激光共聚焦显微镜</td><td>蔡司</td><td> LSM 710 </td><td> 20x/0.8 NA空气或一个40x/1.30无油客观</td></tr></tbody></table>

shape memory polymers for active cell culture

形状记忆聚合物（SMPS）是一类的"智能"材料，有能力从一个固定的，临时的形状改变预先确定的形状后应用的刺激， 如热1-5永久的。 SMP是一个典型的形状记忆周期，首先在比其转变温度T 反 [熔融温度（T 米 ）或玻璃化转变温度（Tg）]更高的高温变形。变形是弹性的性质和主要信息的构成的网络链（以下橡胶弹性理论）的构象熵的减少。变形的SMP，然后冷却到低于其T反温度，同时保持外部的应变或应力常数。在冷却过程中，材料过渡到更严格的状态（半结晶或玻璃状），其中动力学陷阱或"冻结"在这种低熵状态，导致宏观形状固定的材料。触发形状恢复是通过不断加热材料通过T 跨下一个无压力的条件（无约束）。使连锁网络（收复流动）放宽到其热力学的青睐，最大熵的状态，从临时形状的重大变化的永久的形状。 细胞是能够测量的力学性能及其周边环境6。机械细胞之间的相互作用和他们的物理环境控制细胞的行为的机制，通过活跃的研究领域。定义地形基板已成为功能强大的工具，在这些机制的调查。尺度，微观和纳米基板地形模式，已被证明直接单元格对齐方式，细胞粘附和细胞牵引力7-14。这些发现强调基板地形控制和检测机械在细胞培养细胞和它们的物理环境之间的相互作用的潜力，但日期基板一般都被动，不能编程的显着变化，在文化。这个物理瘀有限地形基板，以控制细胞培养的潜力。 在这里，活跃的细胞培养（ACC），SMP的基板介绍，聘请的表面形状记忆提供编程控制基板地形和变形。这些基板展示从一个临时的沟槽地形过渡到第二个，近持平记忆地形的能力。这种地形的变化，可以用来控制细胞标准的细胞培养条件下的行为。

Watch this Scientific Journal Video about Shape Memory Polymers for Active Cell Culture at JoVE.com

Shape Memory Polymers for Active Cell Culture

一个能力地形变化，在文化发展与细胞培养基质的方法是描述。该方法使被称为形状记忆聚合物，有能力记住一个永久的形状的智能材料的使用。这个概念是适应的材料和应​​用的广泛。

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

shape-memory-polymers-for-active-cell-culture

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

13.4K 次观看.  Syracuse Biomaterials Institute. 一个能力地形变化，在文化发展与细胞培养基质的方法是描述。该方法使被称为形状记忆聚合物，有能力记住一个永久的形状的智能材料的使用。这个概念是适应的材料和应​​用的广泛。

视频: Shape Memory Polymers for Active Cell Culture

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

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统:作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

对于中吸入有毒化学测试体外细胞培养模型

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

生物活性，PCL为基础的"自我装配"形状记忆聚合物支架的制备

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

由共轭聚合物活细胞电活动的光控制

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医疗器械细菌高通量鉴定驱蚊聚合物

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...