支持这项工作已部分由下批准号EFRI 0938047美国国家科学基金会，以及由弗吉尼亚理工大学的关键技术和应用科学（ICTAS）的支持。作者想表达感谢伊娃施梅尔茨博士和保罗·罗伯茨博士为他们的那种MOSE-L细胞的礼物。作者承认安吉拉·安德森为她协助细胞培养，Caitlan Swaffar她帮助编辑这个文件，并准备实验，以及所有Bioelectromechanical系统实验室成员。

1。制造原型CDEP微流体设备：概述<ol><li>使用光致抗蚀剂和深反应离子蚀刻（DRIE）蚀刻所需的信道设计（ 图1）到硅片（ 图2）按照以前报道的程序22。沉积薄薄的一层聚四氟乙烯的改善的微型装置从晶片上剥离。 </li></ol><img alt="图1" src="/files/ftp_upload/50634/50634fig1.jpg" /> 图1。低频连续分选装置的示意图，样品通道运行左至右为500μm宽的带锯齿收缩至100μm。两对电极的流体通道构成的源和汇的电极，分别和由20μm厚的PDMS阻挡从样品通道是分开的。 <img alt="图2" src="/files/ftp_upload/50634/50634fig2.jpg" /> 图2。该布标一个CDEP装置ication过程（a）对于60秒，UV光选择性地与光致抗蚀剂通过CDEP设备的几何形状的掩模图案进行曝光（B）光致抗蚀剂是使用显影剂除去。（三）深反应离子蚀刻（DRIE）来蚀刻50微米深的特点，和5分钟的湿法蚀刻以四甲基氢氧化铵（TMAH）的25％，在70℃是用于减少在侧壁上的粗糙度（d）一个特氟隆涂层被添加以改善从设备释放晶片（E）的PDMS倒入可重复使用的晶片邮票脱气后，在100℃F.（F）的PDMS从晶片除去固化45分钟。 PDMS和干净的载玻片暴露于空气等离子处理2分钟，粘合在一起。 <ol start="2"><li>用铝箔包装的晶圆，以防止溢出。在10:1至固化剂，脱气约20分钟，比弹性体的混合聚二甲基硅氧烷（PDMS），倒到晶片上。热在100℃下45分钟，以避免损坏邮票的特富龙涂层。让设备冷却下来。 </li><li>冷却后，除去铝箔，装饰用手术刀片PDMS，并用钝冲床适合于管子的尺寸打孔进入孔在通道的入口/出口。在这里，1.5毫米的钝冲床使用。 </li><li>用漂洗肥皂和水，乙醇，去离子水，和干燥用的空气，分别清洗的玻璃显微镜载片上。用苏格兰魔术胶带清洁PDMS设备。显微镜下检查，以确保通道是干净的。暴露在滑动和PDMS装置空气等离子体2​​分钟。用力按下设备的通道侧到载玻片上，从而避免形成装置和滑动（ 图3）之间的气泡。 </li></ol><img alt="图3" src="/files/ftp_upload/50634/50634fig3.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig3highres.jpg"/> 图3。 </st荣&gt;（a）该硅晶片制造邮票，以及（b）与所述样品和填充有分别的绿色和红色食用色素，流体通道电极成品PDMS-玻璃装置中使用的光刻掩模。参见图1尺寸。 <a href="https://www.jove.com/files/ftp_upload/50634/50634fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 2。制备细胞悬液中的DEP缓冲<ol><li>制备低导电率（100μS/厘米）的缓冲，这将被称为“介电泳缓冲液”（8.5％蔗糖[重量/体积]，0.3％葡萄糖[重量/体积]，0.725％的RPMI [重量/体积]） 16 </li><li>暂停细胞中的DEP缓冲器在3×10 6个细胞/ ml。这里，癌晚期小鼠卵巢表面上皮细胞（MOSE-L）细胞的使用。 </li></ol> 注：由台盼蓝染色排除法细胞活力分析显示活力略有下降早期MOSE（MOSE-E）的细胞在室温下5小时后，悬浮于DEP缓冲液（由95.1-91.6％）。据预测，在可行性类似轻微下降发生于MOSE-L细胞，表明细胞不应该被存储在缓冲DEP长期。 <ol start="3"><li>使用荧光膜渗透性染料，如酶促活化的钙黄绿素AM染细胞。 </li><li>测量染色的细胞悬浮液的导电性。电导率应约100μS/ cm的。如果电导率测量太高，旋转样品下来，重悬在DEP缓冲器以3×10 6个细胞/ ml，并重复电导率测量。 </li></ol> 3。加载CDEP微流控芯片<ol><li>放置在真空下，微流控芯片30分钟。 </li><li>同时，切下一块的柔性管，以跨越从注射泵的距离，显微镜载物台，其中所述微流体芯片将位于。这里，管道的6英寸长是必需的。飞度油管针尖上的注射器。 </li><li>通过将目标收集样品体积通过管的横截面面积估计所需的长度为出口管路。切需要管的长度。 </li><li>绘制细胞悬液注入注射器。检查无气泡存在于管或注射器。 </li><li>在从真空除去芯片，插入出口管路和素样品通道迅速，以避免被截留的空气气泡在通道。吸，以避免破裂的薄（20微米）时，绝缘壁轻轻拍打注射器，虽然它已经观察到，在阻挡可承受近5毫升/小时的恒定的高通量而不破裂。 </li><li>以最优惠的电极渠道，快速地将空的枪头在每个电极流体通道的一个出口。吸取200μl的PBS中含有少量罗丹明B和轻轻拍打到流体引入到电极通道的另一端。保持温和的压力直到PBS与罗丹明B明显进步了空的枪头的尖端。重复每个电极通道。罗丹明B仅用于荧光可视化的流体电极通道。 </li><li>吸后，填充每个枪头储用PBS罗丹明B。避免气泡形成在移液管尖水库，并通过轻轻地上下吹打除去任何可见的气泡。拆下出口管路和适当地丢弃，然后插入一个新的出口管路水库，收集目标卷。 </li></ol> 4。使用CDEP芯片细胞的特征分频点<ol><li>定位在倒置显微镜配备有数码相机（ 图4）的阶段的芯片。 </li></ol><img alt="图4" src="/files/ftp_upload/50634/50634fig4.jpg" fo:content-width="5in" fo:src="/files/ftp_upload/50634/50634fig4highres.jpg"/> 图4。 </stroNG&gt;（a）整体实验装置CDEP实验。该CDEP芯片位于倒置显微镜的平台上。一个摄像头的图像发送到计算机进行录制。注射泵保持恒定的入口流。函数发生器产生被加强了由高电压放大器和连接到所述CDEP芯片通过导线电极的信号。示波器监视发送到芯片的信号。（二）CDEP芯片和流体和电子连接的详细视图。 <a href="https://www.jove.com/files/ftp_upload/50634/50634fig4highres.jpg" target="_blank">点击这里查看大图</a> 。 <ol start="2"><li>安装注射器注射泵和插入线电极插入电极流体通道水库。 </li><li>通过以1毫升/小时的流体泵，以确保注射泵和注射器之间的良好接触。目视检查沟道长度，以保证细胞的畅通和没有气泡或泄漏。红UCE流量0.005毫升/小时。 注：吞吐量高达100毫升/小时31,35已经在其他几何形状的设备已经实现。 </li><li>为了安全起见，由开机函数发生器，高压放大器和示波器，并调整到所需的电压和频率测试电子产品。这里这些参数是200 V和20千赫。经核实可接受的性能，关闭电源。电极连接到高电压放大器。 注：在用于这些实验的高电压下工作的电子时，应遵守相应的电气安全规程。 </li><li>在达到稳流，应用所需的电压在所希望的频率。在该实验中，电压为200伏RMS在5-60千赫之间的频率。 </li><li>与图像处理技术（步骤5）来量化细胞分布在通道和表征交叉频率的细胞应答的记录的视频文件。要做到这一点，保持一个恒定的电压和系统地改变频率。在该实验中，以及随机实验运行之间的开始和结束时，记录控制单元的分布，该细胞的不施加电场的分布。估计的时间让细胞从入口流到监控位置（此处，5分钟）所需的量。设置一个新的频率后，等待该段时间，然后开始录制（这里是2分钟的视频细胞分布的）。 </li></ol> 5。为表征分频点图像处理<ol><li>利用一种计算机脚本，分析每个视频帧由帧来确定相对的y位置（z方向是该信道的垂直深度，y方向是沟道宽度，而x-方向是沟道长度）在每一发荧光的细胞或颗粒的指定的x坐标从在沟道高介电泳力的区域的下游。建立相关显示的图tribution。 </li><li>在每个电压和频率与所述控制分布的中心线比较分布的中心线。对于给定的电压和频率的变化，确定交叉频率，这里的中心线相匹配的控制的y位置。 </li></ol> 6。不同的实验，以进行细胞分选富集或<ol><li>暂停在DEP缓冲器所需的混合物在3×10 6个颗粒/毫升的总浓度。这里，该混合物是MOSE-L细胞中的DEP缓冲器4微米的荧光珠。执行先前描述的步骤，以制备CDEP设备。 </li><li>从混合物进行排序或富集的细胞，确定了靶细胞的交叉频率和如先前所描述的背景颗粒。操作实验的靶细胞和背景粒子的2分频的频率之间的频率，使得粒子的两个群体经历的DEP力在相反ð从根本上来说。要MOSE-L细胞和4微米微珠进行排序，操作上11.90±0.63 kHz的微型装置。 MOSE-L细胞的第一交叉频率最近报道Salmanzadeh 等人 33 </li><li>收集的有序样品中的内容，在收集对象的卷取出出口管路，通过将一个手套的手指在所述出口开口并轻轻拉着管。倒入收集目标体积成储以供进一步分析注：其他样品的回收方法可以包括附加的永久贮存到芯片和由简单的移液除去样品。 </li></ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isolation+of+live/dead+cells+using+contactless+dielectrophoresis+(cDEP).">Selective isolation of live/dead cells using contactless dielectrophoresis (cDEP).</a> Lab Chip. 10, 438-445 (2010).</li><li>Salmanzadeh, A., 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=Dielectrophoretic+differentiation+of+mouse+ovarian+surface+epithelial+cells,+macrophages,+and+fibroblasts+using+contactless+dielectrophoresis.">Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis.</a> Biomicrofluidics. 6, (2012).</li><li>Salmanzadeh, A., 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=Isolation+of+prostate+tumor+initiating+cells+(TICs)+through+their+dielectrophoretic+signature.">Isolation of prostate tumor initiating cells (TICs) through their dielectrophoretic signature.</a> Lab Chip. 12, 182-189 (2012).</li><li>Salmanzadeh, A., Sano, M. B., Shafiee, H., Stremler, M., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Rare+Cancer+Cells+from+Blood+Cells+Using+Dielectrophoresis.">Isolation of Rare Cancer Cells from Blood Cells Using Dielectrophoresis.</a> , (2012).</li><li>Sano, M. B., Caldwell, J. L., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+and+development+of+a+low+frequency+contactless+dielectrophoresis+(cDEP)+platform+to+sort+cancer+cells+from+dilute+whole+blood+samples.">Modeling and development of a low frequency contactless dielectrophoresis (cDEP) platform to sort cancer cells from dilute whole blood samples.</a> Biosens. Bioelectron. 30, 13-20 (2011).</li><li>Salmanzadeh, A., 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=Investigating+Dielectric+Properties+of+Different+Stages+of+Syngeneic+Murine+Ovarian+Cancer+Cells.">Investigating Dielectric Properties of Different Stages of Syngeneic Murine Ovarian Cancer Cells.</a> Biomicrofluidics. , (2013).</li><li>Salmanzadeh, A., Sano, M. B., Shafiee, H., Stremler, M. A., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , 590-593 (2012).</li><li>Sano, M. B., Salmanzadeh, A., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+contactless+dielectrophoresis:+Theoretical+considerations.">Multilayer contactless dielectrophoresis: Theoretical considerations.</a> Electrophoresis. 33, 1938-1946 (2012).</li><li>Millet, L. J., Park, K., Watkins, N. N., Hsia, K. J., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+beads+and+cells+in+multi-channel+microfluidic+devices+using+dielectrophoresis+and+laminar.">Separating beads and cells in multi-channel microfluidic devices using dielectrophoresis and laminar.</a> J. Vis. Exp. (48), e2545 (2011).</li><li>Henslee, E. A., Sano, M. B., Rojas, A. D., Schmelz, E. M., Davalos, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+concentration+of+human+cancer+cells+using+contactless+dielectrophoresis.">Selective concentration of human cancer cells using contactless dielectrophoresis.</a> Electrophoresis. 32, 2523-2529 (2011).</li><li>Salmanzadeh, A., Shafiee, H., Davalos, R. V., Stremler, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+mixing+using+contactless+dielectrophoresis.">Microfluidic mixing using contactless dielectrophoresis.</a> Electrophoresis. 32, 2569-2578 (2011).</li></ol>

拉斐尔·达瓦洛斯博士有一个正在申请专利的非接触式介。

DEP是一个强大的技术来确定粒子的介电性能和引导粒子运动对分拣，隔离，或富集的应用。由于与样品直接接触电极的不利影响，其他人也采取了类似先前提出的方法，以避免接触的方法。例如，巴希尔等人，通过使用由一个薄的玻璃盖玻片从印刷电路板的电极分离的微流体设备的PDMS开发的无接触式的DEP装置，以及该技术也取得了在视频格式。23,36 这里，我们已经表明一个CDEP芯片和流电极使用单一的PDMS衬底的制造中，并在实验方案从细胞和荧光珠的混合物中分离卵巢癌细胞。所提出的技术已成功地用于各种更复杂的和生理学相关的应用程序，包括分选的LIVe和死细胞，28肿瘤启动从前列腺癌细胞的细胞中，30癌细胞从稀红细胞，31,32和乳腺癌37和卵巢癌的阶段中分化29 CDEP也被用于混合颗粒。38这些应用表明，通过使用简单的技术提出，不同的目的，可以简单地通过改变所述通道的几何形状的设计来实现的。 。DEP上的微尺度操纵粒子13对于球形颗粒是有用的，平移的介电电泳力取决于粒子和其悬浮介质的尺寸和电气性能，以及电场梯度的平方： <img alt="式（1）" src="/files/ftp_upload/50634/50634eq1.jpg" /> 式中，εm是悬浮介质的介电常数，r是半径颗粒和Rc [K（ω）]是克劳修斯- Mossotti（CM）因子的实部。在CM的因素是粒子的相对极化率的量度相比，悬浮介质，并确定介电电泳力的方向。它被描述为<img alt="公式2" src="/files/ftp_upload/50634/50634eq2.jpg" /> 哪里<img alt="式（3）" src="/files/ftp_upload/50634/50634eq3.jpg" />和<img alt="公式4" src="/files/ftp_upload/50634/50634eq4.jpg" />是粒子和介质，分别的复数介电常数。复介电常数， <img alt="方程（5）" src="/files/ftp_upload/50634/50634eq5.jpg" /> ，取决于电导率（σ）和频率（ω）。球形颗粒的CM因子-0.5和1之间的理论上的约束。如果CM因子为阴性，所述颗粒经历NDEP因为介质是比粒子更极化，使颗粒从区域移开高电场梯度。如果CM因子为正，则粒子比介质更极化和他们经历pDEP它们走向的高电场梯度的区域。 用于生物颗粒是不均匀的结构，如细胞，克劳修斯 - Mossotti因子可以从该粒子的介电常数的有效值来确定： <img alt="公式6" src="/files/ftp_upload/50634/50634eq6.jpg" /> 哪里<img alt="公式7" src="/files/ftp_upload/50634/50634eq7.jpg" />和<img alt="公式8" src="/files/ftp_upload/50634/50634eq8.jpg" />是细胞的分别的内部，例如在细胞质和细胞膜，有效的复介电常数的复介电常数; r是小区的半径，d为质膜的厚度26 当DEP发生颗粒悬浮在液，相对于流体的粒子的运动将产生在颗粒曳力。确定整体受力作用在粒子时，这个阻力必须加以考虑。对于这里关注的情况下，粘性力占主导地位和粒子被假设球形，体积小，相对较低的速度运动，所以斯托克斯拖动法律提供了一个很好的近似的阻力： <img alt="公式9" src="/files/ftp_upload/50634/50634eq9.jpg" /> 其中η是流体的粘度，U p是粒子的速度， 和 u f是流体，其也可被移动的速度。给定的介电电泳力，已知流体和颗粒性质，以及已知的流速，拖曳力和介电电泳力之间的平衡就可以解决估计粒子速度。剪切速率的细胞经历应低于该阈值，在其中CELL裂解可能发生。 粒子的电气性能特性是必要的预测和控制它们将如何DEP下作出回应。在这项工作中，我们特别利用低频CDEP与MOSE-L细胞，以证明该协议用于确定细胞的第一交叉频率，然后显示的聚苯乙烯珠和MOSE-L细胞连续分选基于它们的相对的DEP的响应。 改变CDEP设备的几何形状会改变电场的空间梯度，允许设备被设计用于高频或低频的操作，并为高选择性和对于一个指定的信元类型排序的效率。此外，高通量设备可以通过制造更宽的信道进行开发，30个信道并行，30或由多层制造35，其中的电极通道的垂直堆叠的上方和下方相对深样品通道。薄膜形成层之间的势垒。初步测试已制成的聚甲基丙烯酸甲酯（PMMA）和聚碳酸酯的设备（PC）薄膜已经证明MOSE-L细胞中的DEP响应。目前正在努力完善多层次的高通量设备和改进，迈向最终的插件和播放平台的外围系统。从基本实验技术呈现扩张，该装置的应用程序和规格可以定制，以适应特殊的需求，如排序与表征，或添加试样槽和半自动化系统的装置。

生物样品中富集和分选粒子往往是必要的用于随后的分析。1比如，从体液中分离稀有细胞在癌症的检测和个体化医学重要的应用。2,3最常用的富集技术是荧光激活细胞分选术（FACS ）4，磁激活细胞分选（MACS），5依赖于表达的表面标志来区分细胞。其他策略包括液力6或7,8惯性分选，光学镊子，9 acoustophoresis，10和介电电泳。11,12介电电泳是在非均匀电场的存在下，偏振粒子的运动。13 DEP已被用于了广泛的应用，2,14包括基于生存能力，15表征细胞的生物电性能分拣单元，16个和排序ING时引起的变化在细胞的生物物理特性。17,18传统的DEP利用内的微流体通道施加电压，并诱导非均匀的电场。13虽然这是一个功能强大的技术进行图案化的平面电极，挑战可能出现，如电极脱层和电解。绝缘体为基础的介电电泳（影像介电泳）19已经解决积垢，电极剥离，电极字段通过诱导的非均匀性在直流电场图案形成绝缘结构空间降解的挑战。影像介电泳已被用于选择性地分离活的和死的细菌细胞，19隔离细菌孢子，20和操纵的DNA，在其他应用21。焦耳加热可能是一个挑战，因为它可以发生作为经常需要高的直流电压的结果。为了改善这些问题，无接触的DEP的微型器件已被开发。22-24 。22独此策略是金属电极更换为充满液体电极通道高导电性的解决方案。这些流体电极两端的薄绝缘屏障，通过AC电压样品通道电容耦合。消除电极与样品接触减少了与DEP为基础的方法，如电解，形成气泡，样品污染和电极脱层的相关问题。其结果是，CDEP因为它支持细胞的样品中的存活率为生物样品特别有用。重要的是，CDEP能保持样品不育。一个芯片就可以在一个细胞培养罩准备和实验可以，而不需要样品接触到金属电极或要求该样本是开放的环境为进行NT。一个简单的贮能固定到芯片插座，以便无菌样品回收。此外，流体的电极由相同的生物相容的聚合物材料（PDMS）的制造作为样品通道可减少产生与金属电极的自定义图案化，需要为制造时的成本高，而且限制了需要专门的洁净室设备的初始图案形成可重复使用的硅片邮票。 由于DEP粒子的运动依赖于粒子和介质，以及该电场的空间梯度的特征。一种粒子和频率依赖因子，称为克劳修斯 - Mossotti（CM）因子，取值范围为-0.5〜1，并确定该介电泳力的方向。的频率，所述CM因子正好为零称为交叉频率。这是在该无介电电泳力作用在粒子和CM因子变化标志点。一个S英格尔分频点惰性固体微球时出现CM因素的变化由负转正25对于哺乳动物细胞中低电导率缓冲液为0.01 S / m的，第一分频点表示从NDEP到pDEP存在近10过渡的顺序- 100千赫，并通过大小，形状，细胞骨架和细胞的膜性能的影响。26,27的第二交叉频率从pDEP转向NDEP制度是10MHz的量级，并且是由影响核-细胞质比率，细胞质电导率和内质网。27 DEP力可以在不流体流动的存在下被应用，但这里我们利用流动流体来实现的悬浮颗粒的连续的分拣。介电电泳力和斯托克斯阻力的综合影响决定了粒子的平移运动。 我们在两个频率范围为开发运行的设备。高FREQUENCŸ设备（100-600千赫）已使用pDEP和细胞实现批量分拣操作，如前列腺肿瘤起始细胞（国际信托），鼠卵巢表面上皮细胞（MOSE）细胞，MDA-MB-231乳腺癌细胞，或住THP -1细胞通过选择性地对绝缘位于样品通道职位俘获所关注的细胞。28-31下限频率（5-100千赫）器件连续工作，并且当在一个频率操作在其中一个人口经历pDEP而背景人群的经验NDEP，可以重定向粒子轨迹来实现排序。32-34在这些低频器件已被用于癌症细胞从红血细胞进行排序，确定变化的渐进MOSE细胞系的介电性能，并阐明的非作用有毒鞘脂处理对倒车积极MOSE细胞的攻击性特点。此外，CDEP微型器件可被设计成在增加的吞吐量进行操作，目前最多为1毫升/小时河31,35 如描述的那样，灵活性和制造工艺的成本低使定制设计的设备的几何形状，其允许呈现的实验步骤是相关于广泛的应用范围。 CDEP的长期目标是实现在临床水平无标记细胞分选和富集，对后续的培养或处理样品回收。这里介绍的方法是一种简单，廉价的方法，从制造到实验，这增加了DEP的可访问性。我们展示了一个准备CDEP芯片和实验协议，实现定性和卵巢癌细胞荧光微球的富集。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning, Midland, MI,USA</td>
			<td>Sylgard 184</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>The Microscope Depot</td>
			<td>76079</td>
			<td>2x3 inch-ground edges</td>
		</tr>
		<tr>
			<td>Microbore PTFE Tubing</td>
			<td>Cole-Parmer Instrument Co, Vernon Hills, IL, USA</td>
			<td>EW-06417-31</td>
			<td>Thin walled 20 gauge, 0.032"ID x 0.056"OD, 100 ft/roll</td>
		</tr>
		<tr>
			<td>Luer-slip plastic syringes</td>
			<td>National Scientific company</td>
			<td>S7510-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Needle tip</td>
			<td>Howard Electronic instruments</td>
			<td>JG20-1.0</td>
			<td>20 Gauge 1.0", ID=0.025" OD=0.036"</td>
		</tr>
		<tr>
			<td>D(+)-Sucrose</td>
			<td>Fisher Scientific, Fair Lawn, NJ</td>
			<td>S3-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D(+)-glucose, reagent ACS, anhydrous</td>
			<td>Acros Organics N.V., Fair Lawn, NJ</td>
			<td>AC410955000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>Quality Biological Inc.</td>
			<td>112-025-101</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calcein AM, Molecular Probes</td>
			<td>Invitrogen Corp. (life technologies), Carlsbad, CA, USA</td>
			<td>C3100MP</td>
			<td>excitation wavelength 488/emission wavelength 516</td>
		</tr>
		<tr>
			<td>Rhodamine B, O</td>
			<td>Science Lab</td>
			<td>SLR1465-100G</td>
			<td>excitation wavelength 540/emission wavelength 625</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (10X)</td>
			<td>Gbiosciences, St. Louis, MO</td>
			<td>RC-147</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leica, inverted light microscope</td>
			<td>Leica Microsystems, Bannockburn, IL, USA</td>
			<td>Leica DMI 6000B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leica DFC420, color camera</td>
			<td>Leica Microsystems, Bannockburn, IL, USA</td>
			<td>Leica DFC420</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>GW Instek, Taipei, Taiwan</td>
			<td>GFG-3015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Wideband power amplifier</td>
			<td>Amp-Line Corp., Oakland Gardens, NY, USA</td>
			<td>AL-50HF-A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HFHV Output Transformer</td>
			<td>&nbsp;</td>
			<td>AL-T50-V25/300-F100K-600K</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>High voltage amplifier</td>
			<td>Trek</td>
			<td>Model 2205</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>USB Modular Oscilloscope, 100 MHz</td>
			<td>AgilentTechnologies</td>
			<td>U2701A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Expanded Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001/002 (115/230V)</td>
			<td>air plasma</td>
		</tr>
		<tr>
			<td>Scotch Magic tape</td>
			<td>3M</td>
			<td>&nbsp;</td>
			<td>any available width is sufficient</td>
		</tr>
		<tr>
			<td>1.5 mm puncher</td>
			<td>Harris Uni-Core</td>
			<td>Z708836-25EA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FluoSpheres Sulfate Microspheres</td>
			<td>Invitrogen</td>
			<td>F8858</td>
			<td>4.0 &mu;m, red fluorescent (excitation wavelength 580/emission wavelength 605)</td>
		</tr>
		<tr>
			<td>AZ 9260 photoresist</td>
			<td>AZ Electronic Materials</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AZ 400 K developer</td>
			<td>AZ Electronic Materials</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tetramethylammonium hydroxide (TMAH) 25%</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>provided by Virginia Tech cleanroom</td>
		</tr>
		<tr>
			<td>Teflon coating</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>applied using DRIE machine</td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>University Wafer</td>
			<td>452</td>
			<td>100 mm diameter, 500 &mu;m thickness, one side polished (SSP)</td>
		</tr>
		<tr>
			<td>Deep Reactive Ion Etching (DRIE)</td>
			<td>Alcatrel</td>
			<td>AMS SDE 100</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

label-free isolation and enrichment of cells through contactless dielectrophoresis

介电电泳（DEP）是由偏振光在非均匀电场粒子经受平移运动的现象，并且可以被用来指示微粒的表面标志物独立的方式运动。传统上，DEP设备包括图案的样品通道的平面金属电极。这种方法可以是昂贵的，并且需要专门的洁净室环境。最近，已开发出一种称为非接触式介电电泳（CDEP）无接触的方式。该方法利用DEP的经典原则，同时避免了流体图案电极和电极和样品之间的直接接触从一个单一的聚二甲基硅氧烷（PDMS）基板的样品通道，并具有应用程序设计进行排序和丰富微粒快速微流体策略。独有的方法，是在电场经由含有高导电性的流体，它从流体中分离电极通道产生的通过薄绝缘阻挡样品通道。因为金属电极不直接接触样品，电解，电极剥离，和样品污染是可以避免的。此外，这使得一种廉价和简单的制造工艺。 CDEP因此非常适合用于操作敏感的生物粒子。因粒子作用在介电电泳力不仅取决于由装置的几何形状可定制的设计中产生的电场的空间梯度，但细胞的内在生物物理特性。因此，CDEP是取决于表面表达，可能在一个人口被可变表达，同时还允许生物粒子的表征，富集，分选和分子生物标志物，避免了无标记技术。 在这里，我们展示了制造和试验用CDEP的基本知识。我们采用软版画解释CDEP芯片的制备简单Ÿ技术。我们将讨论的实验步骤，用于表征颗粒或细胞的频率在该介电电泳力是零的交叉频率。最后，我们证明了使用该技术进行分类的卵巢癌细胞的混合物与荧光微球（珠）。

DEP应定性观察在样品通道，以确认该设备是可操作的，当在电场存在。一种方法来测试DEP的存在是为了远离交​​叉频率是强劲pDEP和NDEP进行了预测（约&gt; 40 kHz到观察pDEP和&lt;10 kHz到观察NDEP对大多数哺乳动物癌细胞在调整频率值与100μS/ cm的电导率缓冲液）。应当指出的是惰性的微球表现出pDEP低于交叉频率和NDEP以上的交叉频率。为锯齿特征几何这里提出，pDEP应在更高的测试频率可以看出对于细胞，由珍珠链和聚焦在该信道的锯齿边缘特征的细胞或颗粒的发生表示，而在较低的测试频率，NDEP应该是明显的，因为细胞被限制在该区域更近的通道的直边。在这些低频率，细胞可能LYS缘于电穿孔，因此样品可能会出现包含更少的细胞，并且那些是可见的，如果使用酶促活化的荧光染料可能会放大或模糊。验证在哪个pDEP和NDEP发生频率允许交叉频率在协议中描述的决心。用于哺乳动物癌细胞，如MOSE-L用在这里，我们观察到NDEP在5千赫（ 图5a）和强pDEP在60千赫（ 图5b）。 MOSE-L细胞的直径为17.7±3.3微米（N = 268个细胞）和珠粒具有4微米的标称直径。相比MOSE细胞在早期发展阶段MOSE-L细胞的介电性能的完整的分析可以在最近的工作由Salmanzadeh 等 33中找到如果pDEP和NDEP不会在这些极端观察到，各种问题可能会发生。该样品的电导率可能过高由于污染物或细胞裂解。 INSUFFICIEnt的电场可能会由于气泡被困在流体通道电极，其防止对接壤的样品通道的通道的一部分的电流传导产生。非定常流可通过截留在注射器或进口管道的气泡引起的，从气泡不保留该设备在真空条件下保持足够的时间，或在从真空，泄漏是由于粘结不良的PDMS的剥离除去不迅速填充所述装置产生的载玻片上，由于用力过猛眼泪在隔膜在泵工作范围的极端填充时的通道，或流速发挥。 除了确定细胞的交叉频率，该技术可用于混合物中，通过MOSE-L细胞和珠的混合物，此处所示的排序。此示例采用负介（NDEP）由4微米微珠频率，其中MOSE-L细胞有正面的介电电泳（pDEP）表现出的优势。我们Øperated设备在200V RMS和10千赫（ 图6a），并观察到两种细胞和珠进行混合，因为他们经历NDEP。在50千赫我们观察MOSE-L细胞的运动到通道的顶部，而珠被限制在通道的下部，使混合物分离成两个部分（ 图6b）。 <img alt="图5" src="/files/ftp_upload/50634/50634fig5.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig5highres.jpg"/> 图5。 MOSE-L细胞的位置，通过改变所施加的电场的频率进行操作。该照片是在最后的锯齿特征在样品通道和所述流体电极分别位于右边的角落。它们被填充有含有PBS若丹明B，其中荧光可视化的频道（一个）MOSE-L细胞经历NDEP在200 V RMS<html>和5千赫。（二）MOSE-L细胞感受珍珠链和pDEP在200 V RMS和60千赫。 <a href="https://www.jove.com/files/ftp_upload/50634/50634fig5highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" src="/files/ftp_upload/50634/50634fig6.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50634/50634fig6highres.jpg"/> 图6。通过最终的锯齿特征MOSE-L细胞（绿色）和4微米的小珠（红）的混合物流入一个低频CDEP装置的样品通道（a）于 200 V和10千赫的细胞和小珠混合。箭头指向隐约出现了可能已经死亡，由于低频率条件下的细胞（B）在200 V和50千赫的细胞经历pDEP，并移动到该信道的特征的边缘，而珠遇到NDEP并保持局限在该区域更靠近直边。p_upload/50634/50634fig6highres.jpg“目标=”_blank“&gt;点击这里查看大图。

Watch this Scientific Journal Video about Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis at JoVE.com

Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

非接触式介电电泳（CDEP）达到通过其固有的介电性能和分拣颗粒的富集。流体通道的电极取代金属电极的传统，以DEP，适合CDEP非破坏无菌表征和生物粒子进行排序。我们将演示如何编写一个CDEP微型设备，并进行细胞鉴定和分选实验。

无标记的分离和细胞的富集通过非接触式介电泳

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to ...

label-free-isolation-enrichment-cells-through-contactless

Research

JoVE Journal

Medicine

15.9K 次观看.  Virginia Tech. 非接触式介电电泳（CDEP）达到通过其固有的介电性能和分拣颗粒的富集。流体通道的电极取代金属电极的传统，以DEP，适合CDEP非破坏无菌表征和生物粒子进行排序。我们将演示如何编写一个CDEP微型设备，并进行细胞鉴定和分选实验。 

视频: Label-free Isolation and Enrichment of Cells Through Contactless Dielectrophoresis

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

科研

医学

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

在卵巢癌肿瘤起始细胞体外富集

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...