作者想感谢乔欧文在生物医学仪器和机加工车间加工和装配莱卡微系统技术通过流商会协助形象细胞流动室部分和马特Maudsley在他的不懈努力。我们非常感激来自杜克灌注服务的凯文柯林斯博士和史蒂夫华莱士流电路设计上的有益的建议，生物医学工程学系。我们还要感谢国家科学基金会研究生研究奖学金计划，支持通过赠款亚历山德拉Jantzen和NIH的支持“自体的EPC的衬里，以改善循环系统的生物相容性协助设备”RC1HL099863 - 01。

1。内皮祖细胞的分离<ol><li>在此之前任何外围人体血液的收集，研究方案提交您的机构审查委员会（IRB），并批准后，获得的志愿捐献者的知情同意（外周血收集和杜克大学IRB批准的EPC隔离和是完全符合美国有关保护人类研究参与者）的监管要求。 </li><li>动物源性内皮祖细胞的工作时，有您的机构的动物保健研究协议的批准和使用委员会（IACUC）。我们所有的猪实验已杜克大学IACUC批准，并按照与人文关怀的最高标准进行。 </li><li>内皮祖细胞的分离，通过从一个同意的志愿者捐助的标准放血技术收集到采血袋充满抗凝citrat 50毫升外周血Ë磷酸葡萄糖和稀释1:1与汉克的缓冲盐溶液（不含氯化钙 ，氯化镁，硫酸镁4）Histopaque等量层，以创造良好定义的层的解决方案。 </li><li>离心机（30分钟，740克，打破低设置），并收集单核细胞（MNC）的层（巴​​菲大衣）。悬浮和洗跨国公司x 3贝科的磷酸盐缓冲盐水（DPBS）10％胎牛血清（FBS），x 10分钟，515克成两个12 -以及完整的EPC生长介质板的电镀前，（MCDB - 131中型与5 ml的200毫米L -谷氨酰胺，10％FBS和临时股东大会，2 SingleQuots）在37 ° C，5％的CO 2。 </li><li>慢慢地改变中期每24小时第7天，然后隔日。 EPC菌落可以被确定后，根据他们的鹅卵石形态平均14天时间。一旦在文化盖荐的12孔表面积的内皮祖细胞，扩大细胞，并确认通过测试EPC身份流式细胞仪表面标志物的存在CD31和CD14，CD45的情况下，正如前面所描述的 6 。可能进行的其他实验包括DII - AC - LDL的细胞形态和标签。 </li><li>对于本文所述实验，扩大分离出内皮祖细胞，直到他们在3个T - 75细胞在培养箱EPC中等文化37烧瓶近汇合℃，5％的CO 2。 </li></ol> 2。剪应力计算<ol><li>我们建议，这些计算之前执行制造室和通道，可以实现的条件有透彻的了解。计算电路中的流速灌流的基础上所需的剪切应力被应用到细胞，根据式（1） 1， </li></ol><img alt="公式1" src="/files/ftp_upload/3349/3349eq1.jpg" /> 其中 Q是所需的流量，τ是目标小号听到应力作用于细胞切线方向，W为流室的宽度，h为流室高度，μ是粘度灌流（流动介质） 。 <ol start="2"><li>使用的媒介（μ）的粘度典型值是0.9 CP（0.009克厘米-1 s - 1时）。请注意，粘度，也可以通过使用高低分子右旋糖酐，如果需要的话 7提出。一个典型的目标剪应力值（τ）使用的是15达因/厘米2，（典型的动脉剪应力）或100达因/ 厘米 2，如果需要超生理剪应力。我们的商会通常在166范围宽1.9厘米，高度（H）（W） - 267微米。 </li></ol> 3。流动腔制造<ol><li>削减日的顶部和底部的板Ë室从6061铝合金7 × 2 × 0.5“和底部的尺寸7 × 2 × 0.25”的顶部尺寸的矩形股票。 </li><li>课间休息的中心部分顶板深0.125“，留下0.950”每月底为1.375“宽x 0.3125”深部流体的水库。 </li><li>钻出1 / 16“宽x 0.625”从底部以渗透到水库流入和流出端的插槽。 </li><li>在每个顶板月底，钻一个软管倒钩渗透液水库位于市中心10/32“跟踪每英寸（TPI），聚丙烯1 / 8洞”。在流入端，创建一个侧面插头与10/32“TPI的通孔聚丙烯1 / 8”软管倒钩。 </li><li>锥度从流体流入玻璃观察窗的顶部底面在0.518度足够流体混合。 </li><li>切成载玻片窗口的顶部观察窗底板。使用进入会议厅的水族馆水泥，胶水载玻片。允许治愈至少24小时。确保观景窗凹进，以便在会议厅内，75 × 25 × 1毫米的显微镜载玻片将坐在冲洗。 </li><li>创建一个O形圈周围的​​顶部和底部板等底面的凹槽，他们之间的间距由0.02除了“插入橡胶O形圈。 </li><li>钻“TPI的孔在顶部和底部室使用8 / 32”平头螺丝10个匹配的8 / 32。 </li></ol> 4。流电路大会<ol><li>组装整个流电路，然后再进行其杀菌。 </li><li> 36“硬管段连接到一个脉冲阻尼（通过1 / 8”连接）。到另一端，连接软油管18“段。 </li><li>将一个1 / 8软油管节“男性在18月底露儿适配器”，在步骤4.2中提到的。将男性露儿一个1 / 8“女性露儿适配器连接露儿到一个新的18”软管段的女性。 </li><li>三个孔钻成250毫升玻璃烧杯盖以适应管。我nsert成一个洞，并通过其他到250毫升的玻璃瓶（如水库）（ 图2）第2洞的软，硬管的自由端的软油管1.5“段（通风口） 。确保硬管到达水库底部的油管从水库流出。 </li><li>消毒蒸汽蒸压上述部分在121 ° C下60分钟。此外蒸压一个完整的流室，3 × 2“段软油管，1男1女1 / 8”露儿适配器，4个半“和6 × 5 / 16”8-32平头螺钉，1镊子，一个75 × 25 × 1毫米的玻璃幻灯片（或其他所需的细胞表面材料），2个手术巾。 </li><li>放入无菌层流罩毛巾。放置到这个无菌领域流电路，流室，4 × 4路活塞，1 × 1路阀，所有的步骤4.5消毒部位。 </li><li>现在，使用无菌手套，连接两个4路活塞。广场上打开的样品端口的上限。 </li&gt; <li>分离流电路的软连接两个管段的男性和女性的露儿适配器插入连接的活塞（图3）。 </li><li>连接软油管插入到水库30毫升注射器，取下注射器活塞。填充瓶125毫升的EPC中（ 图4）。 </li><li>放置一个进风口（ 图4）无菌注射器过滤器。 </li><li>移动到一个培养箱流电路，在37℃，5％的CO 2 。 </li><li>钳进入滚筒泵使用此类型的油管（ 图5）表示的头硬管。确保管路不重叠与滚子泵的安装轨道。标记油管退出上的标记笔两侧的水泵扬程。 </li><li>确保所有的活塞都对样品端口封闭和开放的沿流电路。启动泵。验证流的方向。灌流移动FROM通过硬管的脉冲阻尼玻璃水库。 </li></ol> 5。 EPC荧光标记<ol><li>虽然流电路预热至37 ° C，准备播种到幻灯片的细胞。请注意，如果细胞是不透明的材料，如钛（Ti）的可视化荧光标记是必需的。 </li><li> 1毫米的细胞跟踪橙色（CTO）的原液稀释90μL二甲基亚砜（DMSO）50微克的CTO粉末。确保光线照射（关闭光流罩下工作时），以免受这种混合物。 </li><li>创建首席技术官原液稀释到18毫升的无血清MCDB - 131中型36μL2微米的首席技术官的工作液。 </li><li>在3附近汇合的T - 75细胞培养瓶中，用10毫升（不包括钙或镁）的DPBS冲洗内皮祖细胞。 </li><li> 2微米CTO工作液到每个烧瓶中加入6毫升。 15分钟在37 ° C。冲洗每个容量瓶中，用10 ml DPBS（无钙或镁）× 2。 </li><l我&gt;到每个烧瓶中添加4毫升的胰蛋白酶。在37 ° 3分钟。压制与8毫升的胰蛋白酶中和解决方案。 </li><li>洗离心× 5分钟600克和重悬在5毫升的EPC培养基。 </li></ol> 6。细胞种植前的幻灯片流室大会<ol><li>可用于玻璃，钛，或其他材料制成的幻灯片，和自旋聚合物涂层的滑动面可以修改或增加一层金属。放入无菌矩形四腔细胞培养容器（或使用组织培养幻灯片容量瓶中，如果需要聚苯乙烯表面）的幻灯片。细胞和种子1.5 × 10 6细胞计数在5毫升的EPC中等× 6小时，如果想要到幻灯片的融合细胞层流启动（图11A ）。 </li><li>为了测试细胞扩散和粘附流体切应力下，允许3 × 10 6细胞坚持只有前15分钟开始流（快播）（ <strong&gt;图。 11B）。 </li></ol> 7。流室总成<ol><li>使用无菌手套，一个2“软管段连接到1 / 8”女性露儿适配器。将另外2个男露儿适配器的“软到1 / 8管段”。 </li><li> “女性露儿流入端口适配器连接软油管，将2个”2男露儿软油管流出端口。这些露儿适配器连接4路活塞。其余2“软管件连接端端口连接器脱落的泡沫陷阱，并附加一个1路阀（ 图6） 。 </li><li>用无菌镊子，取出细胞培养容器（或如果使用幻灯片瓶，删除幻灯片烧瓶）的细胞种子幻灯片，并放入流室的底板上的凹槽。确保幻灯片用的种子细胞的一面朝上放置。 </li><li>吸取10毫升温暖的EPC培养基上滑动。允许介质覆盖的幻灯片和流室，BUT不洒在底板上的O形圈的媒介。 </li><li>将流室的底板上，顶板，对准螺丝孔。小心不要引入气泡，在这一步，进入会议厅保持两板平行。 </li><li>螺旋板（电池供电自动螺丝刀的速度，这个过程）。 </li><li>流入泡沫陷阱移除气泡开放流入端的泡沫陷阱端口1路阀，轻轻冲洗与EPC使用10毫升注射器的介质流入管。然后关闭这个活塞和第它（ 图7）。 </li><li>现在删除气泡室通道开放的流出端口4路阀，轻轻冲洗通过流室的EPC的介质，再次使用10毫升注射器。如果此后仍然存在大的气泡，提高室流出45 °角（ 图 8 ）。 </li><li>第4路阀连接并关闭它们关闭。进入孵化器的组装流电路（ 图9）移动流室。 </li><li>暂停流电路泵。关闭方向流动，防止泄漏，并保持无菌室内部流电路活塞。流罩的流室交通流电路。将流动腔流电路。水流方向打开活塞。恢复流程泵。确保在正确的方向流动的灌流。调整流量到所需的剪切应力。 </li><li>流室的流量实验期间，可以从电路中移除图像，通过光或荧光显微镜细胞。为了做到这一点，在活塞，活塞连接断开流电路流室。要保持无菌室，关闭流电路活塞在流动方向和流动腔活塞和活塞前盖，以消除流电路从孵化器（ 图9）。 </li></ol> 8。灌流液样本收集<ol><li>对于容易收集灌流液样本进行分析，（例如，一氧化氮合成），首先暂停泵。 </li><li>关闭位于最接近的脉冲阻尼器的活塞。删除其保护盖，体积小注射器，比如3毫升注射器，插入进样口。保持帽，并确保它不会成为上下颠倒放置污染。 </li><li>关闭活塞流室的方向，保持采样口和电路在脉冲阻尼开放的方向。 </li><li>绘制所需的样本量，如150μL，从电路。关闭之前取出注射器的活塞对样品的端口。存放在一个标记的小瓶和冻结在灌流液样本 - 80℃（一氧化氮的决心在以后的时间点）。 </li><li>重温取样口，并确保所有活塞开始之前开放<html>流。 </li></ol> 9。代表性的成果使用我们的快速种子的方法，人类血源性内皮祖细胞可以接种到钛幻灯片15分钟，坚持下生理（动脉）15达因/厘米2的剪切力。 如图10所示，流量的影响下，从210 ± 11.4微米2 EPCs的传播后，立即播种，657 ± 39.1微米2，3小时后，到1152 ± 55.3微米后24小时15流体剪切应力 2达因/厘米2（P &lt;0.0001，方差分析，组与组之间的差异有统计学显著）。在单元面积的增加，可以通过透明的室光或荧光显微镜成像的同时，圆度计算，根据公式（ 2）6 / files/ftp_upload/3349/3349eq2.jpg“/&gt; 下降至878 × 10-3 ± 5.9 × 10 -3，播种后，立即以671 × 10 -3 ± 19.2 × 10 -3 3小时后，和526 × 10 -3 ± 19.2 × 10 -3后48小时内相同的流体剪切应力曝光（组与组之间的差别又统计学P &lt;0.0001，方差分析显著） 。 图11D说明，内皮祖细胞在流动方向的调整和东方后48小时的流体剪切力在15达因/ 厘米 2后6小时的静态播种期间 ，如图11A所示，其随机取向相比。 我们的方法还允许获得在预定的时间点的分析和/或分泌细胞代谢产物的量化灌流液样本。作为一个代表性的例子，我们描绘了生产TION亚硝酸盐（NO 2 - ）在48小时流动实验与猪内皮祖细胞在图12。我们直接测量替代中等样品收集流电路，在不同的时间点，从12.0 nmol在6小时，12小时的13.1 nmol，16.3 nmol在24标记为NO一氧化氮（NO）的主要氧化产物小时，24.6 nmol后48小时为1 × 10 6细胞在15达因/厘米2 6。 <img alt="图1" src="/files/ftp_upload/3349/3349fig1.jpg" /> 图1。原理概述流电路组装和流量的实验。 （ 一 ）流没有流室的电路组装。这里包括水库，脉冲阻尼器，硬管，软管，4路活塞和连接露儿适配器，以及空气过滤器。 （二）连接通过硬管段的流量水泵扬程，流室。 （三）<html> sertion的流室（四）底板EPC种子幻灯片。流电路组装完成流室。 <img alt="图2" src="/files/ftp_upload/3349/3349fig2.jpg" /> 图2。完全组装灭菌流电路。 <img alt="图3" src="/files/ftp_upload/3349/3349fig3.jpg" /> 图3。组装流电路后与活塞灭菌包括。 <img alt="图4" src="/files/ftp_upload/3349/3349fig4.jpg" /> 图4。灌装125毫升的EPC培养基玻璃水库。 <img alt="图5" src="/files/ftp_upload/3349/3349fig5.jpg" /> 图5。流电路钳位流量水泵扬程前流室插入。 <html> JPG“/&gt; 图6顶式气室盘总成。 <img alt="图7" src="/files/ftp_upload/3349/3349fig7.jpg" /> 图7。法拉盛与EPC培养基中的泡沫陷阱，去除气泡流入厅一侧。 <img alt="图8" src="/files/ftp_upload/3349/3349fig8.jpg" /> 图8。与EPC介质的冲洗室，删除幻灯片和流出的会议厅一侧的气泡。 <img alt="图9" src="/files/ftp_upload/3349/3349fig9.jpg" /> 图9。完全组装插入一个流量实验期间的流动腔流电路。 <img alt="图10" src="/files/ftp_upload/3349/3349fig10.jpg" /> 图10。EPC区（2微米），增加过流实验的时间过程中。 files/ftp_upload/3349/3349fig11.jpg“/&gt; 图11（一 ）HUCB内皮祖细胞接种到纤维连接蛋白的镀膜玻璃幻灯片的光镜图像放大100 × × 6小时前流。 （ 二 ）HUCB内皮祖细胞标记与CTO和快速种子到钛幻灯片× 15分钟前流，用荧光显微镜观察放大100 ×。 （二）（ 三 ）同一钛HUCB内皮祖细胞的幻灯片下，经过3小时的流量在100达因/ 厘米 2，并通过放大100 ×透明室成像。请注意后3个小时的流量的传播的细胞，但仍随机的方向。 （四）同一TI幻灯片，现在流量为100 ×放大倍率暴露后48小时。内皮祖细胞都标有CTO和细胞核与Hoechst染料（蓝色）染色后流。注意细胞在流动方向对齐。 （五）钛相比，猪内皮祖细胞后48小时内的流量和15达因/厘米<sup&gt; 2的剪应力暴露。抗PECAM染色（绿色）和Sytox橙核酸染色细胞核，细胞边界染色。请注意流动方向对齐细胞。 <img alt="图12" src="/files/ftp_upload/3349/3349fig12.jpg" /> 图12图描绘没有猪内皮祖细胞在48小时流动的生产。测得的NO主要氧化产物，亚硝酸盐（NO 2 - ），作为介质流在不同时间点收集的样品中NO的替代指标。 <a href="https://www.jove.com/files/ftp_upload/3349/3349fig13_lg.jpg" target="_blank"><img alt="图13" src="/files/ftp_upload/3349/3349fig13.jpg" /></a> 图13。顶端部分（ 一 ）为0.5“× 2”× 7“和底部的第（二 ）0.25”× 2“× 7”的尺寸，流室是由铝合金6061矩形股票。顶部刨其底面O形圈接触面的0.45“，以确保密封的平整度，上面的部分是其中心部分凹进的0.125”，这让“每一个0.3125结束”X 1.375“储液罐0.95。顶端部分的水库螺纹3/8-24 TPI和0.25“深接收螺纹不锈钢插头。插槽1 / 16“× 0.625”加工成的底部渗透到水库。流入端插件有一个10/32软管倒钩附加到一个活塞“1 / 8聚丙烯通孔”。每个单元的末尾有一个位于市中心螺纹0.25 10/32“深部聚丙烯1 / 8”0.45“× 2”年底软管倒钩TPI的。这锥度的两端使用0.07“线程底部的孔，通过渗透油藏。顶端部分的下部有一个锥形面积0.6875”宽，0.518度为1.460的距离“LH插槽中心。一个单位面积0.6875“宽x深0.0118”和0.590 GLAS结束“混合s（或其他表面上，例如钛幻灯片）休会。这单位必须用玻璃（或其它表面，如钛幻灯片）冲洗一次安装幻灯片。锥度和平面抛光。 0.0118“0.0118”休会凹进的底部表面。继续水库槽上的RH端表面的幻灯片。 O形圈是完全凹进插槽和0.020之间的O型环槽和幻灯片的原始表面留下“幻灯片周围，顶部和底部的单位是十孔钻顶端部分通孔为8 / 32 TPI的沉平头螺钉的底部放在匹配的顶部孔的孔，并有螺纹TPI的8 / 32。底部的单位有一个明确的玻璃滑动窗，其表面的凹进冲洗设在细胞表面载玻片（或钛幻灯片），单位O形环槽，其中包括在与宽度的顶部单元槽底部的幻灯片和内部O形圈的凹槽之间的原始表面留下0.04“。而O - RINGS是红色硅胶AS568A，短跑044的顶部和底部的第046号。

<ol>
	<li>Bhat, V. D., Truskey, G. A., Reichert, W. 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+and+avidin-biotin+as+a+heterogeneous+ligand+system+for+enhanced+endothelial+cell+adhesion.">Fibronectin and avidin-biotin as a heterogeneous ligand system for enhanced endothelial cell adhesion.</a> J. Biomed. Mater. Res. 41, 377-385 (1998).</li><li>Broxmeyer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cord+blood+stem+and+progenitor+cells.">Cord blood stem and progenitor cells.</a> Methods Enzymol. 419, 439-473 (2006).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Heart Association Scientific Sessions, Abstract Oral Sessions, Medical Aspects End Stage Heart Failure: Transplantation and Device Therapies. , (2010).</li><li>Brown, M. A., Wallace, C. S., Angelos, M., Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+umbilical+cord+blood-derived+late+outgrowth+endothelial+progenitor+cells+exposed+to+laminar+shear+stress.">Characterization of umbilical cord blood-derived late outgrowth endothelial progenitor cells exposed to laminar shear stress.</a> Tissue. Eng. Part. A. 15, 3575-3587 (2009).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+titanium+ventricular+assist+device+surfaces+after+gold/palladium+coating+for+scanning+electron+microscopy.">Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy.</a> Microsc. Res. Tech. 73, 71-76 (2010).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biocompatibility+of+titanium+cardiovascular+devices+seeded+with+autologous+blood-derived+endothelial+progenitor+cells:+EPC-seeded+antithrombotic+Ti+Implants.">The biocompatibility of titanium cardiovascular devices seeded with autologous blood-derived endothelial progenitor cells: EPC-seeded antithrombotic Ti Implants.</a> Biomaterials. 32, 10-18 (2011).</li><li>Rinker, K. D., Prabhakar, V., Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+contact+time+and+force+on+monocyte+adhesion+to+vascular+endothelium.">Effect of contact time and force on monocyte adhesion to vascular endothelium.</a> Biophys. J. 80, 1722-1732 (2001).</li><li>Truskey, G. A., Yuan, F., Katz, D. F., Horton, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Transport Phenomena in Biological Systems. , (2004).</li><li>Truskey, G. A., Yuan, F., Katz, D. F., Horton, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Transport Phenomena in Biological Systems. , (2009).</li><li>Allen, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+nitrite+response+and+arterial+reactivity+differentiate+vascular+health+and+performance.">Plasma nitrite response and arterial reactivity differentiate vascular health and performance.</a> Nitric Oxide. 20, 231-237 (2009).</li><li>Xiao, Y., Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+receptor-ligand+affinity+on+the+strength+of+endothelial+cell+adhesion.">Effect of receptor-ligand affinity on the strength of endothelial cell adhesion.</a> Biophys. J. 71, 2869-2884 (1996).</li><li>Conant, C. G., Schwartz, M. A., Nevill, T., Ionescu-Zanetti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+Adhesion+and+Aggregation+Under+Flow+using+Microfluidic+Flow+Cells.">Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells.</a> J. Vis. Exp. (32), e1644-e1644 (2009).</li><li>Conant, C. G., Schwartz, M. A., Ionescu-Zanetti, C. W. e. l. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=plate-coupled+microfluidic+devices+designed+for+facile+image-based+cell+adhesion+and+transmigration+assays.">plate-coupled microfluidic devices designed for facile image-based cell adhesion and transmigration assays.</a> J. Biomol. Screen. 15, 102-106 (2010).</li><li>Conant, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+well-plate+microfluidic+devices+to+conduct+shear-based+thrombosis+assays.">Using well-plate microfluidic devices to conduct shear-based thrombosis assays.</a> J. Lab. Autom. 16, 148-152 (2011).</li><li>Metaxa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide-dependent+stimulation+of+endothelial+cell+proliferation+by+sustained+high+flow.">Nitric oxide-dependent stimulation of endothelial cell proliferation by sustained high flow.</a> Am. J. Physiol. Heart. Circ. Physiol. 295, H736-H742 (2008).</li><li>Radel, C., Carlile-Klusacek, M., Rizzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+caveolae+in+beta1+integrin-mediated+mechanotransduction.">Participation of caveolae in beta1 integrin-mediated mechanotransduction.</a> Biochem. Biophys. Res. Commun. 358, 626-631 (2007).</li><li>Wang, X. L., Fu, A., Spiro, C., Lee, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+of+Vascular+Endothelial+Cells-Effects+of+Laminar+Shear+Stress+and+High+Glucose.">Proteomic Analysis of Vascular Endothelial Cells-Effects of Laminar Shear Stress and High Glucose.</a> J. Proteomics. Bioinform. 2, 445-445 (2009).</li><li>Elfervig, M. K., Minchew, J. T., Francke, E., Tsuzaki, M., Banes, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1beta+sensitizes+intervertebral+disc+annulus+cells+to+fluid-induced+shear+stress.">IL-1beta sensitizes intervertebral disc annulus cells to fluid-induced shear stress.</a> J. Cell. Biochem. 82, 290-298 (2001).</li><li>Archambault, J. M., Elfervig-Wall, M. K., Tsuzaki, M., Herzog, W., Banes, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+tendon+cells+produce+MMP-3+in+response+to+fluid+flow+without+significant+calcium+transients.">Rabbit tendon cells produce MMP-3 in response to fluid flow without significant calcium transients.</a> J. Biomech. 35, 303-309 (2002).</li><li>Patton, J. T., Menter, D. G., Benson, D. M., Nicolson, G. L., McIntire, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computerized+analysis+of+tumor+cells+flowing+in+a+parallel+plate+chamber+to+determine+their+adhesion+stabilization+lag+time.">Computerized analysis of tumor cells flowing in a parallel plate chamber to determine their adhesion stabilization lag time.</a> Cell. Motil. Cytoskeleton. 26, 88-98 (1993).</li><li>Wiese, G., Barthel, S. R., Dimitroff, 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=Analysis+of+Physiologic+E-Selectin-Mediated+Leukocyte+Rolling+on+Microvascular+Endothelium.">Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium.</a> J. Vis. Exp. (24), e1009-e1009 (2009).</li><li>Kaper, H. J., Busscher, H. J., Norde, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+poly(ethylene+oxide)+brushes+on+glass+surfaces+and+adhesion+of+Staphylococcus+epidermidis.">Characterization of poly(ethylene oxide) brushes on glass surfaces and adhesion of Staphylococcus epidermidis.</a> J. Biomater. Sci. Polym. Ed. 14, 313-324 (2003).</li><li>Bakker, D. P., Plaats, A. v. a. n. d. e. r., Verkerke, G. J., Busscher, H. J., van der Mei, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+velocity+profiles+for+different+flow+chamber+designs+used+in+studies+of+microbial+adhesion+to+surfaces.">Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces.</a> Appl. Environ. Microbiol. 69, 6280-6287 (2003).</li><li>Angelos, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+adhesion+of+umbilical+cord+blood+endothelial+progenitor+cells+under+laminar+shear+stress.">Dynamic adhesion of umbilical cord blood endothelial progenitor cells under laminar shear stress.</a> Biophys. J. 99, 3545-3554 (2010).</li><li>Baker, G. R., Sullam, P. M., Levin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+fluorescent+method+to+internally+label+platelets+suitable+for+physiological+measurements.">A simple, fluorescent method to internally label platelets suitable for physiological measurements.</a> Am. J. Hematol. 56, 17-25 (1997).</li></ol>

科尔 - 帕默仪器有限公司赞助的生产和免费访问这个视频文章

我们的流量电路和流室，让我们的主题贴壁细胞，如内皮祖细胞，流体剪切应力定义。由于腔的顶部和底部的透明，细胞粘附和形态可以在实时评估，通过商会本身，或后一个流动的实验和拆卸流室。在这一点上，细胞可以收获无菌条件下，可以重新镀金，或用于收集他们的DNA或RNA等，作进一步的分析。 为了实现层流室的设计必须满足这样几个条件。 首先，流必须层，它可以通过计算其雷诺数（Re），这是惯性力与粘滞力的比例核实。 （如果粘滞力占主导地位 ，再小的流动是层流或“充分发展” -通常 RE &lt; 2300。如果惯性力占主导地位，流动变得越来越随机，直到它是动荡的，是重新 &gt; 4000），我们可以计算出再根据等式（3）8 ， <img alt="公式3" src="/files/ftp_upload/3349/3349eq3.jpg" />其中，ρ为流体密度，Q为流量，μ是粘度，w和 h的会议厅，分别在宽度和高度， 和 D H水力直径，根据方程4月8日， <img alt="公式4" src="/files/ftp_upload/3349/3349eq4.jpg" />雷诺数三流室，不等高度从166 - 267微米，范围从13.9 - 34.6流速彗星alculated获得15达因/厘米2的剪应力。在流速为100达因/厘米2的剪应力计算，分庭的雷诺数范围从90.4 - 234。所有这些雷诺数比2300低得多，并满足层流标准。 二，速度场和剪切应力沿流道（即充分开发），从流体入口的距离，幻灯片必须大于入口的长度，L E的距离无关。这可以得到满意的入口长度计算，根据方程5月9日 。 <img alt="公式（5）" src="/files/ftp_upload/3349/3349eq5.jpg" />上面列出的值，入口长度范围从0.01至0.25厘米。 第三，以确保在横向方向，速度和剪应力不随显著，从一维渠道流值（ΔPH / 2L），H / W的比例必须小于1 。平均壁剪应力二维水流条件下，将根据一维流动壁面剪切应力的95％，H / W必须等于0.10，二维水流条件下的壁面剪切应力壁面剪切应力下的一维流动的97.5％，H / W必须等于0.05。我们设计的流室宽1.7厘米和166 - 267微米的高度尺寸，这些标准是满意的。 压力会随水流方向，如果没有在入口处的横向压力梯度。这可以使用染料或粒子流路进行评估。此外，源源不断实验，脉冲阻尼器插入流电路。脉冲阻尼器的PULSatility引起电路中的滚压泵，并允许我们近似假设源源不断。值得注意的是，应使用的脉冲阻尼器在电路中使用的泵和管兼容，使其可以有效地消除在特定频率辊泵的输出流量脉动。在我们的演示的Masterflex L / S脉冲阻尼器实现时，使用任何的Masterflex L / S系列泵（0 - 600的RPM）的放线和I / P 26管层流。脉动流，一个可编程的泵可用于生成各种波形。 脉动流，一个可编程的泵可用于生成各种波形。 此外，该电路是这样设计的灌流液样本可以很容易地在不同时间点收集细胞或流动介质的污染，而不用担心。在我们的例子中，浓度的NO 2 -化学发光测定一个离子学/的Sievers一氧化氮分析仪（NOA的280的Sievers仪器，博尔德，CO）的先前所描述的 10个。亚硝酸盐的分析中使用的还原剂是碘化钾，醋酸（14.5米乙酸和0.05 M碘化钾），已转换为NO的亚硝酸盐，但不足以减少任何更高，如硝酸盐氮氧化物减排潜力，因而是相对具体为亚硝酸盐。计算的总金额产生的亚硝酸盐是作为产品的生产集中度和电路的总体积，而丢失而采取样品6卷调整。 流实验的成功执行至关重要以下步骤： <ol class="lroman"><li>这是可取的流量设置，以避免在泡沫的形成，因为气泡有可能撕下其表面的细胞。这可避免放置到流室的顶部，底部的一部分，这是最好的照顾通过保持两个相互平行，并降低顶部至底部，在一个没有调整的议案。这样做，可能是当前和/或在流道的浮动的小气泡，将改到泡沫陷阱铝外壳的两端。 </li><li>重要的是要确保在水库流出管到达水库底部。否则，可能被吸进空气，导致油管从水库到腔灌流水平略低于管端落在。 </li><li>研究人员应熟悉泵的流量的方向，使泵不意外在相反的方向运行生效后的流量，这将导致空气被吸成电路和腔。 </li><li>为了避免形成泡沫（由于血清中的变性蛋白），我们建议降低管室水库，导致大约一英寸以上perfusaTE水库内的水平。然而，保持流量中等水平以上，可以验证在实验过程中流入水库。 </li><li>当拧在一起室部分，重要的是要牢牢把握，用一只手的住房，同时用另一只手操作电动螺丝刀。需要注意的是电动螺丝刀的议案将被翻译成扭矩，它有可能“甩腔周围”螺丝拧紧后，。 </li><li>为了避免任何压力波形成电路和解除了他们的表面细胞，确保所有活塞开始流动前开放。 </li><li>特别是较长期的流动研究（&gt; 48小时），我们建议使用油管插入到泵的扬程，以防止破油管的困难，更有弹性。 </li><li>它是可能的，油管泵头由于虐待调整泵的扬程牙齿内部的“迁移”。因此，我们建议密切关注如何在浴缸ING是固定在泵头和庆祝与纪念笔等，你可以很容易发现如果管子是“拉扯”，或在实验中脱落的油管两端。 </li><li>始终仔细检查每一个单一连接器的电路和腔灌注开始之前，以防止由于一个错误的连接在实验灌流泄漏。脉冲减震器的流入和流出的连接器，这一点尤为重要！ </li><li>请记住如果您使用荧光标记，限制光线照射。 </li></ol>一个可能的限制是我们的流动腔的高度是固定的通道高度加工的铝。但是，这并没有验证和调整通道的高度，每次实验前的优势，因此简化了剪应力计算，仅通过调整泵的流量为所需的值。根据您的研究目标，它可能是可取的剪应力不增加泵的转速增加。在这种情况下，我们建议增加灌流液的粘度，如添加葡聚糖中期 11 。 一个流电路的可能限制，介质的大量使用，可问题时，试图量化细胞代谢产物的浓度非常小。虽然这里没有显示，它有可能使用较小的水库和脉冲阻尼器，大大减少了电路体积，降低油管的长度和直径。 此外，还有其他一些市售的系统，可用于细胞培养应用流体切应力。基于微流体系统，如从Fluxion BioFlux系统，装载到作为输入和输出这些渠道12,13,14水库孔板的解决方案在不同的微流体流动渠道，使细胞的同时分析。然而，这些和其他微生物流体系统不兼容标准显微镜载玻片和不允许恢复了进一步的实验，如RT - PCR或免疫印迹，细胞数量足够大。此外，用户友好，成本最低40,000元，并有可能达到了总额超过10万美元，取决于配套设备。 两个macrofluidic系统Flexcell国际公司，Flexcell流光和FlexFlow系统，已成功地用于研究血管内皮细胞15,16,17，人类纤维环细胞18和19流体流动条件下的成纤维细胞。第三个系统，可通过GlycoTech，已利用研究20肿瘤细胞的粘附和白细胞粘附21日至内皮单层。 流光系统允许多张幻灯片，下一次相同的剪应力条件下运行，但缺乏一个观景窗 - 与我们设计 - 不允许进行实时可视化下流动的细胞。 FlexFlow系统有一个观景窗，但需要一个堂堂正正的显微镜，这可能不是在大多数实验室中使用的标准显微镜。此外，FlexFlow系统需要一个细胞涂的盖玻片置于流室倒立时。这排除了可视化荧光细胞上的不透明表面，如钛镀膜玻璃，这是我们在我们的研究表明。最后，专门盖玻片需要购买专门的FlexFlow系统，该系统是在多万美元的价格区间，类似Flexcell流光系统。 GlycoTech提供圆形和矩形平行板流商会，显著较便宜，但不能方便像我们商会蒸压干投丙烯酸制造。值得注意的是，其他流量已描述商会高压灭菌似乎是不切实际的，因为他们需要特殊的微观镜头22,23。 GlycoTech系统采用硅橡胶密封圈，顶板和底板之间的中间人，这将改变重复使用厚度的，因此随着时间的推移（采购后，每10个使用新的制造商建议）改变室高度。我们的铝腔可以与内置的O型圈顶部和底部板的完整的反对，并确保实验之间的不断室高度。最后，真空泵，是必要的，实现了在许多流室设计的密封防漏，包括GlycoTech商会，这是没有必要的设计。 虽然这里没有显示，可以保持流室，在显微镜下，在整个流程细胞的粘附和/或行为的实时成像实验。如果需要的话，这是我们建议使用下腔热灯或加热垫的灌流液的温度保持在37 ° C。毛皮在那里，可以更换辊筒泵用注射器泵，如果没有“再循环”无论细胞或代谢物或研究的药物或制剂所需的 24 。 它也有可能流过不同标记细胞的贴壁细胞，例如，超过一层融合内皮祖细胞荧光血小板（血小板检测Achneck 等 。6,25），细胞与细胞之间的相互作用下流体剪切力，以评估。我们的流动腔结合其他可用流商会，如灌流取样口和一个观景窗，有价值的功能，并有一个倒置或直立显微镜的兼容性的重要优势。它是完全可高压灭菌，并允许在不断室高度和无真空泵的需要，实现了防漏密封反复实验。

<table border="1"><tbody><tr><td> 产品/试剂 </td><td> 公司 </td><td> 产品编号 </td></tr><tr><td> 10毫升注射器</td><td>科尔帕默仪器有限公司</td><td> 07940-12 </td></tr><tr><td> 1路Stopcoks </td><td>科尔帕默仪器有限公司</td><td> 30600-00 </td></tr><tr><td> 30毫升注射器</td><td>屋宇署医疗</td><td> 309650 </td></tr><tr><td> 4路闭锁龙头</td><td>科尔帕默仪器有限公司</td><td> 30600-04 </td></tr><tr><td>铝合金</td><td> N / A </td><td> 6061 </td></tr><tr><td>细胞培养皿（4孔，矩形） </td><td> Thermo Scientific的; NUNC </td><td> 267061 </td></tr><tr><td>细胞追踪橙</td><td> Invitrogen公司</td><td> C34551 </td></tr><tr><td>二甲基亚砜</td><td>研究有机物</td><td> 2162D </td></tr><tr><td> DPBS (-/-)</td><td> Invitrogen公司</td><td> 14190-144 </td></tr><tr><td>临时股东大会，2 Singlequots </td><td>龙沙</td><td> CC - 4176 </td></tr><tr><td>女露儿适配器</td><td>科尔帕默仪器有限公司</td><td> SI - 45500 - 04 </td></tr><tr><td>纤维连接蛋白</td><td>西格玛</td><td> F0895 - 2MG </td></tr><tr><td>玻璃瓶（250毫升），与第</td><td>科尔帕默仪器有限公司</td><td> 34594-24 </td></tr><tr><td>硬油管</td><td>科尔帕默仪器有限公司</td><td> 06508-16 </td></tr><tr><td> HBSS </td><td>西格玛</td><td> H8264 - 500ML </td></tr><tr><td> Histopaque </td><td>西格玛</td><td> H8889 - 500ML </td></tr><tr><td>赫斯特染色解决方案</td><td>西格玛</td><td> H6024 </td></tr><tr><td>胎牛血清胎牛血清</td><td> Thermo Scientific的</td><td> SH30071.01 </td></tr><tr><td> L -谷氨酰胺</td><td>龙沙</td><td> 17 - 605E </td></tr><tr><td>男露儿适配器</td><td>科尔帕默仪器有限公司</td><td> EW - 45505 - 04 </td></tr><tr><td> MCDB - 131，1X中等</td><td> Cellgro </td><td> 15 - 100 - CV </td></tr><tr><td>刺聚丙烯配件</td><td>科尔帕默仪器有限公司</td><td> 06365-90 </td></tr><tr><td> O形圈</td><td> N / A </td><td> AS568A </td></tr><tr><td>脉冲的减震器</td><td>科尔帕默仪器有限公司</td><td> 07596-20 </td></tr><tr><td>泵驱动器（的Masterflex L / S可变高速的经济驱动器） </td><td>科尔帕默仪器有限公司</td><td> 7524-40 </td></tr><tr><td>泵头（易负载的Masterflex泵头） </td><td>科尔帕默仪器有限公司</td><td> 07518-60 </td></tr><tr><td>幻灯片</td><td>科尔帕默仪器有限公司</td><td> 48500-00 </td></tr><tr><td>软油管</td><td>科尔帕默仪器有限公司</td><td> 96400-16 </td> &lt;/ TR&gt; <tr><td>注射器过滤器</td><td>科尔帕默仪器有限公司</td><td> 02915-12 </td></tr><tr><td> Sytox橙核酸染色</td><td> Invitrogen公司</td><td> S - 11368 </td></tr><tr><td>胰蛋白酶EDTA </td><td>龙沙</td><td> CC - 5012 </td></tr><tr><td>胰蛋白酶中和解决方案</td><td>龙沙</td><td> CC - 5002 </td></tr></tbody></table>

parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress

这种方法的总体目标是描述一个技术主题贴壁细胞层流情况，并评估他们的反应以及量化的流体剪切应力1。 我们的流动腔设计和流电路（ 图1）包含一个透明的观赏地区，使细胞粘附和细胞形态的成像测试立即流前（图 11A，B）在不同时间点， 在流动 ， （图11C）后流（ 图11D）。这些实验说明了人类脐带血源性内皮祖细胞（EPCs）和猪内皮祖细胞2,3。 这种方法也适用于其他贴壁细胞类型，如平滑肌细胞（SMCS）或成纤维细胞。 很容易与蒸汽高压灭菌消毒室和电路的所有部分。相对于其他商会，可以恢复后的细胞培养或其他实验，如DNA或RNA的提取，或免疫组化（ 图11E）在无菌条件下的流动实验，如微流体商会，大量细胞（&gt; 100万对细胞的大小而定）或扫描电子显微镜5。剪应力可以调整不同的灌流，流体的粘度，或​​通道的高度和宽度的流量。后者可以减少液量或细胞的需求，同时确保维持一维流动。这是没有必要的试验室之间的高度来衡量，因为室高度不依赖于垫圈的使用，从而大大增加了多个实验的缓解。此外，电路设计，很容易使灌流液样本收集和/或由细胞分泌的流体切应力下暴露的代谢产物定量分析，例如一氧化氮（ 图 12 ）6 </suP&gt;

Watch this Scientific Journal Video about Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress at JoVE.com

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

我们所描述的主题贴壁的细胞在无菌的连续流电路层流剪应力的方法。细胞的黏附，形态可通过透明腔研究，从代谢物的分析和对未来的实验或文化的剪切曝光后的干细胞的电路样本。

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

parallel-plate-flow-chamber-continuous-flow-circuit-to-evaluate

Research

Education

JoVE Core

JoVE Journal

Civil Engineering

Bioengineering

Unit 1

Differential Analysis of Fluid Flow

SCIENTISTS IN ACTION

43.2K 次观看.  Duke University Medical Center. 我们所描述的主题贴壁的细胞在无菌的连续流电路层流剪应力的方法。细胞的黏附，形态可通过透明腔研究，从代谢物的分析和对未来的实验或文化的剪切曝光后的干细胞的电路样本。

视频: Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

使用介电泳分离珠和多通道微流控芯片细胞和层流

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

科研

生物工程

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

细胞的浸入式印刷到改性表面采用连续流动Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

评估白细胞内皮的相互作用下水流条件的<em&gt;离体</em&gt;自动注入微流室法

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

多平行板流室对剪切应力暴露的内皮细胞基因表达分析

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while F&#246;rster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

使用荧光显微镜在剪切流下描述单分子构象变化

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

悬浮中金纳米粒子尺寸的不对称流场-流分数

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

血流成像与超快多普勒

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...