CHO-P细胞从芭芭拉FURIE博士（贝斯以色列女执事医疗中心，哈佛大学医学院）是一种礼物。到JMK这项工作是由一个Movember的前列腺癌基金会挑战奖，以JMK也支持这部分工作为卫生补助HL095722研究所

1。细胞培养<ol><li>人类早幼粒细胞白血病（HL-60）细胞<ol><li>培养HL-60细胞在75cm 2培养瓶中用15毫升的Iscove氏改良的Dulbecco氏培养基（IMDM），补充有20％（V / V）胎牛血清（FBS），1％（V / V）L-谷氨酰胺和1个％（体积/体积）青霉素 - 链霉素。 </li><li>通过吸一半的细胞悬液体积和完整的IMDM培养基取代它改变媒体每3天。 </li><li>为羧基二乙酸酯，琥珀酰亚胺酯（CFSE）染色，离心机的HL-60细胞悬浮液（400×g离心5分钟），重悬在1μM的CFSE溶液（在PBS中预温制得）并孵育15分钟，在37℃下然后离心细胞，吸出上清，重悬的细胞在新鲜培养基中预热30分钟。在PBS中洗涤细胞，然后用对轧制实验（参见图1B为CFSE染色的HL-60细胞上的P-SEL-涂覆的表面的代表图像）。 </li></ol></li></ol><ol start="2"><li>肺微血管内皮细胞（LMVECs） <ol><li>清漆100毫米培养皿用0.1％明胶溶液（在PBS中的体积/体积）并在37℃下进行至少30分钟。 </li><li>在明胶包被的100毫米培养皿中完全内皮细胞生长培养基中培养LMVECs（内皮基础培养基-2（EBM-2）），补充有特定生长添加剂套件，请参阅试剂）。改变媒体在达到80-90％汇合每隔一天和子培养细胞。 </li><li>次级培养，在37℃下用PBS洗涤细胞，然后分离细胞用4ml 1X胰蛋白酶-EDTA的3分钟，中和在完整的EBM-2介质的等体积。细胞悬液转移到15毫升管和centrifugE（400×g离心5分钟）。离心后，沉淀重悬在1ml完整内皮的介质，并用血细胞计数器计数细胞。不要过度通道的细胞，因为这会影响他们的形态和功能使用的通道下7只电池的所有实验。 </li></ol></li></ol><ol start="3"><li>中国仓鼠卵巢-P-选择素（CHO-P）细胞<ol><li> CHO-P细胞，这是CHO细胞稳定转染表达人类的P-SEL，由合作者（贝斯以色列女执事医疗中心，哈佛大学医学院）11,12提供。 </li><li>文化的CHO-P细胞的T175厘米2瓶25毫升的F-12介质。 </li><li>用于传代，洗涤细胞，用10ml的PBS中为4-5秒，然后胰蛋白酶消化在10ml 1X胰蛋白酶-EDTA的为3分钟，在37℃，随后通过中和在完全介质。 </li><li>离心细胞悬液（400×g离心5分钟），小心吸出上清液，重悬细胞沉淀1毫升全媒体，并与细胞计数血球。 </li></ol></li></ol> 2。集成多孔板微流控系统的操作<ol><li>确保所有的设备已正确连接并开启不同的模块：计算机，控制器，倒置显微镜和CCD摄像头。 </li><li>打开成像软件;确保多孔板模块和成像模块被正确显示在屏幕上。 </li><li>管子连接到蒸汽疏水阀（连接到控制器），并​​把它们连接到压力接口。 </li><li>将多孔板在板加热器/适配器。添加试剂到井（在下面描述），并连接接口上的板的顶部。将板成像的自动化阶段。 </li><li>接口附加到所述板的顶部和施加一个气动压力从控制器到孔的上方，驱动通过所述微流体通道中的流体在定义的流率，很容易地控制使用多井板在手动模式下模块屏幕。 </li><li>试剂在整个观察区域，位于所述孔之间的通道流动。微流体通道尺寸为350μm宽×70微米高。线性通道的长度为1毫米，通道的底部包括一个180微米的玻璃盖玻片上，这与明场，相位，荧光和共聚焦显微镜兼容。 </li><li>使用CCD摄像头（流采集，11帧/秒）视频采集，并通过兼容的软件进行分析。 </li></ol> 3。微流体通道与蛋白质底物或细胞单层涂层<ol><li>涂层微流体通道与纤连蛋白或P-/E-selectin <ol><li>制备将1ml 20微克/毫升纤连蛋白在PBS中的溶液。基于信道要被涂敷的数量（使用25-50微升每通道的纤连蛋白）改变音量。 </li><li>加入25-50微升的纤维连接蛋白的解决方案，以每口井。申请的2达因/厘米2的剪切力5分钟灌注的通道。请注意，液体出现在出口以及胎圈。孵育在室温下30-45分钟</li><li>吸从孔中的溶液（不要直接从中间的圆圈，饲料通道吸）1,13。加入200-500微升PBS为出口良好，通过施加2达因/厘米2的剪切流5分钟洗净通道用PBS。该频道现在正确涂有纤维连接蛋白，随时可以使用。 </li><li>涂布用的P-或E-SEL，制备5μg/ ml的溶液在PBS中的所希望的人重组蛋白，和外套，如上所述，用1小时培养，在37℃，以允许表面涂层的通道。 </li></ol></li><li>创作的微流体通道内的CHO-P或LMVEC单层<ol><li>轻轻的胰蛋白酶消化细胞从培养皿中3分钟，淬火用完全介质和离心机的2倍体积（5分钟，在400×g离心）。悬浮细胞10毫升全媒体和离心机（5分钟400 XG）agaiÑ​​。 </li><li>计数细胞，以确定在悬浮液中的细胞浓度。以确保形成一个融合LMVEC单层的通道内，使细胞浓度为15〜20百万个细胞/ ml。对于一个铺满的CHO-P的细胞单层，用50-60百万细胞/ ml。用25-50微升细胞悬浮液为每个信道的 - 用于实验据此确定初始细胞数。 </li><li>添加25-50微升细胞悬浮液中的适当的浓度，以进井。将板在显微镜载物台上，并引入细胞进入通道（2达因/厘米2），直到细胞中观察到的画面充满整个通道上，然后停止流动。 </li><li>用200微升的要么是完全LMVEC或CHO媒体既填补出口和入口。让细胞沉淀，坚持3个小时的孵化器（37℃，5％CO2）。 </li><li>继3小时培养，洗净通道与全媒体（2达因/厘米2，10〜15分钟），除去独立的细胞。电池现在应该会出现完全融合和渠道，现在可以使用了。取决于初始细胞接种密度，可能需要额外的2-3小时的稳定时间，以确保该表面的完全覆盖的细胞。 </li></ol></li></ol> 4。 LMVEC促炎性激活和P-/E-selectin的抗体阻断<ol><li>在LMVEC基础培养基制备以下的TNF-α溶液（10纳克/毫升）。 </li><li>以诱导炎症活化LMVEC在通道中，添加100微升的TNF-α溶液的入口以及与通过施加2达因/厘米2的剪切流5分钟，该溶液引入到通道中。对于控制通道（非活化内皮细胞），加入100μl的LMVEC基本培养基的入口以及与引入通道（2达因/厘米2 5分钟）。通道现在可以滚动试验。 </li><li>要阻止P-SEL和LMVECs和CHO-P细胞的E-SEL，引入中和P-SEL（克隆AK4，5微克/毫升巴中SAL媒体）或E-SEL（克隆P2H3，5微克/毫升的基础培养基）抗体的进入通道，并培育1小时，在37℃下接着，用基础培养基洗涤通道（2达因/厘米2，5分钟）。渠道现在已经准备好了轧制试验。 </li></ol> 5。 HL-60轧含量对基板/细胞单层包覆的微流体通道<ol><li>仔细检查显微镜下通道，以确认信道被适当地涂覆（在涂层与细胞的情况下，一个完全汇合的细胞单层，应观察到的）。 </li><li>以制备的HL-60细胞悬浮液的轧制实验，离心机的HL-60细胞悬浮液（5分钟，在400×g离心），并用基础培养基洗涤一次。计数细胞，重悬在IMDM（基本培养基，含Ca 2 +，Mg 2 +的）来创建一个HL-60细胞悬液500万细胞/ ml。用25-50微升的细胞悬浮液对每个信道进行轧制试验。 </li><li>加入25-50微升细胞suspe的nsion到出口良好，现浇板温度控制的板夹（37°C）和里面的地方显微镜舞台上。接着，通过施加2达因/厘米2的剪切力（细胞应在10-15秒从出口到入口流被观察到的）引入细胞进入通道。 </li><li>检查轧制响应作为剪切应力的函数，减少剪切到0.25达因/厘米2，并获得20-30秒的视频（使用“流采集”功能）在每个所需的剪切力（从0.25逐渐增加剪切高达5达因/厘米2，它也可以使用较高的剪）。 </li><li>使用CCD摄像头（流采集，11帧/秒）视频采集，并通过兼容的软件分析滚动路径和滚动速度。 </li></ol> 6。流式细胞术检测表达表面分子<ol><li>以下胰蛋白酶消化，制备所需的细胞类型（HL-60，CHO-的细胞悬浮液（使用1-2×10 5细胞/样品）P或LMVECs）的PBS（ - / - ），补充有2％FBS中。洗涤细胞两次，并把样品体积至50μl（使用相同的缓冲液中）。 </li><li>孵育各样品具有所需荧光团共轭抗体（参见详细信息附表），在4℃下进行20分钟（用铝箔覆盖）。 </li><li>洗涤细胞两次（相同的缓冲区），并把染色的细胞悬浮液终体积为200微升。使用流式细胞仪来检测表面分子的表达分析的样品。 </li></ol>

<ol>
	<li>Conant, C. G., 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=Well+plate+microfluidic+system+for+investigation+of+dynamic+platelet+behavior+under+variable+shear+loads.">Well plate microfluidic system for investigation of dynamic platelet behavior under variable shear loads.</a> Biotechnol. Bioeng. 108, 2978-2987 (2011).</li><li>Conant, C. G., Schwartz, M. A., 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=Well+plate-coupled+microfluidic+devices+designed+for+facile+image-based+cell+adhesion+and+transmigration+assays.">Well plate-coupled microfluidic devices designed for facile image-based cell adhesion and transmigration assays.</a> J. Biomol. Screen. 15, 102-106 (2010).</li><li>Ankrum, J., Karp, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+therapy:+Two+steps+forward,+one+step+back.">Mesenchymal stem cell therapy: Two steps forward, one step back.</a> Trends Mol. Med. 16, 203-209 (2010).</li><li>Karp, J. M., Leng Teo, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+homing:+the+devil+is+in+the+details.">Mesenchymal stem cell homing: the devil is in the details.</a> Cell Stem Cell. 4, 206-216 (2009).</li><li>Luster, A. D., Alon, R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+migration+in+inflammation:+present+and+future+therapeutic+targets.">Immune cell migration in inflammation: present and future therapeutic targets.</a> Nat. Immunol. 6, 1182-1190 (2005).</li><li>Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+selectins+in+inflammation+and+disease.">The role of selectins in inflammation and disease.</a> Trends Mol. Med. 9, 263-268 (2003).</li><li>Sperandio, M., Pickard, J., Unnikrishnan, S., Acton, S. T., Ley, K. <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+leukocyte+rolling+in+vivo+and+in+vitro.">Analysis of leukocyte rolling in vivo and in vitro.</a> Methods Enzymol. 416 (06), 346-371 (2006).</li><li>Brown, D. C., Larson, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+to+parallel+plate+flow+chambers+to+reduce+reagent+and+cellular+requirements.">Improvements to parallel plate flow chambers to reduce reagent and cellular requirements.</a> BMC Immunol. 2, 9 (2001).</li><li>Lawrence, M. B., McIntire, L. V., Eskin, S. G. <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+flow+on+polymorphonuclear+leukocyte/endothelial+cell+adhesion.">Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion.</a> Blood. 70, 1284-1290 (1987).</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. (10), e1644 (2009).</li><li>Furie, B., Furie, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+platelet+P-selectin+and+microparticle+PSGL-1+in+thrombus+formation.">Role of platelet P-selectin and microparticle PSGL-1 in thrombus formation.</a> Trends Mol. Med. 10, 171-178 (2004).</li><li>Tchernychev, B., Furie, B., Furie, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritoneal+macrophages+express+both+P-selectin+and+PSGL-1.">Peritoneal macrophages express both P-selectin and PSGL-1.</a> J. Cell Biol. 163, 1145-1155 (2003).</li><li>Conant, C. G., 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=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>Larsen, G. R., 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=P-selectin+and+E-selectin.+Distinct+but+overlapping+leukocyte+ligand+specificities.">P-selectin and E-selectin. Distinct but overlapping leukocyte ligand specificities.</a> J. Biol. Chem. 267, 11104-11110 (1992).</li><li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectin+ligands:+will+the+real+ones+please+stand+up.">Selectin ligands: will the real ones please stand up.</a> J. Clin. Invest. 100, S31-S35 (1997).</li><li>Bohnsack, J. F., Chang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+beta+1+integrin+fibronectin+receptors+on+HL60+cells+after+granulocytic+differentiation.">Activation of beta 1 integrin fibronectin receptors on HL60 cells after granulocytic differentiation.</a> Blood. 83, 543-552 (1994).</li><li>Lawrence, M. B., Kansas, G. S., Kunkel, E. J., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold+levels+of+fluid+shear+promote+leukocyte+adhesion+through+selectins+(CD62L,P,E).">Threshold levels of fluid shear promote leukocyte adhesion through selectins (CD62L,P,E).</a> J. Cell Biol. 136, 717-727 (1997).</li><li>Moore, K. L., 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=P-selectin+glycoprotein+ligand-1+mediates+rolling+of+human+neutrophils+on+P-selectin.">P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin.</a> J. Cell Biol. 128, 661-671 (1995).</li><li>Lawrence, M. B., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+roll+on+E-selectin.">Neutrophils roll on E-selectin.</a> J Immunol. 151, 6338-6346 (1993).</li><li>Yao, L., 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=Divergent+inducible+expression+of+P-selectin+and+E-selectin+in+mice+and+primates.">Divergent inducible expression of P-selectin and E-selectin in mice and primates.</a> Blood. 94, 3820-3828 (1999).</li><li>Sackstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoengineering+of+HCELL,+the+human+bone+marrow+homing+receptor:+sweetly+programming+cell+migration.">Glycoengineering of HCELL, the human bone marrow homing receptor: sweetly programming cell migration.</a> Ann. Biomed. Eng. 40, 766-776 (2012).</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. , e1009 (2009).</li><li>Muller, W. A., Luscinskas, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+of+transendothelial+migration+in+vitro.">Assays of transendothelial migration in vitro.</a> Methods Enzymol. 443, 155-176 (2008).</li><li>Bakker, D. P., vander Plaats, A., Verkerke, G. J., Busscher, H. J., vander 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>Benoit, M. R., Conant, C. G., Ionescu-Zanetti, C., Schwartz, M., Matin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+device+for+high-throughput+viability+screening+of+flow+biofilms.">New device for high-throughput viability screening of flow biofilms.</a> Appl. Environ. Microbiol. 76, 4136-4142 (2010).</li><li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectin+ligands.">Selectin ligands.</a> Proc. Natl. Acad. Sci. U.S.A. 91, 7390-7397 (1994).</li><li>Ramos, C. L., 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=Direct+demonstration+of+P-selectin-+and+VCAM-1-dependent+mononuclear+cell+rolling+in+early+atherosclerotic+lesions+of+apolipoprotein+E-deficient+mice.">Direct demonstration of P-selectin- and VCAM-1-dependent mononuclear cell rolling in early atherosclerotic lesions of apolipoprotein E-deficient mice.</a> Circ. Res. 84, 1237-1244 (1999).</li><li>Yago, T., 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=Core+1-derived+O-glycans+are+essential+E-selectin+ligands+on+neutrophils.">Core 1-derived O-glycans are essential E-selectin ligands on neutrophils.</a> Proc. Natl. Acad. Sci. U.S.A. 107, (2010).</li><li>Yago, T., 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=E-selectin+engages+PSGL-1+and+CD44+through+a+common+signaling+pathway+to+induce+integrin+alphaLbeta2-mediated+slow+leukocyte+rolling.">E-selectin engages PSGL-1 and CD44 through a common signaling pathway to induce integrin alphaLbeta2-mediated slow leukocyte rolling.</a> Blood. 116, 485-494 (2010).</li><li>Simone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+rolling+and+adhesion+on+surfaces+in+shear+flow.+A+model+for+an+antibody-based+microfluidic+screening+system.">Cell rolling and adhesion on surfaces in shear flow. A model for an antibody-based microfluidic screening system.</a> Microelectronic Eng. 98, 668-671 (2012).</li><li>Perozziello, G., 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=Microfluidic+devices+modulate+tumor+cell+line+susceptibility+to+NK+cell+recognition.">Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition.</a> Small. 8, 2886-2894 (2012).</li><li>Perozziello, G., 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=Microfluidic+biofunctionalisation+protocols+to+form+multivalent+interactions+for+cell+rolling+and+phenotype+modification+investigations.">Microfluidic biofunctionalisation protocols to form multivalent interactions for cell rolling and phenotype modification investigations.</a> Electrophoresis. , (2013).</li><li>Simone, G., 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=A+facile+in+situ+microfluidic+method+for+creating+multivalent+surfaces:+toward+functional+glycomics.">A facile in situ microfluidic method for creating multivalent surfaces: toward functional glycomics.</a> Lab Chip. 12, 1500-1507 (2012).</li><li>Sarkar, D., 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=Engineered+cell+homing.">Engineered cell homing.</a> Blood. 118, e184-e191 (2011).</li><li>Cheng, Z., 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=Targeted+Migration+of+Mesenchymal+Stem+Cells+Modified+With+CXCR4+Gene+to+Infarcted+Myocardium+Improves+Cardiac+Performance.">Targeted Migration of Mesenchymal Stem Cells Modified With CXCR4 Gene to Infarcted Myocardium Improves Cardiac Performance.</a> Mol. Ther. 16, 571-579 (2008).</li><li>Enoki, C., 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=Enhanced+mesenchymal+cell+engraftment+by+IGF-1+improves+left+ventricular+function+in+rats+undergoing+myocardial+infarction.">Enhanced mesenchymal cell engraftment by IGF-1 improves left ventricular function in rats undergoing myocardial infarction.</a> Int. J. Cardiol. 138, 9-18 (2010).</li></ol>

作者宣称没有利益冲突。

其中外源性细胞为基础的治疗成功翻译的主要挑战是无法有效地提供细胞损伤和炎症，高效率的植入3的站点。细胞轧制代表在细胞归巢的过程中的一个关键步骤，促进血管壁上的细胞的减速，通过血管内皮细胞进入组织5最终导致其牢固粘附和轮回。轧制过程中对候选小区类型的更好的理解可能导致的技术的开发，提高细胞的归巢和朝着改善的基于细胞的疗法显著贡献。 <p class...

一种在基于细胞的疗法的成功临床翻译的主要挑战是低效的交货或全身性输注的细胞靶向至期望的位点3,4。因此，人们不断寻求方法来改善细胞的归巢，并且特别是信元轧制，作为一个策略，以改善细胞疗法。细胞滚动血管在细胞归巢级联，经典的定义被招募到发病部位5白细胞的关键一步。这一步是由内皮细胞选择素之间的特异性相互作用制约， 即 P-和E-选择素（P-和E-SEL），和他们的白细胞5,6表面上柜台的配体。更好地了解和细胞归巢的提高效率，特别是轧制工序，是在寻求新的平台，以提高细胞为基础的治疗具有重要意义。迄今这​​已经通过使用平行平板流动腔（PPFCs），包括两个平板型实现ES与它们之间的垫圈，与位于上板的流入和流出端口，通过该细胞悬浮液是通过使用注射器泵7,8，9灌注。底板的表面可以涂覆有一个相关的细胞单层/基板和下剪切流灌注细胞和表面之间的相互作用，然后探讨7。然而，PPFC是低吞吐量，试剂消耗，以及相当繁琐的方法，用泡沫形成，泄漏，控制不佳的流量呈现主要缺点。 一种替代技术对传统PPFC是一个多穴板的微流体系统中，根据精确的，计算机控制的剪切流动，允许细胞分析的更高的吞吐量性能（比PPFCs高10倍），具有低试剂消耗1,10。细胞轧制实验内的微流体通道中进行的，它可以涂覆有细胞单层或工程衬底和成像USI吴显微镜，采用滚动属性，使用适当的软件易于分析。在本研究中，我们证明此多孔板的微流体系统的通过研究人早幼粒白血病在不同的表面（HL-60）细胞的滚动性能的能力。 HL-60轧像P-和E-SEL，以及对表达不同的轧制受体细胞单层，分析底物。此外，抗体（抗体）阻断被用于证明特定选择素介导的那些表面上的HL-60的滚动运动的直接参与。轧制实验，以增加吞吐量进行的，在稳定的剪切流动，以最少的试剂/细胞消耗，从而允许键滚动参数，如轧制速度，轧细胞的数目，并且滚道特性有效的分析。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cells</td>
		</tr>
		<tr>
			<td>Human Lung Microvascular Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2527</td>
			<td></td>
		</tr>
		<tr>
			<td>P-selectin-expressing Chinese Hamster Ovary Cells (CHO-P)</td>
			<td>Kind gift by Dr. Barbara Furie11,12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HL-60 Cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Cell Culture Reagents</td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 Media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Basal Medium Supplements</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 MV SingleQuots</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM - Iscove&#39;s Modified Dulbecco&#39;s Medium 1x</td>
			<td>Gibco</td>
			<td>12440</td>
			<td></td>
		</tr>
		<tr>
			<td>F-12 (1x) Nutrient Mixture (Ham)</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (P/S)</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (L/G) 200 mM</td>
			<td>Gibco</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>Sa550</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>BD Falcon</td>
			<td>BD-353003</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Cell Culture Dish, Tissue-Culture Treated Polystyrene</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (15 ml polypropylene conical tubes)</td>
			<td>MedSupply Partners</td>
			<td>TC1500</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>BD Falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin Solution (2%)</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>dPBS (without calcium chloride and magnesium chloride)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA Solution (10x)</td>
			<td>Sigma</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Antibodies</td>
		</tr>
		<tr>
			<td>Anti-hE-Selectin/CD62E</td>
			<td>R&#38;D Systems</td>
			<td>BBA21</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated Mouse IgG1</td>
			<td>R&#38;D Systems</td>
			<td>BBA21</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-hP-Selectin</td>
			<td>R&#38;D Systems</td>
			<td>BBA34</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated Mouse IgG1</td>
			<td>R&#38;D Systems</td>
			<td>BBA34</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG&#173;1 &#954; Isotype Control</td>
			<td>BD Bioscience</td>
			<td>555748</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SLeX /CD15s Ab, Clone: 5F18</td>
			<td>Santa Cruz</td>
			<td>SC70545</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Conjugated</td>
			<td>Santa Cruz</td>
			<td>SC70545</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse IgM-FITC Isotype Control</td>
			<td>Santa Cruz</td>
			<td>SC2859</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD162, Clone: KPL-1</td>
			<td>BD Pharmingen</td>
			<td>556055</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse IgG1 k Isotype Control</td>
			<td>BD Pharmingen</td>
			<td>550617</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-P-Selectin Ab (AK4)</td>
			<td>Santa Cruz</td>
			<td>SC19996</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-E-Selectin Ab, Clone P2H3</td>
			<td>Millipore</td>
			<td>MAB2150</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 Isotype Control</td>
			<td>Santa Cruz</td>
			<td>SC3877</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Other Reagents</td>
		</tr>
		<tr>
			<td>Recombinant Human TNF-alpha</td>
			<td>PeproTech</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Trace CFSE Cell Proliferation Kit - For Flow Cytometry</td>
			<td>Invitrogen</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>Human P-selectin-FC recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>137-PS-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Human E-selectin-FC recombinant protein</td>
			<td>R&#38;D Systems</td>
			<td>724-ES-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Human, Plasma</td>
			<td>Invitrogen</td>
			<td>33016-015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Bioflux 1000</td>
			<td>Fluxion Biosciences</td>
			<td>Bioflux Montage was the software used to run the experiments and analyze the data</td>
			<td></td>
		</tr>
		<tr>
			<td>BioFlux 48-well plates</td>
			<td>Fluxion Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 Flow Cytometer</td>
			<td>BD Bioscience</td>
			<td>CFlow Plus was the software used to run the experiments and analyze the data</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti-S</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSnap HQ2 CCD camera</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

systematic analysis of in vitro cell rolling using a multi-well plate microfluidic system

细胞为基础的治疗的一个主要挑战是无法系统地针对大量活菌，高效率的兴趣组织下静脉或动脉内注射。因此，增加细胞归巢目前正在研究作为一项战略，以改善细胞疗法。细胞滚动对血管内皮是在细胞归巢的过程中的重要一步，可以使用平行板流室（PPFC） 在体外进行探测。然而，这是一个非常繁琐的，低通量的检测，控制不良的流动条件。相反，我们使用了一个多穴板的微流体系统，可以使蜂窝轧制性能研究在更高的吞吐量下精确控制的，生理学相关的剪切流动1,2。在本文中，我们表明HL-60（人早幼粒细胞白血病）上的P-和E-选择蛋白包被的表面，以及对细胞单层涂层表面细胞的轧制性能如何可以readilŸ检查。以更好地模拟炎性病症，所述微流体通道的表面上涂布的内皮细胞（EC），然后将其与肿瘤坏死因子-α（TNF-α）激活时，在动态条件下显著增加与HL-60细胞的相互作用。增强的吞吐量和集成的多参数分析的软件平台，允许的参数，如轧制速度和轧路径的快速分析，对评估体外细胞滚动属性重要的优点。使工程设计方法来影响细胞的滚动和归巢的快速，准确的分析，这个平台可以帮助推进外源性细胞为基础的治疗。

HL-60细胞滚上的P-和E-选择素面，但不能在纤维连接蛋白 HL-60细胞被认为是金标准“辊”，因为它们表达多种归巢的配体，包括轧制配体的P-SEL糖蛋白配体-1（PSGL-1）和唾液酸-路易斯X（SLEX）的5,14（ 图1A ）。表面蛋白PSGL-1作为一个支架的四糖SLEX，介导特异性相互作用与P-和E-SEL，这是炎症5,6,15中上调的内皮。为了测试多穴板的微流体系统的功能，许多...

Watch this Scientific Journal Video about Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System at JoVE.com

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

本研究采用多孔板微流体系统，根据有关生理剪切流显著增加通量细胞滚动研究。定小区轧制的多步骤细胞归巢级联和细胞归巢的下列全身递送的细胞的患者的外源性群体的重要性的重要性，该系统提供了潜在的作为筛选平台，以提高基于细胞的疗法。

的系统分析<em&gt;体外</em&gt;细胞滚动使用多孔板微流控系统

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

systematic-analysis-vitro-cell-rolling-using-multi-well-plate

Research

JoVE Journal

Bioengineering

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

11.9K 次观看.  Brigham and Women's Hospital. 本研究采用多孔板微流体系统，根据有关生理剪切流显著增加通量细胞滚动研究。定小区轧制的多步骤细胞归巢级联和细胞归巢的下列全身递送的细胞的患者的外源性群体的重要性的重要性，该系统提供了潜在的作为筛选平台，以提高基于细胞的疗法。 

视频: Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

不对称受体模式研究细胞滚动轨迹

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

多井板膜插入系统中3D 组织模型渗透扩散的测定与模拟方法

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

用 Widefield 高含量分析系统实现荧光标记组织的自动滑动扫描和分割

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

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 ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

利用自动高通量微流控系统对铜绿假单胞菌剪切应力的活细胞分析

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

使用微流体芯片以确定性方式建立基于单细胞的共培养物

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...