The authors have no acknowledgements.

该协议遵循了人类样本的研究机构道德准则和所涉及的所有供体获得知情同意书。从安特卫普大学医院的机构审查委员会获得批准，这里所描述的实验。 注:温度指示总是室温下，除非另有说明。 1.准备流室设置<ol><li>准备车道，油管和引脚<ol><li>涡流的胶原悬浮液剧烈和在由提供者提供到50微克/ ml的终浓度，等渗葡萄糖溶液稀释1/20。 注意:我们使用马肌腱胶原蛋白，主要由I型纤维的。该马I型胶原常被称为"禾木"胶原和为这种类型的测定法9的两个历史以及生物原因金标准。第三人型胶原也可使用，但第Ë蛋白原纤维外套较差，血小板反应不强。其它涂覆的表面也可以使用，例如血管性血友病因子（vWF），纤维蛋白原，纤连蛋白，层粘连蛋白，玻连蛋白，血小板反应蛋白-1或这些16的组合。 </li><li>以从供应商的容器新的一次性生物芯片。这里使用的生物芯片的尺寸单位为mm 3 0.4W点¯x0.1H点¯x20L。 </li><li>吸管0.8微升到微流体生物芯片上的芯片和标记为出口的一端车道（多个）。确保车道填充5/6用含有1.1.1制备涂覆溶液中的胶原蛋白。确保有没有气泡。 注意:通道被部分地涂覆，以避免胶原纤维堆积在通道的入口（见讨论）。 </li><li>孵育在4℃下，在湿润的和封闭的容器4小时或过夜。 </li><li>吹打封闭缓冲液（1.0％（重量/体积）牛血清白蛋白的方框涂覆频道ð0.1％（重量/体积）葡萄糖在10mM 4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）缓冲的盐水（HBS; 0.9％（重量/体积）氯化钠，pH值7.4）在其另一端与标记作为入口。确保该车道被完全填充有封闭缓冲避免气泡。 </li><li>在等长（12厘米）的切割管道。使用每通道之一，各管一个引脚连接。例如，一个实验比较两种条件下，一式两份运行将需要四个管道伸展和四个引脚来制备。 </li><li>冲洗使用注射器和一＆G针或所附连接用蒸馏水油管。 </li><li>饱和用封闭缓冲液的管。商店中最少1小时的封闭和加湿容器。 </li></ol></li><li>准备泵和阀组<ol><li>冲洗泵和用蒸馏水歧管，以消除气泡。 </li><li>吸出封闭缓冲液出使用在出口处的固定的10微升末端生物芯片车道（多个）。清洁的表面用精密无尘擦拭酒精变性生物芯片去除印刷品和灰尘。 </li><li>修正了在自动化显微镜阶段的生物芯片。如果多于一个车道在一个运行同时使用的，八车道歧管分离器连接至生物芯片的插座。 注意:八车道歧管分离器是一个硬件（ 图S1）连接到所述泵和生物芯片。它允许在生物芯片或车道可以是运营商定义在所附的软件的组合的所有可用（8）泳道的操作。 </li><li>使用管道针修复他们在生物芯片的入口。将管接头（没有针）的1.5毫升的锥形试管填充用HBS的另一端。冲洗所有管道和它们与使用泵1毫升HBS连接通道，以便除去剩余封闭缓冲液和不良粘附胶原蛋白。 </li></ol></li></ol> 2.血液样品的制备<ol><li>收集和分离新鲜全血从健康志愿者。17 <ol><li>收集的血液的第一毫升在含有乙二胺四乙酸（EDTA）作为抗凝剂真空管和只用自动血液分析仪使用该样本为全血计数（CBC）。 </li><li>收集的血液在使用真空管合适的抗凝剂的体积。对于流动腔实验标准抗凝剂是肝素或水蛭素时血纤维蛋白形成不是研究方案和柠檬酸钠时，它是的一部分。 注意:肝素用作在结果中所描述的所有实验中的抗凝血剂。 注意:血液的量取决于实验次数被执行。大约1管（7ml）中为3条车道。 </li><li>放置试管旋转器上挂起的血液重建。 注意:该测定应放血的3小时内完成。 </li><li>在250G离心15分钟，以制备富血小板血浆（PRP）。不要使用离心机休息，防止松散填充颗粒的干扰。 <ol><li>当收集多个管子，池血在一个锥形离心管中。 注意:离心可以做更慢或长或短，取决于PRP产量和差动单元"污染"优选的。 </li></ol></li><li>卸下并丢弃PRP和白膜层产生浓缩红细胞少数血小板。 注:在聚集的红细胞部分的血小板计数为13±5×10 3％微升（平均值±SD，N = 12），平均在我们手中。 </li></ol></li><li>重建血<ol><li>在37℃下解冻AB血型（恒河猴D阴性）等离子体进行5分钟，每4毫升20秒。 </li><li>确定使用自动血液分析仪在2.1.5中制备的浓缩红细胞的血细胞比容。 </li><li>确定血库的血小板浓度来制备浓缩血小板塔T将用于重建上述红细胞馏分。 </li><li>计算聚集的红血细胞和血小板浓缩，将产生40％的血细胞比容和250×10 3血小板/微升1ml样品中的体积。 注:细胞的其它靶滴度可任意设定，取决于研究协议。 </li><li>转移使用裁剪吸管尖和添加血小板浓缩直至达到1毫升的样品体积堆积的红血细胞和血浆入新鲜试管。 注意:根据所研究的变量，将血浆部分应该是所有冲调样品在等于因为等离子体具有对血栓形成速率的显著影响。例如，反复从不同供体或不同的抗凝血剂作出可能影响结果冻融或血浆。 </li><li>轻轻颠倒混合重组血液和执行CBC。 </li><li>制备其中血小板馏分的体积置换的"空白"对照样品用相同体积的0.9％（M / V）氯化钠的水，以确定内源性血小板中使用CBC重构血液中的浓度（ 即非血库血小板）。 </li></ol></li><li>贴标<ol><li>移液管1毫升含有1微升的5mM钙黄绿素AM（5μM的最终浓度）的试管中重构血液。 注:其他细胞的染料可以使用14。 </li><li>通过颠倒轻轻混匀。 </li><li>孵育在37℃，使用前5分钟。 </li></ol></li></ol> 3.灌注试验<ol><li>专注于车道的底部附着在胶原纤维的目标。理想情况下，用于此聚焦战略相衬或微分干涉对比（DIC）的设置。在实验软件选择"选定的小片区域的设置当前Z&#39;以数字修正所选Z-位置。 </li><li>在的米实验软件选择的车道（XY）感兴趣区域（ROI）的区域icroscope将在实验过程中被记录。 注:投资回报率可以是灌注车道内随意选择任何表面区域。最好是不进行分析血栓形成接近一个车道的入口和出口，以便避免在该区域中的可变流动剖面的副作用，即使这是比较小的。在ROI表面积应包含血小板的显著数或血栓，以允许信号的流平性。在这个协议中的投资回报率是得到0.62 平方毫米的2厘米长车道中间的三个相等大小的侧面并排图像的数字缝合集合体。 </li><li>轻轻颠倒混合样品并在自动阶段这些旁生物芯片的位置。 </li><li>放置与该生物芯片在含有重组血样试管入口连接的管路。 </li><li>启动用于链接到试管那些信道（根据需要或其它剪切应力），在50达因/厘米2的泵含有用泵的软件重构血样。 注:其他的剪切应力可以被使用。 </li><li>记录的图像，每15秒，用显微镜的采集和实验软件实时5分钟。 注:其他的时间序列可以根据实验设置使用。 注意:我们一般使用放大100倍（10X物镜和10X透镜），但更高（或更低）放大率可以方便地用来作为替代。 </li></ol> 4.洗出<ol><li>洗出所有管路连接于出口，并使用蒸馏水连接到多信道歧管或泵，接着次氯酸钠（漂白剂）0.5％（体积/体积），最后的0.1M NaOH水溶液。放弃固定在生物芯片的入口作为危险废物管道。 </li></ol> 5.数据分析<ol><li>确定与该图像分析软件血栓生长动力学。下列命令专用于ZEN2012。 <ol&gt; <li>打开插件图像分析来确定血小板的表面覆盖。 </li><li>坐落在分析交互式标签来定义一个积极的信号相关 ​​的像素亮度， 即在坚持血小板或血小板粘附荧光阈值。 </li><li>使用创建的表自动生成的电子表格将包含那些"对象"包含的像素与所选择的阈值之间的信号的单独的表面积（以μm2）。这是对每个时间点进行。 注意:一旦荧光阈值已选择，分析软件会自动检测视野&#39;对象&#39;是符合标准。这些对象是血栓，小血小板聚集体或单个血小板并覆盖像素的数量。每个对象在电子表格中单独列出。 </li><li>以XML格式保存这些电子表格和电子表格程序打开它们进一步计算。 </li><li>由求和所选对象的总的表面积和由测量字段（微米2）的总面积除以结果。这将产生的相对表面覆盖率（％）。这样做的每一个时间点。 </li><li>在灌注时间函数图，这些表面覆盖范围，并通过线性回归计算斜率，产生特定条件血栓生长动力学。 </li></ol></li></ol>

<ol>
	<li>Broos, K., Feys, H. B., De Meyer, S. F., Vanhoorelbeke, K., Deckmyn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+at+work+in+primary+hemostasis.">Platelets at work in primary hemostasis.</a> Blood Rev. 25, 155-167 (2011).</li><li>Stalker, T. J., 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=Hierarchical+organization+in+the+hemostatic+response+and+its+relationship+to+the+platelet-signaling+network.">Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network.</a> Blood. 121, 1875-1885 (2013).</li><li>Sakariassen, K. S., Bolhuis, P. A., Sixma, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+blood+platelet+adhesion+to+artery+subendothelium+is+mediated+by+factor+VIII-Von+Willebrand+factor+bound+to+the+subendothelium.">Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-Von Willebrand factor bound to the subendothelium.</a> Nature. 279, 636-638 (1979).</li><li>Savage, B., Saldivar, E., Ruggeri, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+platelet+adhesion+by+arrest+onto+fibrinogen+or+translocation+on+von+Willebrand+factor.">Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor.</a> Cell. 84, 289-297 (1996).</li><li>Westein, E., de Witt, S., Lamers, M., Cosemans, J. M., Heemskerk, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+in+vitro+thrombus+formation+with+novel+microfluidic+devices.">Monitoring in vitro thrombus formation with novel microfluidic devices.</a> Platelets. 23, 501-509 (2012).</li><li>Varga-Szabo, 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=The+calcium+sensor+STIM1+is+an+essential+mediator+of+arterial+thrombosis+and+ischemic+brain+infarction.">The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction.</a> J Exp Med. 205, 1583-1591 (2008).</li><li>Westein, E., 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=Atherosclerotic+geometries+exacerbate+pathological+thrombus+formation+poststenosis+in+a+von+Willebrand+factor-dependent+manner.">Atherosclerotic geometries exacerbate pathological thrombus formation poststenosis in a von Willebrand factor-dependent manner.</a> Proc Natl Acad Sci USA. , (2013).</li><li>Grabowski, E. F., Yam, K., Gerace, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+hemostasis+in+flowing+blood.">Evaluation of hemostasis in flowing blood.</a> Am J Hematol. 87 (Suppl 1), S51-S55 (2012).</li><li>Roest, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+chamber-based+assays+to+measure+thrombus+formation+in+vitro:+requirements+for+standardization.">Flow chamber-based assays to measure thrombus formation in vitro: requirements for standardization.</a> J Thromb Haemost. 9, 2322-2324 (2011).</li><li>Heemskerk, J. W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+surfaces+to+measure+thrombus+formation+under+flow:+possibilities+for+standardization.">Collagen surfaces to measure thrombus formation under flow: possibilities for standardization.</a> J Thromb Haemost. 9, 856-858 (2011).</li><li>Shrivastava, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+platelet+storage+lesion.">The platelet storage lesion.</a> Transfus Apher Sci. 41, 105-113 (2009).</li><li>Miyaji, 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=Decreased+platelet+aggregation+of+platelet+concentrate+during+storage+recovers+in+the+body+after+transfusion.">Decreased platelet aggregation of platelet concentrate during storage recovers in the body after transfusion.</a> Transfusion. 44, 891-899 (2004).</li><li>Goodrich, R. P., 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=Correlation+of+in+vitro+platelet+quality+measurements+with+in+vivo+platelet+viability+in+human+subjects.">Correlation of in vitro platelet quality measurements with in vivo platelet viability in human subjects.</a> Vox Sang. 90, 279-285 (2006).</li><li>Van Aelst, B., 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=Riboflavin+and+amotosalen+photochemical+treatments+of+platelet+concentrates+reduce+thrombus+formation+kinetics+in+vitro.">Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro.</a> Vox Sang. 108, 328-339 (2015).</li><li>Van Aelst, B., Devloo, R., Vandekerckhove, P., Compernolle, V., Feys, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultraviolet+c+light+pathogen+inactivation+treatment+of+platelet+concentrates+preserves+integrin+activation+but+affects+thrombus+formation+kinetics+on+collagen+in+vitro.">Ultraviolet c light pathogen inactivation treatment of platelet concentrates preserves integrin activation but affects thrombus formation kinetics on collagen in vitro.</a> Transfusion. , (2015).</li><li>de Witt, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+platelet+function+defects+by+multi-parameter+assessment+of+thrombus+formation.">Identification of platelet function defects by multi-parameter assessment of thrombus formation.</a> Nat Commun. 5, 4257 (2014).</li><li>Cazenave, J. P., 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=Preparation+of+washed+platelet+suspensions+from+human+and+rodent+blood.">Preparation of washed platelet suspensions from human and rodent blood.</a> Methods Mol Biol. 272, 13-28 (2004).</li><li>Jackson, S. P., Nesbitt, W. S., Westein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+platelet+thrombus+formation.">Dynamics of platelet thrombus formation.</a> J Thromb Haemost. 7 (Suppl 1), 17-20 (2009).</li><li>Diedrich, B., Remberger, M., Shanwell, A., Svahn, B. M., Ringden, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+randomized+trial+of+a+prophylactic+platelet+transfusion+trigger+of+10+x+10(9)+per+L+versus+30+x+10(9)+per+L+in+allogeneic+hematopoietic+progenitor+cell+transplant+recipients.">A prospective randomized trial of a prophylactic platelet transfusion trigger of 10 x 10(9) per L versus 30 x 10(9) per L in allogeneic hematopoietic progenitor cell transplant recipients.</a> Transfusion. 45, 1064-1072 (2005).</li><li>Neeves, K. B., 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=Sources+of+variability+in+platelet+accumulation+on+type+1+fibrillar+collagen+in+microfluidic+flow+assays.">Sources of variability in platelet accumulation on type 1 fibrillar collagen in microfluidic flow assays.</a> PloS one . 8, e54680 (2013).</li><li>Born, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+of+blood+platelets+by+adenosine+diphosphate+and+its+reversal.">Aggregation of blood platelets by adenosine diphosphate and its reversal.</a> Nature. 194, 927-929 (1962).</li><li>Cattaneo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+Standardization+of+Light+Transmission+Aggregometry:+A+Consensus+of+the+Working+Party+from+the+Platelet+Physiology+Subcommittee+of+SSC/ISTH.">Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.</a> J Thromb Haemost. 11, 1183-1189 (2013).</li><li>Deckmyn, H., Feys, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+for+quality+control+of+platelets+for+transfusion.">Assays for quality control of platelets for transfusion.</a> ISBT Sci Series. 8, 221-224 (2013).</li><li>Andre, P., 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=Anticoagulants+(thrombin+inhibitors)+and+aspirin+synergize+with+P2Y12+receptor+antagonism+in+thrombosis.">Anticoagulants (thrombin inhibitors) and aspirin synergize with P2Y12 receptor antagonism in thrombosis.</a> Circulation. 108, 2697-2703 (2003).</li><li>Casari, 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=von+Willebrand+factor+mutation+promotes+thrombocytopathy+by+inhibiting+integrin+alphaIIbbeta3.">von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin alphaIIbbeta3.</a> J Clin Invest. 123, 5071-5081 (2013).</li><li>Gitz, E., 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=Improved+platelet+survival+after+cold+storage+by+prevention+of+Glycoprotein+Ib&#945;+clustering+in+lipid+rafts.">Improved platelet survival after cold storage by prevention of Glycoprotein Ib&#945; clustering in lipid rafts.</a> Haematologica. , (2012).</li><li>Maurer-Spurej, E., Labrie, A., Brown, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+Quality+Testing+of+Blood+Platelet+Transfusions+with+Dynamic+Light+Scattering.">Routine Quality Testing of Blood Platelet Transfusions with Dynamic Light Scattering.</a> Part Part Syst Char. 25, 99-104 (2008).</li><li>Van Kruchten, R., Cosemans, J. M., Heemskerk, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+whole+blood+thrombus+formation+using+parallel-plate+flow+chambers+-+a+practical+guide.">Measurement of whole blood thrombus formation using parallel-plate flow chambers - a practical guide.</a> Platelets. 23, 229-242 (2012).</li><li>Zwaginga, J. J., 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=Flow-based+assays+for+global+assessment+of+hemostasis.+Part+2:+current+methods+and+considerations+for+the+future.">Flow-based assays for global assessment of hemostasis. Part 2: current methods and considerations for the future.</a> J Thromb Haemost. 4, 2716-2717 (2006).</li><li>Zwaginga, J. J., 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=Flow-based+assays+for+global+assessment+of+hemostasis.+Part+1:+Biorheologic+considerations.">Flow-based assays for global assessment of hemostasis. Part 1: Biorheologic considerations.</a> J Thromb Haemost. 4, 2486-2487 (2006).</li></ol>

The authors have nothing to disclose.

微流体流室的实验是研究在流动的血液中血小板的功能一个很好的工具，用于在不同的实验环境，以评估在体外止血。尽管可怜的实验室间标准化9，我们证明了我们的实验室内的实验误差是可以接受的。这允许一个给定的研究中比较可靠（配对）的样品。这是使用血小板存储病变，这是血小板保存在血库条件11的不利后果的有案可稽现象验证。此外，我们最近公布的三个可用的病原体灭活技术在PC上血小板功能在微流控流动腔血液之后的14,15重建的影响。 血小板有不同的反应时，生物物理和-化学参数变化20。因此，剪切应力，细胞数和细胞composi化，温度，涂层，抗凝血剂和许多其它因素可根据所研究的问题该协议内进行修改。该协议仅使用市售硬件和软件工具，允许其他实验室以进行类似的测定。对基础研究的目的这可能是一个缺点，特别是因为可用的硬件比定制的设置功能较少。本身该法是强大的，但重复性从生物学和时间变化受到影响。因此，测定样品需要尽可能配对并研究尺寸应足够大。样品也需要时间来进行配对，因为重建血液只能存放时间短。 虽然血小板功能的研究微流室都推动了研究领域，应谨慎采取过度解读对血液流变学血小板依赖。第一，流动室的矩形形状没有生理，卜T中最好的，我们必须允许光学聚焦于不断增长的血栓。第二，血管不是由塑料制成，血管弹性的对血小板功能的影响，因此无法在这个协议中进行研究。三，心脏搏动引起流动，而注射泵更线性的（虽然也略有搏动）。最后，纤维状I型胶原蛋白是下气流用于血小板研究的标准材料，这是动物来源的。值得注意的是但是，纤维状I型胶原和血小板功能的临床结果在很多情况下很好的相关性如由几十年（透光）经验凝集21,22。 有许多实验来研究血小板功能23。大多数这些解决一个或几个血小板功能，而把血小板工作，止血（模型）。这里展示了血栓的形成实时成像是最具包容性的，到今天为止。这意味着解放军的大多数方面telet对血管损伤的响应都包括在内。独特的优点是血液流动的包容和所有血细胞的存在。该测定是为抗血小板治疗24以及产生异常血小板功能25基因改变目前使用的药物敏感。这表明它作为血小板功能的相关指标值。该测定的综合性质仍然也意味着它是比那些测定法测定血小板的响应的特定功能的分析更小。因此，通过可以通过流室测定挑上浓缩血小板血库操作的影响，但是解读是什么原因导致他们，则需要额外的分析。例如，我们的数据表明，温度显著影响在微流体流动腔室血小板粘附。但是，更多的详细的分析表明，冷藏血小板改变形状和集群GPIbα启动受体26。 等[16]最近的研究证明基板定义的相关性血小板系统生物学。此外，在基片的功能变化剪切速率的组合是重要的，因为与固定的VWF血小板结合将要求高剪切速率，而这是用于结合到胶原不太重要。因此，根据所研究的问题，选择可以在什么基片制成，它们各自的流速将被包括在内。 总之，我们提出了一个协议，用于微流体流室实验来研究血小板功能血库和输血医学的背景下。标准化工作正在进行9,10,28-30，大部分的这些建议都包含在提交的协议。血液的重建为模型输血，但额外的验证工作应注明相关临床结果。

止血需要的细胞，蛋白质，离子和组织在有限的时空背景下1组合和受规管活动。不受控制的活性可能导致出血或血栓形成和发病率或死亡率有关凝血障碍的光谱。一种微流体流室的实验是一个具有挑战性的技术， 在体外模拟止血。这种方法允许的参与止血与血小板的主导作用过程中的复杂的相互作用的研究。 血管损伤后，血小板粘附到暴露的内皮下基质（糖）的蛋白质，以防止失血。以下粘附，血小板激活和聚集响应于自动和旁分泌信号并最终导致形成血小板网络的，由纤维蛋白稳定化，并导致一公司，伤口密封血栓2。与大多数其他的血小板功能检测，experi与流动室ments顾及血流的物理参数，因此，流变学对参与细胞和生物分子的3,4的影响。 流动腔实验通过改变影响止血（分）关键参数过程，包括粘合剂基质，流变和流动剖面，蜂窝组合物，毒素或药物，离子强度等等的存在产生在止血和血栓形成里程碑式的见解。在过去的二十年中，低通量需要大样本量（10-100毫升）流动腔实验已经发展到微流控室往往由小平行板室，并包括现代技术控制在墙上剪切条件下5灌注全血。 Microscaling已显著增加检测的吞吐量主要是因为硬件设置简化并需要更少的（血）量，使实验更加方便和versati勒。例如，来自小实验动物的血液，现在可以无需牺牲动物使用。因此，转基因小鼠的血样已经在帮助促进或抑制止血的关键分子的鉴定，并在新的基本见解6。 专门研究实验室还经常使用，例如定制流室从聚二甲基硅氧烷（PDMS）7上，可以通过软件blueprinted石印模具聚合。所得室是便宜，一次性和可容易地拆卸为事后分析。此外，基本上船只，包括分叉或急转弯的任何设计可以建立在命令。这种优势也是其缺点，因为标准化已经与流室实验的首要问题，而PDMS定制室都没有帮助这一点。在这个特殊问题的顶部，涂层（条件），荧光探针，抗凝，临时erature和时间采样和分析之间都很差标准化8。这些变量标准化是具有挑战性的，但仍然需要允许实验室间的结果进行比较。本主题是血栓与止血国际社会在对生物流变学9,10科学和标准化分委员会的主要议题。 血小板浓缩物（PC）进行输血在从引起血小板减少和/或出血的各种疾病的患者。但血小板在个人计算机是众所周知的脱敏，特别是在放置时间11功能，劣化进程链接到老化和通常被称为血小板储存损伤。据说有时，这种血小板输血一次发行12恢复，但对于这方面的证据是稀缺的。此外，血小板构成的PC的功能不是常规测试，因为这样的测定之间的关系的治疗或预防效力是13不清楚。微流体流动腔室提供给调查在个人电脑的血小板功能，以优化收集和发行之间操作的链的装置。它是PC，因为我们先前公布的14,15和这里描述的直接（配对）比较强大的研究工具。

<table border="0" cellpadding="0" cellspacing="0" width="1113">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer tube with EDTA&#160;</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368856</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Heparin</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368480</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Sodium Citrate</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">366575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hirudin Blood tube</td>
			<td style="width:220px;">Roche</td>
			<td style="width:268px;">6675751 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer Eclipse</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368650</td>
			<td>Blood collection needle with preattached holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 100-1000</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">740290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 2-200</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">739280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 1-10</td>
			<td style="width:220px;">Eppendorf</td>
			<td style="width:268px;">A08928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tube 5mL</td>
			<td style="width:220px;">Simport</td>
			<td style="width:268px;">11691380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 15mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">1888271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 50mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL Syringe</td>
			<td style="width:220px;">BD</td>
			<td style="width:268px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precision wipes</td>
			<td style="width:220px;">Kimtech</td>
			<td style="width:268px;">5511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Fluoro+ Biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">188V8CF-400-100-02P10</td>
			<td>Named in figure S1 A as &#39;Biochip&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Tubing</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">TUBING-TYGON-B1IC-B1OC-ROLL 100F</td>
			<td>Named in figure S1 B as &#39;Disposable tubing&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Needles</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">SS-P-B1IC-B1OC-PACK200</td>
			<td>Named in figure S1 B as &#39;Pin&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Connectors for single inlet cables of biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">CONNECTORS-B1IC-PACK100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multiflow8 connect</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">MF8-CONNECT-BIC3-N-THROMBOSIS</td>
			<td>Named in figure S1 B as &#39;Reusable tubing&#39; and &#39;Splitter&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Humidified box</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">HUMID-BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microfluidic pump</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">N/A</td>
			<td>Venaflux Assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Horm Collagen</td>
			<td style="width:220px;">Takeda/Nycomed</td>
			<td style="width:268px;">1130630</td>
			<td style="width:409px;">Native equine tendon collagen (type I) 
			Isotonic glucose solution to dilute collagen is supplemented</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEPES buffered saline (HBS)</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blocking buffer</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcein AM</td>
			<td style="width:220px;">Molecular probes</td>
			<td style="width:268px;">C1430</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach 10%</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.1M NaOH</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Denaturated alcohol</td>
			<td style="width:220px;">Fiers</td>
			<td style="width:268px;">T0011.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mirus Evo Nanopump</td>
			<td>Cellix</td>
			<td>188-MIRUS-PUMP-EVO</td>
			<td>with Multiflow8. Named in figure S1 A as &#39;Pump&#39; and &#39;Manifold&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer Z1</td>
			<td>equipped with a colibri-LED and high resolution CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:268px;">N/A</td>
			<td>ZEN 2012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematology analyzer</td>
			<td>Sysmex</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge</td>
			<td>Eppendorf</td>
			<td>521-0095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Platelet incubater</td>
			<td style="width:220px;">Helmer</td>
			<td style="width:268px;">PF-48i</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubation water bath</td>
			<td>GFL</td>
			<td>1013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette</td>
			<td>Brand</td>
			<td>A03429</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube Roller</td>
			<td>Ratek</td>
			<td>BTR5-12V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile docking device</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">TSCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing Sealer</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">AC-155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:220px;">VWR</td>
			<td style="width:268px;">58816-121</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic flow chambers using reconstituted blood to model hemostasis and platelet transfusion in vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

为了证明批内变异，三个相同的重构的全血样品，在胶原涂覆的表面（ 图1）同时灌注。这导致了8.7％的变异系数。这一统计数字表明，接受内部试验和实验室内的变化允许相关样本之间可靠的比较。 商业流室的我们在这里描述的入口垂直于测量室，这可引起轻微的湍流，而不是在该点的层流。特别是在无抗凝实验这可能会导致由于血液和固定激动剂之间的增加的接触时间的堵塞。堵塞的入口可以混淆在腔室下游的读出。因此，安装用部分涂覆流动室（ 图2）离开优化入口缺乏血小板激动剂，从而避免主次止血不及时激活。粘合面的部分涂层而且成功地在该领域16被其他研究团体。此外，这种简单实用的技巧是一种资产，研究"过渡"区，在那里流过非反应型（无涂层）表面的血液继续通过反应（涂层）部分。 输血通过用缺陷型分量重构血液模拟。到这个目的，抗凝全血从健康供体通过差速离心和PC血小板呈现血小板中加入以增加血小板计数。在这里，一个重要的问题是什么血小板计数为目标的呢？在大多数情况下，现实生活中的输血不导致在血小板患者血小板计数正常化。而高于某一阈值的目的是为，虽然确切的目标值是不明确的19。为了理解不同于在血液重构的上下文中的微流体流动室成果血小板计数的影响下，几个reconstitutions用减少血小板浓度（ 图3）进行的。正如预期的，低级的血小板计数导致的灌注时间函数少的粘合。因此，一个给定的研究中，血小板计数应规范允许样品条件20之间的比较。起码的事实是有当血小板计数低不然而并不意味着该测定不能用于测量血小板样品中血小板沉积因为显微镜和相机的设置可以适于提高灵敏度粘着较少。最后，在大多数情况下，用于重构的血小板样品含有这类似于在大多数输血苛刻患者19的情况其余自体血小板。这是CURRently不清楚，什么自体血小板的作用是与异体输血的血小板的背景下，但为今后的研究一个有趣的问题。 继健康的自愿献血者的血液采集，全血通常冷却到和之前成分制剂保持在恒定室温。血小板对温度变化敏感然而， 图4示出了采血后和灌注过程中的温度下降的效果。血栓被发现相比于相同的（成对）样品至更慢建立当血液被冷却到室温（ 图4A）保持在37℃，在整个研究中（ 图4B）。 在循环，血小板结合在升高的壁剪切应力条件血管损伤位点。在微流控流动腔Ç不同的剪切速率与VWF oated /纤维蛋白原导致在终点总血小板粘附（ 图5A）的差异和血栓生长动力学（ 图5B）。 最后，以证明流如何微流体腔室可以被用于研究用于输血的PC，在PC存贮的功能血栓生长动力学进行了研究。在样品制备和实验设置所有变量在整个研究期间进行了标准化。因此，唯一的可变参数是电脑储存时间。 图6表明在减少储存时间的函数血栓增长表明血小板储存损伤的止血体外的作用。 <img alt="图1" src="/files/ftp_upload/53823/53823fig1.jpg" /> 图1:在微流体流动腔实验批内变异微流体FLO<html>瓦特室实验在50达因/平方厘米上固定胶原进行。所有三个相同的重构全血样品中平行，并在同一时间进行灌注。结果显示为范围（威威）在灌注时功能的表面覆盖。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53823/53823fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53823/53823fig2.jpg" /> 图2:用相差显微镜观察局部涂通道 （A）胶原纤维被发现仅在涂层区。 （B）在50达因泵送钙黄绿素AM标记的重构的全血样品5分钟灌注后快照/厘米2所示。两个图像是在100X放大倍数下拍摄。<html> ig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53823/53823fig3.jpg" /> 图3:血栓生长动力学依赖于重组血小板计数 （A）血用血库的血小板再造浓缩产生不同的血小板浓度:245×10 3 /μL（●），42×10 3 /μL（■）和12 ×10 3 /μL（▲）。物在50达因/平方厘米的剪切应力同时进行所有三个样品用胶原涂覆的通道的微流体流动室实验。 （B）在A组中描绘的原始数据进行线性回归计算的斜坡。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53823/53823fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 内容】"FO:保together.within页="1"&gt; <img alt="图4" src="/files/ftp_upload/53823/53823fig4.jpg" /> 图4:温度和微流体流动腔室中肝素真空采血管采集的全血保存在室温下，（A）或在37℃（B）的水浴15分钟。在50达因的剪切应力在固定化胶原微流体灌注/进行厘米2。在结束点（5分钟灌注）描绘捕捉镜头。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53823/53823fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53823/53823fig5.jpg" /> 图5:剪切应力对血小板粘附到固定的VWF /纤维蛋白原的作用 （A）中。 四个相同，钙黄绿标有AM，再造全血样品被灌注在一个VWF /纤维蛋白原表面覆盖可变剪切应力:4.5达因/平方厘米（●），50达因/平方厘米（■），90达因/平方厘米（▲）和225达因/平方厘米（♦）。 3分钟灌注后，快照拍摄所有四个通道。在（A）中描述的样本时间的函数（B）表面覆盖范围显示出血栓生长动力学差异。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53823/53823fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53823/53823fig6.jpg" /> 图6:血栓形成血小板中的储存时间函数集中所有微流体流动室实验在胶原涂覆的表面在标准条件下，在50达因/厘米2的剪切速率进行。血液是reconstituted与上一日三（●）重复测试相同的浓缩血小板样本，七（■）和十（▲）后捐赠。在灌注时间的功能被描绘表面覆盖范围。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53823/53823fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="的身影1" src="/files/ftp_upload/53823/53823sfig1.jpg" /> 图S1:微流体流动室硬件设置 （A）腔8氟+生物芯片被安装在显微镜的自动阶段并经由柔性管连接到注射器泵与歧管泵送功能分成八个单独的通道。 （B）再生血液流经右侧一次性管导入生物芯片（入口）的所选择的信道。一次性管通过stainl固定在入口与安装提供ESS钢一次性针。 （B）中的血流量是从右到左，并收集在长的柔性管材。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53823/53823sfig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro at JoVE.com

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

microfluidic-flow-chambers-using-reconstituted-blood-to-model

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

9.3K 次观看.  Belgium Red Cross-Flanders. Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro. 

视频: Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

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

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 ...

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

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 interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

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

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

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 ...

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 ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

在生理体外模型和病理血流应用的血管细胞重塑调查

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

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 ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

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 ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

微流体流钱伯斯再造使用血止血建模和血小板输注

摘要

探索更多视频

此视频中的章节

微流控装置重新创建一个肿瘤微环境