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>

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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...

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><tbody><tr><td>BD vacutainer tube with EDTA&amp;nbsp;</td><td>Becton, Dickinson and Company</td><td>368856</td><td></td></tr><tr><td>BD vacutainer tube with Heparin</td><td>Becton, Dickinson and Company</td><td>368480</td><td></td></tr><tr><td>BD vacutainer tube with Sodium Citrate</td><td>Becton, Dickinson and Company</td><td>366575</td><td></td></tr><tr><td>Hirudin Blood tube</td><td>Roche</td><td>6675751 001</td><td></td></tr><tr><td>BD vacutainer Eclipse</td><td>Becton, Dickinson and Company</td><td>368650</td><td>Blood collection needle with preattached holder</td></tr><tr><td>Pipette tips 100-1,000</td><td>Greiner bio-one</td><td>740290</td><td></td></tr><tr><td>Pipette tips 2-200</td><td>Greiner bio-one</td><td>739280</td><td></td></tr><tr><td>Pipette tips 1-10</td><td>Eppendorf</td><td>A08928</td><td></td></tr><tr><td>Tube 5 ml</td><td>Simport</td><td>11691380</td><td></td></tr><tr><td>Conical tube 15 ml</td><td>Greiner bio-one</td><td>1888271</td><td></td></tr><tr><td>Conical tube 50 ml</td><td>Greiner bio-one</td><td>227261</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>Precision wipes</td><td>Kimtech</td><td>5511</td><td></td></tr><tr><td>Vena8 Fluoro+ Biochips</td><td>Cellix</td><td>188V8CF-400-100-02P10</td><td>Named in &lt;strong&gt;Figure S1&lt;/strong&gt; A as &#039;Biochip&#039;</td></tr><tr><td>Vena8 Tubing</td><td>Cellix</td><td>TUBING-TYGON-B1IC-B1OC-ROLL 100F</td><td>Named in &lt;strong&gt;Figure S1&lt;/strong&gt; B as &#039;Disposable tubing&#039;</td></tr><tr><td>Vena8 Needles</td><td>Cellix</td><td>SS-P-B1IC-B1OC-PACK200</td><td>Named in &lt;strong&gt;Figure S1&lt;/strong&gt; B as &#039;Pin&#039;</td></tr><tr><td>Connectors for single inlet cables of biochips</td><td>Cellix</td><td>CONNECTORS-B1IC-PACK100</td><td></td></tr><tr><td>Multiflow8 connect</td><td>Cellix</td><td>MF8-CONNECT-BIC3-N-THROMBOSIS</td><td>Named in &lt;strong&gt;Figure S1&lt;/strong&gt; B as &#039;Reusable tubing&#039; and &#039;Splitter&#039;</td></tr><tr><td>Humidified box</td><td>Cellix</td><td>HUMID-BOX</td><td></td></tr><tr><td>Software microfluidic pump</td><td>Cellix</td><td>N/A</td><td>Venaflux Assay</td></tr><tr><td>Horm Collagen</td><td>Takeda/Nycomed</td><td>1130630</td><td>Native equine tendon collagen (type I)&lt;br/&gt; Isotonic glucose solution to dilute collagen is supplemented</td></tr><tr><td>HEPES buffered saline (HBS)</td><td>in house preparation</td><td>in house preparation</td><td>10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4)</td></tr><tr><td>Blocking buffer</td><td>in house preparation</td><td>in house preparation</td><td>1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS</td></tr><tr><td>Calcein AM</td><td>Molecular probes</td><td>C1430</td><td></td></tr><tr><td>Bleach 10%</td><td>in house preparation</td><td>in house preparation</td><td></td></tr><tr><td>0.1 M NaOH</td><td>in house preparation</td><td>in house preparation</td><td></td></tr><tr><td>Denaturated alcohol</td><td>Fiers</td><td>T0011.5</td><td></td></tr><tr><td>Mirus Evo Nanopump</td><td>Cellix</td><td>188-MIRUS-PUMP-EVO</td><td>with Multiflow8. Named in &lt;strong&gt;Figure S1&lt;/strong&gt; A as &#039;Pump&#039; and &#039;Manifold&#039;</td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axio Observer Z1</td><td>equipped with a colibri-LED and high resolution CCD camera</td></tr><tr><td>Software microscope&amp;nbsp;</td><td>Zeiss</td><td>N/A</td><td>ZEN 2012</td></tr><tr><td>Hematology analyzer</td><td>Sysmex</td><td>N/A</td><td></td></tr><tr><td>Table Top Centrifuge</td><td>Eppendorf</td><td>521-0095</td><td></td></tr><tr><td>Platelet incubater</td><td>Helmer</td><td>PF-48i</td><td></td></tr><tr><td>Incubation water bath</td><td>GFL</td><td>1013</td><td></td></tr><tr><td>Pipette</td><td>Brand</td><td>A03429</td><td></td></tr><tr><td>Tube Roller</td><td>Ratek</td><td>BTR5-12V</td><td></td></tr><tr><td>Sterile docking device</td><td>Terumo BCT</td><td>TSCD</td><td></td></tr><tr><td>Tubing Sealer</td><td>Terumo BCT</td><td>AC-155</td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td>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?...

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

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 ...

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

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

平行板流动腔和评估内皮祖细胞层流剪应力下连续流电路

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

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

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 ...

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 ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

实时研究全血-内皮相互作用和血栓动力学的体外微流体疾病模型

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

免疫与感染

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.

研究血管医疗器械血细胞相容性和宿主细胞活化的体外血流循环模型

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive ...

医学

<meta charset="utf8"></head><body>评估创伤和输血中血小板功能的平台

Microfluidics in Assessing Platelet Function

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

<meta charset="utf8"></head><body>微流体评估血小板功能

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

微流体评估血小板功能

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying ...

微流体流钱伯斯再造使用血止血建模和血小板输注

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微流控装置重新创建一个肿瘤微环境