这项研究是由基金会研究和比利时红十字会龙龙血液服务的发展提供支持。我们承认"Bijzonder onderzoeksfond"（BOF - 专项科研基金）提供的财政支持来自根特大学授予合同号BOF30290744项目。

该协议如下对人样品研究机构伦理指南，以及从所涉及的所有供体获得知情同意书。从安特卫普大学医院（批准文号16/10/120）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.微流控设置<ol><li> 制备微流体流动室的通道，管道和连接销 <ol><li>悬浮胶原库存小瓶通过涡旋混合，然后在由制造商所提供的50微克/毫升的最终浓度，等渗葡萄糖溶液稀释。 注: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的金标准，对历史和生物学方面的原因。人类III型胶原也可使用，但这些纤维涂层较差，血小板反应并不强。其它涂覆的表面也可以使用，如血管性血友病因子，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3的组合。 </li><li>以一个新的，一次性的微流体生物芯片与八连胜，水货渠道和尺寸在毫米，0.4宽x 0.1高x 20 L.吸取0.8μL胶原蛋白进入生物芯片的通道（S），另一端标记这是的出口。填只是沟道长度的5/6，使得胶原纤维不会在通道入口涂覆，因为这会导致堵塞，这妨碍有效的灌注。 </li><li>孵育生物芯片，在4℃下，在湿润的和密封的容器中至少4小时。 </li><li>为了与接触途径研究组织因子（外在）介导的凝​​血组合（固有），外套与渠道科亚根和重组人组织因子（rhTF）。 <ol><li>在10mM 4-（2-羟乙基）稀释rhTF -1-哌嗪乙磺酸（HEPES）缓冲盐水（HBS; 0.9％（重量/体积）氯化钠，pH值7.4，1:3000，储备浓度:4.5纳米）。 </li><li>除去经由出口胶原涂布溶液。吸管0.8 rhTFμL的在HBS（原料浓度，终浓度稀释1/500:下午9点）进入通道的出口，填充通道长度，类似胶原涂层策略5/6。 </li><li>孵育生物芯片用于在湿润和密封的容器中另外的30分钟。 </li></ol></li><li>填用封闭缓冲液涂覆的通道（1.0％（重量/体积）牛血清白蛋白和0.1％（重量在HBS / v）葡萄糖）中，从另一端部（标记为入口）开始。填充在混合生物芯片相同的封闭缓冲液Y形通道的数目相等。 <ol><li>确保所有的通道和通道臂完全充满封闭缓冲液。既孵育生物芯片S表示在加湿并密封的容器至少1小时。 </li></ol></li><li>对于正在使用的每个通道，准备了8厘米长，1个2厘米长，而且长一46厘米两种油管。放置一个销在三个较小管子的一端与在最长油管的两端。 </li></ol></li><li> 准备清洗泵 <ol><li>冲洗所有的泵流体，并用蒸馏水连接管。 </li><li>除油生物芯片的铸造用精密无尘擦拭和变性酒精清除打印和灰尘。 </li><li>填写与他们连接到生物芯片前，阻止缓冲区中的所有管道。 </li><li>固定在自动显微镜舞台上涂生物芯片。固定混合生物芯片在相同的高度使用实验室剪式千斤顶显微镜阶段。 </li><li>连接涂层的生物芯片，使用时间最长的管道，有46厘米长的混合生物芯片。确保两个生物芯片是在一定距离，以使管子形成了一个灵活的，但直线。 </li&gt; <li>附加注射器连接销插入冲洗泵的鲁尔兼容管。其连接到2厘米管子的自由端和固定的另一端，以涂覆生物芯片的插座。 </li><li>固定两个8厘米管道使用其销混合生物芯片入口。把各管的自由端在HBS的小瓶。冲洗所有管路，用1毫升哈佛商学院的所有通道。 注:HBS从药瓶由于漂洗泵的拉力流经8厘米管道到混合芯片，并通过46厘米管子到涂覆芯片（从未涂覆流到涂覆的部分）。此步骤除去阻断缓冲液并粘附不佳胶原（或rhTF在双面生物芯片）。 </li></ol></li><li> 准备灌注泵 <ol><li>打开灌注泵的驱动程序软件。在初始化软件中的注射器图标双击水泵。 </li><li>选择将要使用的注射器的类型:1毫升注射器凝血buffer和2mL注射器重构血液。 </li></ol></li><li> 注射器连接到生物芯片构造 <ol><li>断开漂洗泵和所有管路，除所述一个连接两个生物芯片。断开的管被在下面的步骤再次使用。 </li><li> 2厘米管连接，其连接器的注射器针到厚壁废液管的鲁尔锁。填充厚壁管材用注射器标准的胃蛋白酶溶液。 注:加入胃蛋白酶的废液管抑制和裂解灌注后凝固，从而防止系统的堵塞在后端。 </li><li>固定厚壁管与科赫尔钳的开口端。放置与漂白下方适当的废液容器流过以收集。 </li><li>固定其销到涂覆生物芯片的出口的管道。 </li></ol></li></ol> 2.血液样品的制备<ol><li>从健康志愿者采集的血液，并分离其组分4 <ol><li>在真空乙二胺四乙酸（EDTA）容器收集血液。仅对于使用自动血液分析仪进行全血计数（CBC）使用该样本。 </li><li>收集在疏散柠檬酸钠容器血。 5毫升的血液每信道是必需的。 </li><li>放置在一个水平振动器未决重建血液样品（S）。 注:该法应放血的3小时内完成。浓缩血小板和全血样本ABO / RhD血型组相匹配。 </li><li>离心13分钟，在250 xg离心以制备富血小板血浆（PRP）。不要使用离心机制动，防止松散填充颗粒的干扰。 </li><li>转移PRP到一个新的圆锥形离心管中。离心在4500 xg离心（与制动）（PPP）10分钟以沉淀血小板和制备贫血小板血浆。丢弃高原ELET沉淀。孵育在PPP的水浴中在37℃。 </li><li>由穿孔原始一个具有21g的针的底部传送聚集的红血细胞到新鲜的锥形管中。 注:在堆积的红细胞级分的残余血小板计数为11±4×10 3每μL（平均值±SD，N = 10）。 </li></ol></li><li> 重建血 <ol><li>确定使用自动血液分析仪在步骤2.1.6制备的浓缩红细胞的血细胞比容。 </li><li>确定的血小板浓缩物，将使用自动血液分析仪用于分析的血小板浓度。 </li><li>计算堆积的红血细胞，血小板浓缩物，和PPP的体积，将产生40％的血细胞比容和250×10 3血小板/μl在2.5毫升最终体积。混合这些准备复溶血液和执行CBC。 注:细胞的其它靶滴度可以使用，这取决于第Ë研究问题。 </li><li>制备其中血小板浓缩液的体积用相同体积的生理盐水代替确定的"内源性"血小板（ 即非血库血小板）的浓度的"空白"对照样品。 </li></ol></li><li> 标签重组的血液血小板和纤维蛋白原 <ol><li>吸管13的Alexa Fluor 405标记的纤维蛋白原（70微克/毫升终浓度）在试管10.7毫克/毫升溶液的微升和添加的重组血1毫升。 注:纤维蛋白原使用根据制造商的手册的市售的试剂盒进行标记。 </li><li>混合另1毫升重新构成的血液中含有1mM DIOC 6（1μM最终浓度）的2微升或含有5mM钙黄绿素AM（5μM的最终浓度）的2微升的试管中。此添加到含有纤维蛋白原血管，给予2毫升血小板的最终体积和纤维蛋白原 - S的tained血。 注:染料的选择可取决于所研究的问题或研究者5的偏好。要知道，任何染料的激发能引起光化学文物。 </li><li>轻轻颠倒混合。在37℃孵育样品10分钟。 </li></ol></li><li> 准备凝血缓冲 <ol><li>准备包含10毫米氯化钙2和3.75毫米氯化镁2 HBS凝血缓冲。制备1毫升凝固缓冲器用于一个信道。 </li><li>过滤-消毒通过一个0.2微米的过滤器凝血缓冲器。预温暖这个至37℃。 </li></ol></li></ol> 3.灌注试验<ol><li>聚焦显微镜光学器件以附着在通道的底部的胶原纤维。使用相衬或微分干涉对比（DIC）。在实验软件，选择"选定的小片区域的设置当前Z"到所选的对焦数字修复。 </li><li>在使用显微镜的采集软件所选声道定义感兴趣区域（ROI）的区域。 注:投资回报率可能是一个灌注通道内随意选择任何表面区域。它不宜进行分析，以便血栓形成和纤维蛋白生成靠近入口和出口，以避免获取不均匀分布血栓的图像。在ROI表面积应包含血栓的一个显著数目以允许平整出由个体的血小板信号变异性。在通道的xy平面上的ROI的位置总是固定的，之前灌注确定。如果需要的话，信道的未涂覆部分可被包括在分析中，以确定是否血小板非特异性结合到生物芯片的塑料。 </li><li>准备灌注样品<ol><li>装入含有1毫升预热的凝血缓冲剂在灌注泵1毫升注射器。 </li><li>轻轻倒转和负载2米混合预热重构血L的一个2毫升的注射器。 </li><li>确保所有的空气从两个注射器拆除，每个连接到使用注射器连接器引脚8厘米的管道。 </li><li>素的连接器和高速（400微升/分钟）都注射器管和当一切填充有凝结缓冲液或血液停止。 注意:通过吸管，混凝缓冲和重新配制的血液会立即开始实验时，避免了导入空气对灌注腔混合在混合生物芯片的Y形通道。 </li></ol></li><li>用销，固定两管道的另一端，以在混合生物芯片的Y形通道的分割腿。底部腿用于灌注凝血缓冲器和用于重构血液的上腿。 </li><li>通过以4.4微升/分钟凝血缓冲和重组血44微升/分钟开始灌注开始实验。在废液管允许出口perfu取出钳科赫尔锡永。 注:流速可以根据所研究的问题而改变。启动秒表，以确定实验开始和实时图像采集的开始之间的时间。 </li><li>图像记录每10秒使用显微镜的采集与实验软件实时30分钟。开始录制时的重组血液和凝血缓冲混合进入安装在显微镜舞台上涂生物芯片。 注:其他的时间序列可以根据实验装置中使用。使用放大100倍（10X物镜和10X镜头）。 </li></ol> 4.终止实验<ol><li>终止泵和图像采集后30分钟或更早，如果信号饱和。拆下所有管路和注射器和丢弃它们作为生物危害性的废物。 </li></ol> 5.数据分析<ol><li> 确定血栓增长（绿色荧光）和纤维蛋白形成（紫色fluorescence）与图像分析软件动力学。下面的命令是针对这里所使用的软件: <ol><li>打开插件处理:缝合缝合每个时间点的一侧由端的图像。 </li><li>打开插件图像分析来确定血小板的表面覆盖。 </li><li>打开插件措施。通过绘制包括所有的血栓和纤维蛋白纤维的一个矩形区域定义投资回报率;在这个协议中，投资回报率是一个矩形测量637毫米2。 选择标签所有视图包括所有记录的时间点。 </li><li>使用创建的表自动生成的电子表格将包含各时间点的选定矩形区域的平均荧光强度值。 </li><li> XML格式保存的电子表格。 </li></ol></li><li> 打开进一步计算的电子表格程序电子表格。 <ol><li>减去初始（backgrou次）的所有数据值。 </li><li>绘制荧光信号的灌注时间为绿色（血小板）和紫（纤维蛋白）染料的功能。 注:考虑到泵的初始化和图像采集的实际出发点之间的时间。血栓形成一般是在此模型中的两个步骤的过程:（1）"慢"血小板粘附（ 即，粘附）到固定胶原随后（2）凝固驱动血栓生长在这两个血小板结合"快速"的增加（ 即，累加）和纤维蛋白沉积（ 即，凝固）（ 图1）。 </li><li>计算血小板粘附和通过线性回归累积的斜率;该斜率产生在其形成的各个阶段血小板血栓生长动力学。 </li><li>计算的血纤维蛋白凝血的斜率和推断此线截取的X轴，在确定发病的时刻。 </li></ol></li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rate-limiting+roles+of+the+tenase+complex+of+factors+VIII+and+IX+in+platelet+procoagulant+activity+and+formation+of+platelet-fibrin+thrombi+under+flow.">Rate-limiting roles of the tenase complex of factors VIII and IX in platelet procoagulant activity and formation of platelet-fibrin thrombi under flow.</a> Haematologica. 100 (6), 748-756 (2015).</li><li>McDonald, J. 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=Fabrication+of+microfluidic+systems+in+poly(dimethylsiloxane).">Fabrication of microfluidic systems in poly(dimethylsiloxane).</a> Electrophoresis. 21 (1), 27-40 (2000).</li><li>Gutierrez, 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=Microfluidic+devices+for+studies+of+shear-dependent+platelet+adhesion.">Microfluidic devices for studies of shear-dependent platelet adhesion.</a> Lab. Chip. 8 (9), 1486-1495 (2008).</li><li>Hartman, R. L., McMullen, J. P., Jensen, K. 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V. a. n., 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=Platelet-+and+erythrocyte-derived+microparticles+trigger+thrombin+generation+via+factor+XIIa.">Platelet- and erythrocyte-derived microparticles trigger thrombin generation via factor XIIa.</a> J. Thromb. Haemost. 10 (7), 1355-1362 (2012).</li><li>Nickel, K. F., 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+polyphosphate-factor+XII+pathway+drives+coagulation+in+prostate+cancer-associated+thrombosis.">The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis.</a> Blood. 126 (11), 1379-1389 (2015).</li><li>Okorie, U. M., Denney, W. S., Chatterjee, M. S., Neeves, K. B., Diamond, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+surface+tissue+factor+thresholds+that+trigger+coagulation+at+venous+and+arterial+shear+rates:+amplification+of+100+fM+circulating+tissue+factor+requires+flow.">Determination of surface tissue factor thresholds that trigger coagulation at venous and arterial shear rates: amplification of 100 fM circulating tissue factor requires flow.</a> Blood. 111 (7), 3507-3513 (2008).</li><li>Shen, F., Kastrup, C. J., Liu, Y., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold+response+of+initiation+of+blood+coagulation+by+tissue+factor+in+patterned+microfluidic+capillaries+is+controlled+by+shear+rate.">Threshold response of initiation of blood coagulation by tissue factor in patterned microfluidic capillaries is controlled by shear rate.</a> Arterioscler. Thromb. Vasc. Biol. 28 (11), 2035-2041 (2008).</li></ol>

作者什么都没有透露。

血小板输血是浓缩规定对血小板的患者，以防止或止血。其临床的影响最近强调了急性白血病12的历史回顾，回顾，增加儿科患者在六七十年代的生存率分别为主要归因于（血小板）输血医学的改进。血小板浓缩物也用于阻止在急性创伤或外科手术过程中止血。在这些条件下，片晶具有优良的促凝血场所迅速引发止血是优选的，但血小板浓缩物从自愿捐献者捐献制备，不像药理药物，是由制备标准化只，不通过（化学）组成。因此，在血库领域的几个问题是没有答案，包括那些捐赠的最佳方式，储存条件和输血的做法。在复数的领域atelet（输血）生物学，从循环细胞清除了很多问题，输注血小板的贡献止血，或它们的免疫学也仍然没有答案。 血小板功能分析众多的实验室技术是可用的，但这些大多涉及血小板功能的一个单一的方面，如聚集或脱粒13。要广泛评估血小板和浓缩血小板，止血全面的模型是必不可少的，而这些必须包括流体力学。血液流变是关键的止血14,15或血栓形成16中正确地解释血小板的行为，即使抗凝防止钙和/或凝血酶参与柠檬酸盐或肝素，分别为2的存在。研究凝血和血小板功能之间的相互影响下的流17，18，正常凝血酶生成是必需的，因此，游离Ca 2+为好。在这些条件下学习止血实验装置是复杂的，因为措施，以防止"伪"或凝固的不受控制的活化应采取尽可能。此外，许多专门的研究小组使用定制的硬件19，20，这会导致大量的实验室间的变异5。这种方法的典型局限性是使用矩形容器，非脉动流型材，和非人类表面涂层5。我们在这里描述的测定使用市售的工具，因此可适合用于标准化。 因为一旦血液不人体内包含凝血级联反应是很容易被激活，控制复钙是一个关键的步骤。 ŧõ达到这个目的，添加Ca 2+需要被推迟到刚刚灌注在活性胶原表面之前，因为当大量静态血液被供给的 Ca 2+缓冲，凝血难免发生，最终堵塞管路和偏置数据解释（数据未显示）。对这个问题的解决是分别泵送的 Ca 2+缓冲液和血液中，从而允许它的方式来分析室灌注期间通过对流和扩散力混合。完全混合是在混合和分析室之间管道的46厘米的段来实现的。这个长度是为试剂的最佳停留时间计算来混合基于在使用的流量灌注过程中的质量和对流运输21的基本法则（1000秒-1）。这种方法被证明可控性和可重复性。 凝血联系激活有时被视为简单的一件神器解释凝固时间时要避免血液取样和。因此，我们证明，在不存在抑制剂的，接触引起的凝固开始于灌注16.7分钟（±3.7分钟）。在CTI的存在下抑制FXIIa介导的接触活化，凝血未在任意限定的实验（30分钟）的过程中开始。实验的该窗口足够长，以辨别是亲或抗血栓形成的样品。尽管凝血通过接触激活可以是血液接触人工表面不想要的结果，我们的数据表明血小板明显的生物剂量效应。这对于输血医学重要的，因为最近的调查结果FXII，血小板聚磷酸盐10，磷脂酰丝氨酸分布22和血小板微粒23实际上重估接触途径凝血作为一个重要的贡献者，止血，E特别是在血栓形成24的情况下。例如，血小板输注患者变量产量或库存的血小板的变量磷脂酰丝氨酸表达可能从而诱发变异的疗效，如果显示成功凝血取决于这些因素。 通过共固定脂化rhTF与胶原蛋白，外源性凝血途径或TF途径中，对胶原血小板沉积期间激活。这扩展了检测，以最全面的方式，为带来血液与TF的细胞和组织接触受伤的典范。我们的数据显示，胶原和TF的共固定化降低凝血发病的时刻，但不改变凝结的速率。值得注意的是，rhTF的固定化在表面上的量在该模型25，26的一个重要变量，应该给定的研究中被标准化。在presenCTI的CE，所述TF途径可以专门研究（未示出），因为可以防止接触活化。 总之，我们的实验装置结合了血小板的血液血小板存入银行重组和钙依赖的血小板沉积和纤维蛋白形成在流体动力学流止血模型输血的典范。此测定法将被用来回答关于编组血小板促凝性质问题，并影响血小板浓缩物制备技术对本。

小学和中学止血最初概念化为两个比较可分离的生化过程。初级止血被视为在动脉流动条件的一个主要因素，与血小板关键作用，而次级止血被视为支配下静脉血流凝固，使蛋白酶级联以形成不溶性纤维蛋白。在过去的几十年里大幅重塑这一传统观点，承认血小板活化和凝血相互依存，在所有（非极端）流体力学让人误解的生理和病理过程中止血同样重要的过程。这一观点已被翻译到诊所，制定全面衡量全血凝血试验正越来越多地使用，尽管在相关性，应用性和实用性1许多剩余的问题。 我们最近发表的血小板的功能分析集中使用在输血2的体外模型涂覆有纤维状胶原微流体流动室，用含有红细胞，血浆和血小板的正常量的样品。此方法使用肝素或水蛭素抗凝固处理的血液，这意味着没有或仲止血，这是依赖于凝血酶生成和离子化钙（Ca 2+）的有限的参与。为了支持凝血酶形成，从而包括在微流止血的方方面面，我们在同一技术平台上开发的补充方法，包括现在游离钙离子 。以这种方式，血小板沉积和纤维蛋白形成，可以研究，再次在血小板输注的模型，以评估血小板浓缩质量。 在该方法中，血小板和纤维蛋白原标记与已完全分离的发射光谱的荧光团。双色，实时视频显微镜然后允许的分析初级血小板粘附，以及凝固起始和纤维蛋白沉积。在这种类型的测定法的一个重要的变量是再钙化，由于添加Ca 2+的静态血液之前，灌注，势必造成在容器接触启动凝血。这将引发偏读出因灌注前的油管和生物芯片入口堵塞。因此，在我们的方法中，柠檬酸盐抗凝的血液和钙离子含缓冲通过一个Y形入口室的上腿分别泵送。各组分进入涂覆有单独的或与重组人组织因子（rhTF）组合胶原分析腔室之前灌注过程中混入。通过调整独立的泵的流速和Ca 2+的浓度的缓冲液中，最终血液样品的10％的稀释度进行。 使用这种扩展协议，我们展示凝块的贡献化因子XII（FXII）和血小板浓度接触下流动途径凝血启动。我们的数据还表明，通过涂布rhTF沿着胶原，组织因子途径可以伴随测量。

<table border="0" cellpadding="0" cellspacing="0" width="1210">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">1 mL Syringe</td>
			<td style="width:220px;">BD</td>
			<td style="width:256px;">A00434</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">10 mL Syringe</td>
			<td style="width:220px;">BD</td>
			<td style="width:256px;">309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">2 mL Syringe</td>
			<td style="width:220px;">BD</td>
			<td style="width:256px;">307727</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Alexa Fluor 405</td>
			<td style="width:220px;">Life Technologies</td>
			<td style="width:256px;">A30100</td>
			<td style="width:443px;">The manufacturer&#39;s manual was followed for labeling of fibrinogen</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">BD vacutainer Eclipse</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:256px;">368650</td>
			<td>Blood collection needle with preattached holder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">BD vacutainer tube with EDTA&#160;</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:256px;">368856</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">BD vacutainer tube with Sodium Citrate</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:256px;">366575</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Blocking buffer</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:256px;">in house preparation</td>
			<td style="width:443px;">1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Calcein AM</td>
			<td style="width:220px;">Molecular probes</td>
			<td style="width:256px;">C1430</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Coagulation buffer</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:256px;">in house preparation</td>
			<td style="width:443px;">10mM CaCl2 and 3.75 mM MgCl2 in HBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Conical tube 15mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:256px;">1888271</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Conical tube 50mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:256px;">227261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Corn trypsin inhibitor (CTI)</td>
			<td style="width:220px;">Enzyme Research Laboratories</td>
			<td style="width:256px;">CTI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Denaturated alcohol</td>
			<td style="width:220px;">Fiers</td>
			<td style="width:256px;">T0011.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">DiOC6</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:256px;">318426</td>
			<td style="width:443px;">3,3&#8242;-Dihexyloxacarbocyanine iodide - 1mM solution in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Exigo microfluidic pump</td>
			<td>Cellix</td>
			<td>EXIGO-PC-FS7.0-MF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Fibrinogen</td>
			<td style="width:220px;">Sigma-Aldrich</td>
			<td style="width:256px;">F3879</td>
			<td style="width:443px;">Labelled in house with AF 405 - stock concentration 10.7 mg/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Hematology analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">HEPES buffered saline (HBS)</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:256px;">in house preparation</td>
			<td style="width:443px;">10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Horm Collagen</td>
			<td style="width:220px;">Takeda/Nycomed</td>
			<td style="width:256px;">1130630</td>
			<td style="width:443px;">Native equine tendon collagen (type I) 
			Isotonic glucose solution to dilute collagen is supplemented</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Humidified box</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:256px;">HUMID-BOX</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Incubation water bath</td>
			<td>GFL</td>
			<td>1013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">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="20">
			<td height="20" style="height:21px;">Mirus Evo Nanopump</td>
			<td>Cellix</td>
			<td>188-MIRUS-PUMP-EVO</td>
			<td>with Multiflow8</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Pipette</td>
			<td>Brand</td>
			<td>A03429</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Pipette tips 100-1000</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:256px;">740290</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Pipette tips 1-10</td>
			<td style="width:220px;">Eppendorf</td>
			<td style="width:256px;">A08928</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Pipette tips 2-200</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:256px;">739280</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Platelet concentrate orbital shaker</td>
			<td style="width:220px;">Helmer</td>
			<td style="width:256px;">PF-48i</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Precision wipes</td>
			<td style="width:220px;">Kimtech</td>
			<td style="width:256px;">5511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Pepsin solution&#160;</td>
			<td style="width:220px;">Hanna instrument&#160;</td>
			<td style="width:256px;">HI7073L&#160;</td>
			<td style="width:443px;">2g pepsin per 75 ml solution, Protein cleaning solution with pepsin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Recombinant Human Tissue Factor Innovin</td>
			<td style="width:220px;">Dade Berhing</td>
			<td style="width:256px;">B4212-40</td>
			<td style="width:443px;">rhTF with synthetic phospholipids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Software microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:256px;">N/A</td>
			<td>ZEN 2012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Sterile docking device</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:256px;">TSCD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Table Top Centrifuge</td>
			<td>Eppendorf</td>
			<td>521-0095</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Tube Roller</td>
			<td>Ratek</td>
			<td>BTR5-12V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Tubing Sealer</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:256px;">AC-155</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Vena8 syringe connector pin</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:256px;">CONNECTORS-B1IC-PACK100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Vena8 Fluoro+ Biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:256px;">188V8CF-400-100-02P10</td>
			<td>Coated biochip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Vena8 Needles</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:256px;">SS-P-B1IC-B1OC-PACK200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Vena8 Tubing</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:256px;">TUBING-TYGON-B1IC-B1OC-ROLL 100F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">VenaDelta Y1 Biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:256px;">VDY1-400-100-01-02</td>
			<td>Mixing biochip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:291px;">Vortex mixer</td>
			<td style="width:220px;">VWR</td>
			<td style="width:256px;">58816-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">ZEN2012 software</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:256px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a microfluidic flow chamber model for platelet transfusion and hemostasis measures platelet deposition and fibrin formation in real-time

止血的微流体模型评估流体剪切的条件下，血小板的功能，但在抗凝血剂的存在下，该分析被限制为仅血小板沉积。钙离子依赖凝血和血小板功能之间的错综复杂的关系，需要分析前的血液细心和控制再钙化。我们设置使用一个Y形的混合通道，它提供集中的钙 /镁离子缓冲刚刚灌注前的血液流动，从而实现快速复钙样品无瘀。两者之间的水库流量速度的10倍的差距最小的稀释。该复钙血液然后在胶原包被的分析腔室的灌注，和差异化标记允许使用荧光视频显微镜既血小板和纤维蛋白沉积的实时成像。该系统只使用可商购的工具，增加标准化的机率。救援人员到场的重建mbocytopenic血液与血小板篇章集中。此外机型血小板输注，证明了其在这一研究领域的使用。示例性数据表明，凝固起始和纤维蛋白沉积为线性依赖于血小板浓度，证实在我们的模型中原发性和继发止血之间的关系。在16分钟灌注的时限，接触激活未能成行，尽管钙化正常的Ca 2+和镁离子水平。当凝血凝血因子XIIa用玉米胰蛋白酶抑制剂抑制，这一时间范围甚至更长的时间，指示其中在血小板促凝性质的变化可以评估相当动态范围。胶原组织因子的共固定化显著减少到发病凝血的时间，而不是它的速率。研究组织因子和/或接触的途径中的选项增加该测定的通用性和实用性。

实时的原始数据的分析在图1中说明。首先，血小板粘附在反应性表面，导致在记录绿色荧光（ 图1A，i）的稳定增加，称为附着力 。在这一阶段中，几乎没有紫色荧光，这表明纤维蛋白没有或仅略微形成（ 图1B）。在凝固开始时，紫荧光纤维蛋白沉积迅速（iii）中，并在这段时间内，在大约相同的速率血小板绿色荧光的增加，此处指定为血小板积累 （ⅱ）。因此，一个单一实验返回荧光增加三种速率（I，II，和iii）用于在该血小板和纤维蛋白沉积发生在此模型中的速度的替代标记。此外，凝血发作（ 矩的发病 （IV））的时刻外推，这是血小板的决定因素促凝潜力。 在没有TF的凝血启动很慢，主要是通过所在的内在tenase复合物通过由新华富时和/或FXII直接激活FIX 6的激活FX的接触途径运行。为了证明FXII依赖性，玉米胰蛋白酶抑制剂的4μM的（CTI）加入以抑制活化的FXII（FXIIa）7。这种抑制没有影响血小板粘附（ 图2A），但凝固没有启动用于灌注实验中（ 图2B和补充视频1）的任意定义的总的持续时间。 在体外 ，接触途径可通过异物如玻璃，或在临床试验使用诸如高岭土矿物材料来启动。 在体内 ，活化的血小板提供的负电荷8 &lt;/ SUP&gt;通过酸性磷脂9，如磷脂酰丝氨酸，和/或通过多磷酸盐的释放（息肉）10，11的膜曝光。我们的微流体实时测定模仿了后者，因为，在一个范围内的血小板的浓度，止血反应取决于血小板计数（ 图3）。通过增加重构样品中血小板的数量，粘附率（ 图3A），积累（ 图3B，绿色），和凝血（ 图3B，紫色）线性增加。此刻-的发作由血小板浓度增加显著缩短（ 图3C），这表明需要（激活）沉积血小板的阈值数量来触发凝血。 在体内的组织损伤，然而，携带TF的细胞将我通过在钙离子的存在下的FVIIa-TF外在tenase复合物nitiate血液凝固。这在我们的实验装置由模仿后涂敷脂化rhTF的含胶原的灌注腔室。在涂有仅使用或与rhTF组合胶原信道的配对分析，凝血发病是显著更快（ 图4A）。血小板粘附率为不是线性的，所以可以不执行线性回归（数据未示出）。既凝血和血小板积累率不条件（ 图4B-4C中 ， 补充视频2）之间不同。 <img alt="图1" src="/files/ftp_upload/55351/55351fig1.jpg" /> 图1:在微流体灌注腔血小板粘附和凝固的回归分析。该图阐明了从实时荧光原始数据得出的参数<html>在一个反应​​性表面复钙血流期间获得的。 （A）平均绿色荧光强度显示的灌注时间功能的血小板沉积。该曲线描述与（i）开头的双峰过程缓慢增加线性血小板粘附接着（ⅱ）快速增长的线性累积 。曲线的两个线性部分倒退，这些斜率描述的由血小板荧光血栓形成的两个速率; （ⅰ）粘合性和（ii）的积累。 （B）的平均紫色荧光的强度示出了在时间的函数的血纤维蛋白的沉积。在血小板粘附，紫色荧光是基本上不存在，而它发展迅速凝固以下开始。的（ⅳ） 矩的发作被定义为具有由此处定为凝固血纤维蛋白荧光（ⅲ）血栓形成的第二相的外推线性回归的x轴的截距。EF ="http://ecsource.jove.com/files/ftp_upload/55351/55351fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55351/55351fig2.jpg" /> 图2:在没有的TF，凝血在胶原包被灌注流动腔取决于FXIIa。含有CTI或对照缓冲液（CONTROL）再生血液微流体灌注物在1000秒的剪切速率，共30分钟进行。 （A）血小板附着率（秒-1）是不依赖于CTI。 （B）中为CTI处理的样品的力矩的发病超出了实验（示为虚线）的30分钟限制，表明该参数的FXIIa的依赖性。酒吧和点是平均值，晶须标准偏差。统计分析是由配对t检验。 （NS =不显著，N≥3）。 <a href="http://ecsource.jove.com/files/ftp_upload/55351/55351fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55351/55351fig3.jpg" /> 图3:根据流混凝取决于血小板数。微流体灌注实验在胶原包被的腔室与重新构成的血液中的 Ca 2+的存在下和不同的血小板浓度（正≥3）中进行。 （A）的速率血小板积累（绿色）和凝血（紫色），和（C）矩的发病的血小板粘附（B）的比率的被示出为在重组血小板浓度的功能。点是平均值，晶须标准偏差。 <a href="http://ecsource.jove.com/files/ftp_upload/55351/55351fig3large.jpg" target="_blank">请点击此处查看大VERSI</a>这个数字的。 <img alt="图4" src="/files/ftp_upload/55351/55351fig4.jpg" /> 图4:这两个TF和接触凝血途径的激活显著缩短矩的发病凝固。重组血，在钙离子的存在下，通过涂以单独或与胶原蛋白和rhTF研究单独的接触通路或接触和TF途径的组合胶原腔室灌注。 （ 一 ）矩的发病凝固在含TF-流动腔显著缩短。 （B）的凝固和（C）中的血小板的蓄积速度凝固的速度不仅或胶原和rhTF涂覆流动室胶原之间显著不同。酒吧和点是平均值;晶须是标准偏差。统计分析是由配对t检验。 （NS =不显著，* P &lt;<html> 0.05，N≥3）。 <a href="http://ecsource.jove.com/files/ftp_upload/55351/55351fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="视频1" src="/files/ftp_upload/55351/55351vid1.jpg" /> 补充视频1:在没有的TF，凝血在胶原包被灌注流动腔取决于FXIIa。接触的通路的作用是在胶原包被灌注流动室来确定。在没有CTI的血小板（绿色）和纤维蛋白（原）（紫）沉积（A），它覆盖荧光图像序列。 （B）相同的顺序为A组，但与绿色通道关闭。血小板（绿色）和纤维蛋白（原）的CTI的存在下（紫色）沉积的（C）的覆荧光图像序列。 （D）的相同序列面板C，但用的绿色通道关闭。ecsource.jove.com/files/ftp_upload/55351/Supplementary_video_1.avi"目标="_空白"&gt;请点击这里下载该视频。 <img alt="视频2" src="/files/ftp_upload/55351/55351vid2.jpg" /> 补充视频2:两个TF的激活和接触途径显著缩短凝血矩的发病。研究的TF的作用，被用于涂以单独或与胶原蛋白和rhTF胶原流动室。 （A）的叠加的血小板（绿色）和纤维蛋白（原）的仅涂覆有胶原流动室（紫色）沉积的荧光图像序列。 （B）相同的顺序为A组，但与绿色通道关闭。血小板（绿色）和纤维蛋白（原）的涂覆有两个胶原和rhTF流动室（紫色）沉积的（C）的覆荧光图像序列。 （D）的相同序列面板C，但用的绿色通道SWITC建置关闭。 <a href="http://ecsource.jove.com/files/ftp_upload/55351/Supplementary_video_2.avi" target="_blank">请点击这里下载该视频。</a>

Watch this Scientific Journal Video about A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time at JoVE.com

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

本文介绍了止血的实验模型，同时测量血小板功能和凝血。血小板和纤维蛋白的荧光实时测量，并且血小板附着率，凝固速度，和凝血的发病被确定。该模型被用于确定下在浓缩物用于输血流动血小板促凝性能。

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 ...

a-microfluidic-flow-chamber-model-for-platelet-transfusion-hemostasis

Nanyang Technological University

Research

JoVE Journal

Bioengineering

13.7K 次观看.  Belgian Red Cross-Flanders. 本文介绍了止血的实验模型，同时测量血小板功能和凝血。血小板和纤维蛋白的荧光实时测量，并且血小板附着率，凝固速度，和凝血的发病被确定。该模型被用于确定下在浓缩物用于输血流动血小板促凝性能。 

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

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 ...

科研

生物工程

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

微京尼平沉积技术的微模式化血管肌肉薄膜的扩展文化

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

白细胞 - 富血小板纤维蛋白，一种新型生物材料的表征

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

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.

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 Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

在微生物发酵过程中制造一种纳菲安涂层、还原石墨烯 Oxide/Polyaniline 化学电阻传感器, 以实时监测 ph 值

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...