我们感谢查克·斯科特和科学公司从CBS公司（ <a href="http://www.cbsscientific.com/" target="_blank">www.cbsscientific.com</a> ）维克多麦克奈特工程援助和制造3D室，气体交换部，并插入。这项工作是由美国国立卫生研究院（的补助R21DK067084和R43CA141782 SKK）的支持。

1。准备细胞生长的胶原涂层上，下刀片<ol><li>计算实验所需的插入的数量。将每个插入在一个单独的无菌培养皿（35×10毫米），并通过辐照灭菌（11戈瑞）。 </li><li>准备涂层膜的胶原溶液:50微克/毫升，154微升每个刀片（插入表面面积1.54厘米2）。 </li><li>将插入膜（不要让枪头接触的表面）的表面上，然后小心加入剩下的等分，形成一个大的降了一圈的胶原蛋白溶液数滴（每滴约20微升）膜表面。确保胶原溶液保持半球形，从膜表面不流失。 </li><li>量带盖子的陪替氏培养皿，在平坦表面上，并在室温下孵育1小时。 </li><li>吸胶原溶液，一次洗膜覆盖160微升PBS。 </li><li> ASPIRA德PBS及更换盖子。如果使用的同一天，培养皿放置在室温下在干燥的地方。否则，在4℃下放置烹调和使用插入于7天。 </li></ol> 2。文化内皮细胞上插入<ol><li>准备EC悬挂所需的培养基。确保细胞活力&gt; 98％，并立即使用。 </li></ol> 注意:对于人脐静脉EC（人脐静脉内皮细胞），4×10 5细胞于160微升每个刀片被使用。 <ol start="2"><li>培养皿准备在第1条中的胶原蛋白涂层刀片;不删除插入菜。放置几小滴的细胞悬液（每滴约20微升）在一个圆圈的胶原涂层膜的表面上（不要让枪头触摸表面），然后小心地加入剩余的细胞悬液形成一个大的下降。保证细胞悬液仍然HEMIS的<html> pherical，从膜表面不流失。 </li><li>更换盖子，并小心地将细胞在5％CO 2培养箱内培养，并培育中的菜肴（无晃动），在37℃下30分钟，以使细胞附着的膜。 </li><li>轻轻地加入5 ml的培养基中，以确保插入保持上盘底部，并完全覆盖介质每个培养皿中。更换盖子，小心翼翼地把菜肴在孵化器。在37°C过夜培养细胞。 </li><li>使用相同的方法来准备第二个（可选）低插入细胞局部微环境，这将是放在下面的插入含有EC层。 </li></ol> 注意:如果较低的插入件包括在实验中，上部和下部插入第一彼此连接之前，将插入到3D的移动设备（ 图4B）。 E"&gt; 3。组装三维流室设备<ol><li>拧在一起的上板和下板，连接板，使用管材的气体交换单元，将组装的移动设备到一个干净的塑料袋中，然后灭菌袋和内容的照射（11 Gy的）。 </li><li>进入无菌组织培养罩，喷用70％乙醇中取出细胞培养孵化器，将托盘式货架，擦去，然后，覆盖无菌卫生巾用大托盘。 </li><li>将照射引擎盖进塑料袋，取出设备，从包里无菌孵化器托盘上。将设备连接到提供与油管蠕动泵。程序的一个最佳的速度为0.2毫升/分钟的泵。 </li></ol> 注意:要连接泵的电源插座，电线挤出通过孔在孵化器的背面。周围使用强盗插头电源线，以确保该洞是密闭的。 <ol start="4"><li&gt;广场5 - 7毫升到无菌的15毫升管（"入口"）所需的培养基。 </li></ol> 4。总理的3D流室设备<ol><li>从插座上拔下入口管路拉动金属针从油管（使用小型无菌卫生巾保持无菌的油管）。使用的金属针作为一个"入口"和作为"出口"的管道连接的终端连接油管。将金属针到15毫升管入口与媒体，将出口到空的15毫升管。 </li><li>打开泵，所以流量是逆时针的，并设置在0.2毫升/分钟的流速。允许绘制介质从15毫升入口管到管，当介质停泵油管到达终点之前它连接到设备（ 图2，停止＃1）的负压。 </li><li>拧下该装置的上板和下板。小心地取下上层板和地点的Medi 1,500 - 1,550微升UM（无论是单独或介质加上实验趋化因子）进入下水井。确保井包含足够的介质中以形成一个半球，达到约1 - 2毫米以上下板。 </li><li>传送准备插入，从陪替氏培养皿用无菌镊子下板的孔中，确保没有气泡下方的插入。吸任何形式的媒体中出现的下板的表面上，以确保在板保持干燥。将上盘下盘，并重新放回板用螺丝连接。 </li><li>立即开启蠕动泵，并允许介质流经3D模型的移动设备。提高设备的出口端，并保持在这个位置上，以防止在板之间的空间内形成气泡室。 </li><li>允许介质填充的腔室和管路连接腔室的气体交换单元。立即停泵，介质达到之前的气体eXchange的单元（ 图2，停止＃2）。将3毫升的气体交换装置的媒体插入，打开泵上的，并允许气泡逸出的气体交换单元。提高气体交换单元，以允许介质填充管排出的气体交换单元，当介质达到停泵的出口管的端部（ 图2，停止＃3）之前的2 - 3厘米。 </li><li>连接入口的金属针插座使用小型无菌巾。重新编程的泵，以便在相反的方向（顺时针方向）的介质流朝向气体交换单元，并确保任何除去气泡一旦达到气体交换单元。检查无气泡保留在系统中。 </li><li>重新设计的泵使介质流量逆时针。加入100微升的试验细胞悬浮液的气体交换单元。关闭气体交换单元的盖子，将托盘进入孵化器的工作系统，并允许测试细胞circulate在三维的移动设备所需的时间，在37℃。 </li></ol> 注意:用70％乙醇清洗泵的电源线，并把它的孵化器内。 5。终止测试，并收集样本进行化验<ol><li>从孵化器的工作系统和地方在引擎盖上取下纸盘。停泵，断开入口和出口两端放置到空无菌的15毫升管。 </li><li>打开泵，并从系统中收集的细胞悬液细胞悬液在冰上，如果合适的话，直到使用。 </li><li>拆卸室拆下螺丝和提升上板。从下盘取出插入他们转移到培养皿。 </li></ol> 注:插入可染色，可视化和原位细胞计数（结晶紫在DH 2 O）0.05％胰酶消化收集EC进一步测试。 <ol start="4"><li>移除介质和从下部井的细胞成管，以210×g离心5分钟。除去上清液，然后随意洗涤和悬浮细胞，并根据具体的实验目标（在下面进一步讨论）处理细胞。 </li></ol>

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级联生命科学公司拥有独家权利，美国专利号7927867 B2"设备，用于评估在体外细胞迁移流动条件下和其用途的方法"。 SKK流室装置的3D技术的发明者和科学的共同创始人和股东级联生命科学公司

的3D模型的移动设备可以是一种有用的技术，在各个领域，包括干细胞生物学，血管生物学，血细胞，肿瘤，自身免疫性疾病和炎症的研究。的3D模型的移动设备将是特别有用的研究造血，间充质，神经和其他干细胞的分子机制调节每一个步骤的干细胞外渗级联。研究细胞迁移的研究人员也可以使用这个设备，以检查不同的T细胞和B淋巴细胞，单核细胞，嗜中性粒细胞，嗜酸性粒细胞，NK细胞，和其它游走细胞迁徙机制。在血管生物学EC的能力，以支持细胞的招募和模仿血脑屏障体外局部微环境的效果评估，该设备将是有益的。对于重点疾病的发病机理的研究人员，该设备可以被用来研究细胞毒性T细胞的迁移到胰腺在I型糖尿病，英里运移进入肺部的哮喘患者中，迁移的嗜中性粒细胞，单核细胞，淋巴细胞到炎症部位的各种疾病，细胞招聘伤口愈合站点，与新生血管的细胞毒性T细胞的相互作用，嗜酸性粒细胞和招聘成肿瘤细胞毒性T细胞。 新颖的3D设备也将是一个有用的工具，转化科学和临床前药物开发和筛选。例如，该设备可以被用于优化制备用于静脉内给药的治疗的细胞悬浮液，测试招聘受伤组织与正常组织相比，治疗细胞，并审查的特定器官或组织的内皮细胞和微环境中的作用的过程。药物开发中，该装置可以用于测试候选药物靶肿瘤细胞和阻止外渗级联反应的每一步，测试作为药物传递的细胞迁移Ý车辆肿瘤部位，测试药物，调节人体器官特异性的血管内皮细胞或局部组织的特定的微环境的功能，并进行测试的药物的组合，调节不同的步骤回归级联。最后，当被转换为高吞吐量的系统，该设备可以用于筛选的小分子，肽，抗体，该抗体介导细胞的运输目标分子。 技术细节和技巧 流动条件下的滚动和粘附外渗级联的关键步骤，并作出显著贡献到体内细胞迁移的效率。值得注意的是，这些措施不利于在体外的静态检测。然而，静态轮回检测可作为阴性对照实验中，测试剪切应力的影响，对细胞的存活和功能包括EC的能力，以支持低亲和力相互作用（轧）和粘附牢固。介质的实验中的3D模型的移动设备的选择是重要的。媒体包含不同的生长因子可能影响循环细胞的生存，尤其是在孵化时间长。此外，该浓度的Ca 2 +和Mg 2 +的 ，这可能会影响粘合剂的相互作用的生理流动条件下，有很大的不同商业培养基。规划与3D模型的移动设备的实验时，应考虑此技术细节在下面讨论。 EC单层的选择 签名EC细胞表面受各种参数的影响，包括物种和组织中的起源和细胞培养条件，应考虑到设计的实验。一些常用的EC包括来自肺微血管EC（LDMVEC），骨髓来源的EC（BMDEC），脑源性EC（BDEC），和人脐静脉内皮细胞。EC的特性也各不相同，他们的起源。例如，组成BMDEC快递选择素和VCAM-1，这是负责的滚动和粘附，而BDEC，LDMVEC，和人脐静脉内皮细胞不表达这些分子在正常情况下。因此，插入涂有BDEC LDMVEC，和人脐静脉内皮细胞与TNF-α（10毫微克/毫升，4小时），或其他因素，以模仿炎症和诱导表达的归巢分子进行预处理。 局部微环境试验串扰 有越来越大的兴趣，了解局部微环境调节功能EC和参与细胞外渗。我们的研究结果表明，插入下方的EC单层基质细胞单层显着增加循环细胞外渗。这一发现表明，EC和基质之间的串扰提高欧共体的能力，以支持循环细胞外渗。不仅如此版，插入不同的微环境（ 例如，间充质干细胞，肺成纤维细胞，肿瘤细胞，星形胶质细胞）的细胞，可有助于模仿特定的血管床。特别是，脑源性EC胶质细胞和星形胶质细胞的组合可能是一个有用的方法来模仿血 - 脑屏障。同样，细胞取自患者或正常细胞在体外操纵，可以帮助患病模仿一个特定的微环境。 在我们的研究中，我们使用较低的插入基质细胞生长至50％汇合。尽管最佳的插入件上的微环境的细胞密度将取决于研究的目标，这可以很容易地操纵和控制。 选择剪切应力速率 0.8达因/厘米2的剪切应力，在这里使用，因为这已被证明由Von安德里安等 。的剪切应力是在骨髓中microvasculature，其中造血祖细胞的正常生理条件下15退出流通，并进入组织。与此相反，观察到更高水平的剪切应力在较大的容器，是有限的细胞外渗。因此，高的剪切应力率可以作为额外的控制各种细胞类型的实验研究外渗。 剪切力作用下的细胞的生存取决于几个因素，包括细胞类型和体外细胞治疗（察洛娃等人，未出版的观察），并且在测试过程中，因此，应该仔细评估。可评价细胞的敏感性不同水平的剪切应力（剪应力阻力），通过编程的蠕动泵，以增加从0.8达因/厘米2到6达因/厘米2的剪切应力速率增量。在这些测试中，细胞可以定期收集从气体交换室监测细胞死亡，使用作为说，如台盼蓝排斥，膜联蛋白V和PI染色，细胞凋亡标记的表达。 如果试验是设计用来测试体外基因工程细胞的细胞是来自于薄壁的剪切应力的阻力，它是建议，包括额外的阳性对照，如血源性细胞，在实验中。 选择插入孔径和ECM涂层 刀片有很多的孔隙体积（3，5，和8微米）。孔径大小的选择将依赖于循环的试验细胞的大小和性质的，这也是静态Transwell小室测定的情况下。建议用大孔径（8微米）插入到测试的大细胞，如上皮来源的肿瘤细胞外渗。白细胞或造血干细胞更常见的测试与5微米的孔插入，最好将其保留测试的s外渗的3微米孔径的插入小光伏电池。然而，单独测试单元尺寸是不是唯一重要的因素，我们建议测试插入几个孔径。 在我们最初的研究中，我们测试了各种ECM他们的能力，以支持经济增长的EC，插入和承受剪切应力&gt; 4小时。 ECM测试包括基底膜，聚-D-赖氨酸，纤连蛋白，层粘连蛋白，键入I型胶原，水凝胶，元角质I，II，III，IV，和Extracel。 EC生长提供了最好的结果，得到与Extracel，纤连蛋白和胶原蛋白。然而，在我们的手中，在0.8微克/厘米2 Extracel显着降低轮回测试细胞穿过细胞膜。因此，我们现在使用的纤连蛋白（10微克/厘米2）或胶原蛋白（5微克/厘米2）的涂层的插入。然而，细胞外基质的最优选择可能会影响不同的EC和transmigratory的性能的测试电池。应进行的初步实验测试完整性性EC单层生长在选定的ECM。例如，预包被的地方插入EC单层在装有培养基的培养皿中，放置在摇动器上的菜创建剪切应力（低设定），并置于37℃和5％CO 2的12小时。使用结晶紫染色，可以评估的完整性EC暴露后的剪切应力。 选择最佳测试细胞浓度的 测试细胞退出流通的能力取决于诸多特点，具体到每个细胞类型。要产生统计上显着的结果，循环细胞密度必须进行优化，以确保有足够数量的细胞迁移到阳性对照井。因此，我们建议执行初始测试理解之间的关系的细胞数加载到系统中，并与细胞密度的范围内（ 例如，10 3个/ ml〜10 7个/ ml）细胞经过轮回。 此外，循环的最佳浓度可从不同来源获得的相同的细胞类型不同。例如，CD34 +细胞是含有造血干细胞和提交的谱系特异性祖细胞的细胞种群异构。他们可从骨髓动员的外周血和脐带血。来自这三个来源的CD34 +细胞具有不同的归位和嫁接能力16-18，因此在3D模型的移动设备测试这些细胞的条件满量程的研究之前应进行优化。 选择最佳的细胞循环时间 最佳的流通时间应根据经验来确定对每个小区不同。循环所需的最短时间可以从静态迁移实验的结果推算，但是这可能是EXTEN副执行干事当细胞受到剪切应力。试点研究测试循环次数在4和96小时之间。 气体交换部 气体交换单元的主要目的，是在介质中，通过3D的移动设备中循环，类似于标准的组织培养孵化器中的设置以保持最佳的CO 2浓度。气体交换的单元，也可用于加入的试验细胞的化合物，并在测试过程中，收集探针和样品。 评价测试细胞数 循环细胞可以在不同时间取样，在实验过程中通过气体交换单元。在实验结束时，留在循环的细胞可收集从插座拔出。在实验结束时，当3D设备拆卸转生细胞可以从较低的井收获和收集f或进一步分析。如果输入细胞是未标注，将的转生细胞可以与台盼蓝染色，死/活细胞显微镜列举。或者，输入单元可以被标记为与许多"小区跟踪器"，然后加载到3D模型的移动设备可用于活细胞成像的染料之一，在这种情况下，收获transmigrated应定量荧光裂解细胞。 最佳样本大小的选择 以允许统计分析，必须选择的样本大小，将提供约80％的功率，以检测在两组的平均百分比的变化进行测试时，在使用一个双面的t检验的显着性水平为0.05的假设差异。根据我们的经验与骨髓细胞，我们使用三种实验条件下的井每为3D设备实验。然而，最佳样本大小将随实验的目标，应确定ë键mpirically。可以使用多元回归统计测试，双面t-检验，方差分析，以验证结果的统计相关性。 趋化因子的选择 的所有轮回试验，趋化因子的选择将取决于测试单元的属性和研究目标。 SDF-1是一个完善的造血干细胞的趋化。在我们手中，浓度为50 ng / ml的SDF-1的最佳刺激骨髓来源的造血祖细胞的外渗。我们还使用C3a和碱性成纤维细胞生长因子间充质干细胞的趋化因子19。不过，我们建议一个全面的研究之前，滴定跨滴定每个趋化因子和趋化因子的组合，以确定最佳的浓度范围。对于某些类型的细胞，趋化因子的组合可能是有益的。如果特定的测试细胞的趋化因子是未知或不可用，CON刺激淋巴细胞，成纤维细胞和其他细胞的ditioned媒体可以用来作为源的趋化因子。 读出的参数的选择 3D设备的多功能性将扩大类型的轮回可以进行实验，实验的成功将取决于周到的考虑和设计参数读出。作为一个规则，从上部隔室（循环）收集细胞，细胞计数在同一时间，较低的（静态的）隔室应检测的可行性。有些细胞具有较低的微血管中观察到的生理剪切应力的耐受性。在这种情况下，死细胞的数量将增加在上层车厢。贴壁细胞的数量逮捕（或被困）EC层可以进行评估，测试细胞特异表达的免疫细胞化学标记。可以使用特异的抗体CD31和vWF检测到EC和允许测试细胞和EC之间的歧视。此外，在每次试验结束时，应监测EC单层的完整性。 收集到的细胞进行分析的类型将取决于研究者的实验目标，但可以包括芯片和定量PCR，流式细胞仪分析表面分子的表达，用流式细胞仪和免疫印迹的信号通路的激活，和因子分析基因表达的变化采用ELISA法的分泌。功能测试是一个巨大的阵列可能收集到的细胞在体外 ，证明了我们自己的工作中，我们培养转生列举骨髓细胞的造血祖细胞（ 图3）。收集到的样本也可在体内试验中测试了多种。 一个重要的参数，可以帮助预测测试细胞在体内的行为，是倾向于一些细胞ENCY形成聚集体，在不同的速率下的剪切应力。例如，治疗的细胞，在3D模型的移动设备进行聚集物的形成可能会产生较大的倾向，形成团块静脉内给药后， 在体内引起急性血管阻塞（察洛娃等人，未出版的观察）。聚集物的形成，可以通过采样过程中或完成后的三维装置实验测试细胞显微镜监测。

细胞外渗循环细胞退出的过程，血液在体内许多生理反应的重要组成部分。组织再生的过程也是非常重要的，例如，当从组织进入血管（静脉注射）治疗细胞动员迁移通过脉管系统和退出循环的损伤或变性的网站。外渗也是许多疾病，包括炎症，免疫排斥反应，自身免疫，肿瘤转移的发病机制的重要组成部分。 外渗是一个复杂的多步骤的过程，涉及循环的生理流动条件下的细胞与内皮细胞（EC）的相互作用。该方法包括:（一）细胞滚动，（二）坚定EC腔表面的附着力，及（c）在整个欧共体轮回。重要的是，每一步的外渗级联的一个子集的c受埃尔型特异性和种属特异的分子。然而，详细的分子机制，规范外渗特定细胞亚群没有很好的理解，主要是由于血液中的扼要条件的技术难度。这里描述的新颖的三维装置旨在克服这些技术挑战，并允许进行实验，这将提高我们理解生物学的细胞迁移。 分子介导的细胞迁移是重要的治疗目标。控制特定类型的细胞迁移的分子事件，一个详细的了解，将有助于确定新的目标治疗促进或抑制外渗。例如，干预措施，提高治疗性干细胞迁移（无论是来自成人，新生儿，甚至从胎儿组织）向受伤或退化的网站将是伟大的事业在组织再生。有越来越多的兴趣， 在体外生成治疗性干细胞，包括多能干细胞，在体外操纵成年干细胞（扩展，操控基因，并用不同的酶预处理）1。因此，有需要新技术之前的干细胞用于治疗目的的质量评价。在其他参数中，细胞必须是能够有效地退出循环，因为多焦点失调的治疗可能需要静脉内给药的干细胞，2。事实上，干细胞生物学当前的挑战之一是克服干细胞组织损伤3-6家网站，突出需要解决这种差距在我们的理解干细胞迁移的效率极低。策略，与此相反，通过针对特定的归巢分子嵌段细胞迁移将是有用的，在用于治疗炎症和自身免疫性疾病以及转移性癌症。因此，了解基础科学转化医学和药物发现以及相关的分子机制，调解循环细胞和欧盟之间的相互作用，在细胞迁移和外渗。 目前有一些方法可用来研究细胞迁移的不同方面。然而，这些方法的缺点是可以克服的，与新的三维的移动设备。 动物模型: 如免疫功能低下小鼠和基因操作小鼠的动物模型中，已经有用的工具来研究在体内的人类细胞的迁移。然而，对这些模型的一个显着的缺点是人体细胞的相互作用黏附分子对小鼠EC不足，部​​分原因是由于在许多细胞表面分子的种属特异的序列差异。因此，使用的啮齿动物研究人体细胞迁移是不可能真实地反映人体器官特异性血管床的事件。此外， 在体内模型不适合高通量筛选候选药物。传统的体内模型中，使用来研究细胞的归巢不区分外渗级联反应的不同步骤，使得难以识别和瞄准新的归巢分子。活体显微镜方法的开发，以解决这方面的需要，并一直翔实，然而，这个技术是非常时间和劳动力密集的7,8。 静轮回化验: Transwell小室或Boyden小室测定的多孔膜和细胞迁移横跨在迁移研究中被广泛使​​用。该试剂盒具有的优点是，它可以用来不仅要学习的细胞运动和细胞外基质（ECM）的相互作用，也趋化因子介导的细胞迁移整个EC单层生长在多孔膜。不幸的是，在静态条件下不同表型和功能的EC显着那些在生理流动9,10。 Transwell小实验趋事件发生不能忠实地模仿剪切应力下细胞迁移和欧盟之间的相互作用。此外，"滚动"归位级联的步骤，增加了一个额外的维度的选择性的整个过程，不会发生在静态Transwell小室测定。因此，尽管这个技术确实让整个欧共体单层趋化因子介导的细胞迁移的定量评价，它是有限的，因为它不能提供模拟体内血流的剪切应力。 剪切力作用下的分析: 壁面切应力是众所周知的发挥重要的作用，调节EC功能9,10。剪切力诱导信号级联反应，迅速激活TRANSCription因素，EC 11,12的差异表达基因。此信息导致发展下一代的粘附实验-平行的层流室和毛细血管-研究滚动和粘附细胞EC的剪切应力的条件下，7,13。这些实验的限制因素是，他们只能测量滚动和粘附，但不区分会去轮回的贴壁细胞和贴壁细胞即"抓"上的EC单层不会轮回。此外，这些实验无法测量细胞走向趋化因子梯度迁移。 一些报告描述了贴壁细胞的能力抓取EC单层生长条件下的流量14玻片上下方。这种抓取技术的局限性包括:分析只有有限数量的细胞，它是非定量分析细胞上爬行矩阵和大公升面，但不是朝趋化梯度迁移，它不允许转生细胞进行分离。因此，虽然这个实验中施加剪切应力的EC单层的优点在于，它不能量化迁移，细胞朝趋化因子梯度。 这里描述的新颖的3D技术克服了这些缺点，由（上层车厢）与静态趋化因子梯度（下室）相结合的剪切力，并允许外渗级联的每个步骤（ 图1）的定量评价。该设备还可以用于研究如何EC和微环境之间的串扰影响的氨基甲酸乙酯的能力，以支持循环细胞外渗。以下协议描述了一步一步的方法，使用3D流室装置。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td></tr><tr><td>PVC tubing (bore 1.42, wall 0.8)</td><td>Watson-Marlow Limitid</td><td>981.0142.000</td><td></td></tr><tr><td>3D Chamber</td><td>C.B.S.Scientific</td><td>FC-1000</td><td></td></tr><tr><td>Gas exchange unit</td><td>C.B.S.Scientific</td><td>FCR-1000</td><td></td></tr><tr><td>Inserts</td><td>C.B.S.Scientific</td><td>FCI-1005U</td><td></td></tr><tr><td>Collagen Bovine,Type I</td><td>BD Biosciences</td><td>354231</td><td></td></tr><tr><td>Hydrochloric Acid 0.1M</td><td>VWR</td><td>BDH3200-1</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14190</td><td></td></tr><tr><td>HUVEC</td><td>Lonza</td><td>CC- 2517</td><td></td></tr><tr><td>EGM Bullet Kit (cc - 3121 &amp;amp; cc - 4133)</td><td>Lonza</td><td>CC - 3124</td><td></td></tr><tr><td>Trypsin Neutralizing Solution</td><td>Lonza</td><td>CC-5002</td><td></td></tr><tr><td>Trypsin</td><td>Lonza</td><td>CC-5012</td><td></td></tr><tr><td>HEPES</td><td>Lonza</td><td>CC-5024</td><td></td></tr><tr><td>SDF-1</td><td>R7D System</td><td>460-SD</td><td></td></tr><tr><td>Extracel Trial 2.5 Kit</td><td>Glycosam Byosystems</td><td>GS211</td><td></td></tr><tr><td>Fibronectin, human , nature</td><td>BD Biosciences</td><td>356008</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Millipore</td><td>A-003-E</td><td></td></tr><tr><td>BD Matrigel</td><td>BD Biosciences</td><td>354277</td><td></td></tr><tr><td>Human Brain Microvascular Endothelial Cells</td><td>ScienCell</td><td>1000</td><td></td></tr><tr><td>Endotelial Cell Medium (ECM) for brain cells</td><td>ScienCell</td><td>1001</td><td></td></tr><tr><td>Trypan Blue</td><td>StemCells Tecnologies</td><td>7050</td><td></td></tr><tr><td>TNF-alfa, 10 &amp;mu;g</td><td>Invitrogen</td><td>PHC3015</td><td></td></tr><tr><td>VCAM-1, mouse anti-human, CD106-FITC</td><td>Southern Bioteck</td><td>9519-02</td><td></td></tr><tr><td>Propidium Iodide Staining Solutuion</td><td>BD Pharmingen</td><td># 556463</td><td></td></tr><tr><td>Frozen Human Cord Blood CD34&lt;sup&gt;+&lt;/sup&gt; cells</td><td>StemCell</td><td>CB007F</td><td></td></tr><tr><td>Frozen Human Cord Blood CD34&lt;sup&gt;+&lt;/sup&gt; cells</td><td>Zenbio</td><td>SER - CD34-F</td><td></td></tr><tr><td>MethoCult GF M3434</td><td>StemCells Tecnologies</td><td>GFM3434</td><td></td></tr><tr><td>Mouse Methylcellulose complete media</td><td>R&amp;amp;D</td><td>HSC007</td><td></td></tr><tr><td>Anti-Von Willibrand Factor antibody</td><td>abcam</td><td>C# ab11713</td><td></td></tr><tr><td>Petry dish (35 x 10 mm)</td><td>VWR</td><td>CA25373-041</td><td></td></tr><tr><td>15 ml tubes</td><td>VWR Fisher</td><td>21008 - 918</td><td></td></tr><tr><td>Tissue culture flasks 75cm&lt;sup&gt;2&lt;/sup&gt;</td><td>VWR</td><td>BD353136</td><td></td></tr><tr><td>Cristal violet</td><td>Sigma</td><td>C-3886-25G</td><td></td></tr><tr><td>Dissecting forceps, curved</td><td>VWR</td><td>82027-384</td><td></td></tr><tr><td>1,000 &amp;mu;l Pipette tips</td><td>VWR</td><td>83007-380</td><td></td></tr><tr><td>1 - 200 &amp;mu;l Pipette tips</td><td>VWR</td><td>37001-530</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Peristaltic pump</td><td>Watson-Marlow Limitid</td><td>403U/VM3</td><td></td></tr></tbody></table>

a novel three-dimensional flow chamber device to study chemokine-directed extravasation of cells circulating under physiological flow conditions

从血液循环细胞外渗，在许多生理和病理过程，包括干细胞的归巢与肿瘤转移中起着核心的作用。的三维流室装置（以下称的3D模型的移动设备）是一种新型的体外技术，再现生理剪切应力，并允许细胞外渗级联的每个步骤进行量化。的3D模型的移动设备包括感兴趣的细胞循环剪切应力下的上部隔室和下部隔室还包含趋化因子的静态井。两个室分开的多孔插入涂有单层内皮细胞​​（EC）。可选的第二个插入细胞微环境，可以立即放置下方的EC层。的气体交换单元可以保持最佳的CO 2张力，并提供一个接入点加入或退出细胞或化合物在的实验。测试细胞在所需的剪切应力（流率），通过蠕动泵控制循环中的上格。在实验结束时，循环和迁移的细胞被​​收集用于进一步的分析。的3D模型的移动设备可用于检测细胞上滚动和粘附到EC响应趋化因子，抗剪切应力，簇的形成，细胞的存活在剪切力作用下，轮回。此外，可选的第二个插入允许检查EC和微环境的细胞之间的串扰的影响。的平移的3D模型的移动设备的应用包括测试候选药物靶细胞迁移和预测静脉注射后的细胞在体内的行为。因此，新颖的3D设备是一个灵活和廉价的工具来研究的分子机制，介导细胞外渗。

骨髓来源的EC线STR-12的小鼠骨髓生长在插入有5微米的孔隙。欧盟增长的速度是在显微镜下监视和欧盟分别为100％汇合时，插入井转入下格的3D设备。之前立即将插入隔室的下部，在孔中填充有单独的培养基（阴性对照），或与基质细胞衍生因子-1（SDF-1，5毫微克/毫升和50毫微克/毫升）的培养基。此后，将三维的移动设备的组装和腔室被填充有介质，如在协议中所述。分发设备的上格的试验细胞，新鲜收获的小鼠骨髓细胞（3.5×10 6个细胞每室）。甲定义为0.8达因/厘米2的剪切应力施加蠕动泵的速度设定在0.2毫升/分钟。整个工作系统中，然后在5％CO 2培养箱中在37℃下的ceLLS被允许流通，与EC单层4小时。该时间结束时，循环细胞，腔室被拆卸，除去插入在协议中​​所述。转生收获细胞，从下部井，洗涤，再悬浮于新鲜培养基中，并转移到补充造血生长因子，集落形成细胞（CFC）测定（ 图3）的甲基纤维素培养。正如预期的那样，我们发现一个显着更高的CFC整个欧共体单层迁移到含有50毫微克/毫升SDF-1井不是井含5毫微克/毫升SDF-1或中等。 正如我们前面所述， 体外技术可用来研究细胞迁移中的电流没有能够测试EC的能力，以支持细胞迁移外渗局部微环境的效果。为了说明如何可以做到这一点的3D设备，我们检查外渗循环EC和隔着一层一层骨髓基质干细胞的造血干细胞。对于这一点，第二个（低级）插入含有基质细胞层并置上插入下板（ 图4）中包含EC单层。实验，然后如上所述进行转生从孔收获细胞，并计数。结果表明，插入一个额外的层下方的EC单层基质细胞向SDF-1的造血干细胞的迁移（ 图4）显着增加。这一发现是一致的概念，局部微循环细胞组织招聘。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50959/50959fig1highres.jpg" src="/files/ftp_upload/50959/50959fig1.jpg" /> <stroNG&gt;图1。三维流室设备。甲:密封3D的移动设备包括分开的两个隔室由一个可更换的多孔膜（PM）。欧共体上生长的膜，然后形成的上部和下部隔室之间的屏障。定义的剪切应力施加到三维的移动设备使用一个恒定的输液蠕动泵的灌注介质的上格。循环中细胞无相互作用（NIC），互动瞬时与欧共体（&#39;滚&#39;RC），或坚持EC（AC）。贴壁细胞亚群轮回整个欧共体层设备（TM）的低静态车厢B:俯视图图（上图）的3D设备。的水平剖面图（下图）显示的位置上的膜（红色），较低的膜（绿色），和底部的井（蓝色）。:isometriC查看说明不可分割的组成部分的设备:四个螺丝，腈纶毛条块，上腔密封，上部和较低的膜，底部的O形圈，3孔压克力底块，D:照片的3D流腔室相连的蠕动泵和气体交换单元。该系统是组装在无菌罩中，然后转移到一个细胞培养孵化器。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50959/50959fig2highres.jpg" src="/files/ftp_upload/50959/50959fig2.jpg" /> 图2。的3D设备系统的示意图，图中示出三站需要进行系统装配。培养基中从进气口被吸入，由蠕动泵所产生的负压。通过停止第1介质的流动被切断，以允许插入件被放置到设备。后气室是封闭的，该泵是SWITCHED再次立即室填充有介质插入件上生长的细胞，以防止干燥。停止第2允许介质被放置在所述气体交换单元。允许流的入口和出口的连接要被停止的停止＃3。在顺时针或逆时针的方向的流动，可以通过气体交换单元，以去除气泡，通过使用一个开关在泵上。 <img alt="图3" fo:content-width="5.75in" fo:src="/files/ftp_upload/50959/50959fig3highres.jpg" src="/files/ftp_upload/50959/50959fig3.jpg" /> 图3。生理剪切应力条件下的3D模型的移动设备支持造血祖细胞的SDF-1-介导的轮回。答:三组装设备（每片含3下格井）和并行运行。介质中单独或含有SDF-1的5或50纳克/毫升的培养基中，加入到隔室的下部井。小鼠博东北骨髓细胞在4小时的设备被允许流通。之后，每个设备都被拆卸后转生朝向SDF-1（黄色）的细胞，收集从下部井，计数，并补充造血生长因子（GF）的甲基纤维素培养。 14天后，在显微镜下取得殖民地B:从下腔恢复祖细胞集落形成细胞（CFC）数显示为平均值±SD三口井条件。 * P &lt;0.05。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50959/50959fig4highres.jpg" src="/files/ftp_upload/50959/50959fig4.jpg" /> 图4。微环境对造血细胞和内皮细胞之间的相互作用的效果。答:该图显示了第二的位置多孔膜培养STromal下方的上部与EC膜成纤维细胞（SF）。其他的缩写的说明图1A，B:培养SF插入下方的上部膜的附加 ​​的膜（低级插入）C:三个设备（每片含3个井）并行运行。介质中单独或培养基中SDF-1，在50 ng / ml的SDF-1的溶液中加入到隔室的下部井。阴性对照室被删去SC，SDF-1，或两者兼而有之。骨髓细胞被允许在37℃下4小时循环之后，每个设备都被拆卸和转生细胞中收集并计数从隔室的下部井。转生回收细胞的数量的平均值±SD每个条件的三个井所示。 * P &lt;0.05。

Watch this Scientific Journal Video about A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions at JoVE.com

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

的三维流室装置是一种新型的<em&gt;在体外</em技术的定量和阶梯状的生理剪切应力条件下的循环癌细胞的外渗级联评价。因此，该设备填补基本的，临床前和临床研究的细胞迁移的迫切需要。

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

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

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Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

18.3K 次观看.  Torrey Pines Institute for Molecular Studies. 的三维流室装置是一种新型的在体外

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

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

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

三维细胞培养模式的测量肿瘤细胞浸润间质流体流动的影响

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

研究微加工通道细胞迁移的

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

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

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

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

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

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

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

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

平面梯度扩散系统的调查趋化三维胶原基质

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

免疫与感染

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

小鼠边缘区 b 细胞剪切流诱导迁移的体外分析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

定制室测定细胞迁移

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...