This work was supported in part by the European Commission (FP7 Program) through a Career Integration Grant (PCIG09-GA-2011-293604), the Israel Science Foundation (Grant nr. 990/12) and the Technion Center of Excellence in Environmental Health and Exposure Science (TCEEH). Microfabrication of microfluidic chips was conducted at the Micro-Nano Fabrication Unit (MNFU) of the Technion and supported by a seed grant from the Russel Berrie Institute of Nanotechnology (RBNI) at Technion. The authors thank Avshalom Shai for assistance during deep reactive ion etching (DRIE) and Molly Mulligan and Philipp Hofemeier for helpful discussions.

1.主制造<ol><li>使用绝缘体（SOI）晶片上的硅的深反应离子蚀刻（DRIE），为在原作品32，33中所述来制造一个主硅晶片。 注:DRIE优选标准的SU-8的微加工，由于高宽比特征（40微米，宽90微米的深沟槽）。 </li></ol> 2.铸造和微流体装置的密封<ol><li>在一个10混合的PDMS和固化剂:内部的清洁的小容器1的重量比如塑料称量皿。 </li><li>脱气混合物在真空下干燥器直至所有气泡被除去。 注:所有的后续步骤准备足够的PDMS。这里的下方，首字母缩写"PDMS"总是指脱气的10:1 PDMS:这是在步骤2.1和2.2编写的固化剂的混合物。 </li><li>的脱气混合物倾到的主晶片上方约1mm的高度。脱气再次用于至少40分钟以除去所有的气泡的晶片上方，并尽量减少在晶片下方的气泡。 注:确保晶片是尽可能靠近到板的底部。如果有必要按晶圆轻轻用搅拌2棍棒和德加再次底部。 </li><li>烘烤在65℃下在自然对流烘箱20分钟。 注:20分钟后，将PDMS硬化和几乎完全固化。而较长焙烧时间是可能的烘烤20分钟节省了时间，提高了第二PDMS层的粘附（参见下文），以第一个。 </li><li>文件用细砂纸以改善粘附至PDMS的塑料2毫升注射器的筒部。此外，使用砂纸通过放置在平坦表面上的沙纸和滑动在它上面的注射器筒的基部弄平注射筒的底部。清洁使用压缩空气注射器。 </li><li>放置在注射器的筒段与所述拉第一PDMS层的顶部RGE开口朝向PDMS的表面上，并倾的PDMS上的第一个的顶部上的第二层到约5毫米的高度，并在干燥器中再次脱气的PDMS。 注:第二PDMS层应该从筒体周围小容器倾倒，并且不应当在它进入。 </li><li>烤整个设置在65℃下，在自然对流炉中在至少2小时。 注意:没有必要在固化过程以保持桶中发生的，因为PDMS按压筒的广基的重量中牢固地保持桶。 </li><li>通过使用手术刀主晶片的图案化区域周围的PDMS模具切割。切割时，手术刀应弱触摸晶片的表面上。然后，轻轻插入薄的工具，如用手术刀产生的缺口晶圆镊子，并剥离从主晶片投的PDMS。 </li><li>放置上铸造用铝箔覆盖与图案化侧的软表面朝上（ 即 ，桶应该从表中的边缘挂起），并使用1毫米活检穿孔在腔室入口和通道入口冲床在PDMS中的孔。 </li><li>外套干净的载玻片上用（脱气）10:1的PDMS:使用以3000rpm进行30秒编程的旋涂机的固化剂混合物中，并在65℃下烘烤&gt; 1小时。然后，清洁幻灯片和PDMS使用透明胶带蒙上。 </li><li>用O 2等离子体处理的PDMS模具和PDMS涂覆载玻片的表面上（ 例如，使用手持式电晕）中1分钟，然后轻轻地表面压在一起并且在65℃过夜（O / N）烘。 </li></ol> 3.设备灌装和开动<ol><li>混合与水和甘油水中的悬浮的荧光聚苯乙烯粒子在玻璃小瓶以获得具有0.25％64/36（体积/体积）甘油/水混合物（重量/重量）的颗粒.. </li><li>放置在通道入口上方的甘油溶液一滴和DI笏一滴器上的腔室入口，然后将干燥器和真空〜5分钟内的装置。 注:释放真空等待在甘油溶液和去离子水的液滴，以弹出形成气泡之前。在真空释放的液体被吸入装置内部的空隙。如果残留空气保持在通道内，通过施加外部压力的流体（ 例如 ，使用注射器）和使空气扩散到PDMS消除它。 </li><li>注入约2ml DI水进入前腔室（ 即，针筒筒体， 图2b），直到其完全充满水。再涂用19号钝注射器尖端顶室，切另一个钝19号注射器尖端的前端与插入此尖端到侧室入口。两个注射器提示连接到经由薄聚四氟乙烯管和一T形连接器的1ml注射器。 注意:确保1 ml注射器，聚四氟乙烯管，T形连接器和顶部室（2毫升注射器巴尔EL）都装满水，无气泡。这可以通过打开的连接点，通过管道的空部分推动水和重新连接的连接点来实现。 </li><li>的1ml注射器连接到注射器泵预编程在1秒以模仿例如一个安静的潮气呼吸周期（与周期T = 4秒）线性斜坡， 即构成，从零至1.8毫升/分钟，从1.8毫升/分钟至-1.8毫升/分钟在2秒和从-1.8毫升/分钟回零在1秒。 </li></ol> 4.流量可视化实验:微型粒子图像测速（μPIV） <ol><li>当设备被致动，获得一系列9 - 12锁相，双帧使用微粒子图像测速（μPIV）系统，包括例如一个双帧多重曝光的CCD的颗粒种子流的图像相机（ 例如 ，1600×1200个像素以获得足够的分辨率），一个双脉冲Nd-YAG激光器（波长:532纳米，输出能量:400毫焦，脉冲持续时间:4纳秒），并在倒置显微镜。 注:这种系统是能够获得具有时间滞后帧对的向下到第一和第二帧之间的几微秒。实现锁相双帧图像，这是获得在例如一个双帧系列有用的。，10赫兹（帧对由0.1秒从彼此分开）。然后，数据可以被重新安排，是由一个全周期的时间（这里T = 4秒）分隔的所有帧对形成一个新的时间序列。图像采集应而修改每个帧中对第一和第二帧之间的延迟时间被重复若干次（ 即 ，100微秒到0.1秒）肺泡腔内解决不同的流动区域。 注:关于图像采集系统的最佳组合替代设置（ 即摄像机）和照明光源（ 如激光）图像等微流也可用34，35。 </li><li>使用求和的相关一个算法计算得到的流场的锁相速度矢量地图从图像系列为每个使用的时间滞后。重复此过程数次，在每一帧中一对肺泡腔内解决不同的流动区域的第一和第二帧之间的变化的滞后时间。接下来，使用一个数据分析程序通过平均重叠的数据点33缝合的各个流程映射到的流动模式的完整和高详细地图在一起。 </li></ol>

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The authors have nothing to disclose.

<html>这里介绍的微流体平台腺泡的一个重要特点是它重现生理逼真的呼吸运动是引起生理流动分布和速度腺泡内管和肺泡内的能力。因为微流体通道具有相对低的长宽比产生（ 即 ， 宽 深 / 小时 ≈3.9，其中，w，d是所述管道的宽度，h是所述管道高度），所测量的流量显示更多柱塞样流动特性相比预期的抛物线流量分布，将存在于循环通道。尽管如此，所测量的速度是公生理范围内;它被发现的特征量纲雷诺数，比较惯性粘滞力，产生一个最大的约0.01对应于中到远端腺泡区域，下面的半经验估算2。 内容"&gt;这里，雷诺数定义为重新= U 的x， 最大 深 高 /ν 甘油溶液 ， 其中 U 的x，max是在最大流量的瞬间穿过管道中平面的平均流向速度，D h为管道和ν 甘油溶液的水力直径是用于流量可视化将其匹配于空气中的〜24℃（ν 空气的运动粘度的甘油溶液的运动粘度= 1.55×10 -5米2 /秒，ν 甘油溶液 = 1.51×10 -5米2 /秒）。另外，通过左右两个因素作为来自预期每个分叉后观察到在流幅值下降腺泡模型的二叉分枝模式。即，该级联流速是腺泡的一个重要特征在气道树流动。 附近（ 图4）肺泡腔内的流动分布显示，乳腺导管速度是逐步走向更深腺泡代递减。此外，流动量值沿肺泡导致流速是相对于导管大小较慢内肺泡两到三个数量级的开口急剧下降;这种流拓扑在若干数值研究1,9，15以前报道此外，流动型态显著改变从一个腺泡代到另一个，如在模拟7预测，15:而生成1设有其大致与一致的再循环区肺泡的中心（图4左），第3代的特征在于它是朝向的近侧移位的再循环区肺泡与更开放的流线模式（图4，中间）。最后，没有回流区径向流线在装置世代5（图4，右侧）观察到。以最好的作者的知识，这是第一次广泛的肺泡流动模式的存在是实验抓获。 该方法的成功依赖于微细加工协议的几个关键步骤。首先，为了防止从主晶片在释放撕裂晶片的表面应具有直壁的刻蚀图形的薄的PDMS壁并且不能坚持固化的PDMS。因此，强烈建议以产生使用SOI晶片的DRIE如Fishler 等人描述的晶片。 （2013年）。这样的主晶片是耐用，可以通过很容易地涂有防粘层如Fishler 等人所述硅烷化的表面上。（2013）或通过确保吨帽在DRIE过程的最后一步是用CF 4钝化。另一个关键步骤是提交（步骤2.5）和嵌入（步骤2.6）注射器筒打造顶级室。气泡注射器基座和第一PDMS层可大大降低制造装置的完整性和耐久性之间。为了防止气泡的形成，这是至关重要的注射器筒的底部是平的，均匀地提起。 虽然目前的设计允许只使用一个主晶片的两层器件的制造中，修饰方法可以包括创建包含一个圆形凹槽，以形成顶室的附加的PDMS层。对于第二个PDMS层的附加主芯片设有圆形脊可以使用标准的SU-8光刻制造。该协议的额外修饰可以包括用于PDMS键合不同的方法，该方法不要求一个电晕。附着的PDMS模具到玻璃滑动，第一涂层如在协议的步骤2.10中描述，但使用5载玻片:1，而不是10:1的PDMS:固化剂的重量比。烘烤涂覆的玻璃15分钟在65℃下在一个自然的对流烘箱，按PDMS模具涂覆玻璃的PDMS，并且在自然对流烘箱中在65℃烘烤过夜。 液体泄漏的从PDMS模具和玻璃可以采取下列措施之间的接合面的场合:（1）确保电晕是在处理过程中产生电火花，如果没有，提高输出电压，（2）延长与电晕和（3）使用的PDMS模具接合到玻璃替代方法，处理时间（见上文第）。通常水可以通过薄聚四氟乙烯管向腔室入口的连接泄漏。为了规避这样的泄漏，确保19号钝尖的注射器是用来聚四氟乙烯管连接到进气口。如果PDMS模具和日之间的水泄漏È顶部室（2毫升注射器筒）确保注射器筒体的基部被正确地提交（参见步骤2.5中协议），并且PDMS的第二层倾足够高（约5毫米第一PDMS层上方）。 需要注意的是壁变形的程度高度依赖于PDMS的机械性能。器件的制备过程中的微小变化可能导致不同的设备之间的所测量速度的相当大的可变性。为了确保最大的可重复性使用恒定的制备条件（湿度，烘烤时间等 ）。此外，设备致动期间的体积变化的微调可以通过使用相差显微镜可视化通道的顶表面和调节注射泵的速度斜坡使得信道的顶表面被偏转到所需的距离来实现如由显微镜载物台的Z运动测量。 一个重要的limita当前技术的灰是肺的精确形态特征（ 例如，解剖学，形态学）不能被精确地再现。的确，腺泡模型的平面设计不捕获例如外的平面腺泡分岔和肺泡到导管容积的比大于在体内测量值37低得多。此外，简化的微流体几何仅捕获完整腺泡的一小部分。尽管有这些限制，本模型能够直接在真解剖长度尺度以再现预期的流动模式和速度，并因此表示为腺泡传输现象一个有价值的测试平台。 最后，肺腺泡的功能微流体模型显示巨大潜力，作为呼吸腺泡定量调查流动模仿的呼吸方式体外工具。在这里，简单腺泡模型包括5克膨胀和收缩蜂窝状管道，从而再现一些预期的重要的基本流的属性的enerations到肺部的腺泡区域之内。流动可视化，采用微PIV，肺泡内的腔体为复杂的循环，并沿腺泡树辐射状肺泡流量范围的第一次实验证据。此微流控方法允许下一个相对简单的程序与移动壁复杂腺泡结构的制造，并提供一个有吸引力的替代按比例增加腺泡车型。特别是，在一对一的比例递送模型的主要优点，真正吸入腺泡粒子动力学可以不进行动态相似性匹配进一步需要调查。

在末节呼吸流量动态的细致的量化，肺部蜂窝状的区域是朝着在肺腺泡理解气流混合，并预测在最深的气道吸入1-3气溶胶的命运至关重要。在寻 ​​求改进和靶向给药吸入治疗剂的局部肺站点4,5以及用于全身递送新策略一方面寻址吸入颗粒污染物的危害或相反时，这后一方面是特别关注的。 迄今为止，在深肺腺泡区域呼吸流量已经典型地使用计算流体动力学（CFD），或者与按比例增加的实验模型中体外以下液力相似性匹配调查在硅片 。在过去的几十年中，CFD方法已经被越来越多地应用于研究腺泡流动现象，从SINGLË肺泡模型6,7和蜂窝状的管道8-12更详细的在硅片模型捕捉解剖学逼真腺泡树形结构与蜂窝状的管道多代，最多几百个人肺泡13-15。 总之，数字的努力已经在上随后腺泡气流模式的呼吸运动过程中的作用和室壁运动的影响力脱落光的关键。在没有呼吸运动，静态肺泡功能循环的腔表现出腺泡管和肺泡6,7之间的空气对流不交流中流动;换句话说，肺泡流将被完全从流动腺泡树木内分离空气的交换将来自扩散机制唯一结果。与肺泡域的环状扩张的存在，然而，肺泡流拓扑大幅度修改和地区环境部门一道内部肺泡lting流动模式紧密并列沿着腺泡树的肺泡的位置（ 例如 ，近端与远端世代）。 特别是，已经假设在肺泡流动模式强烈肺泡的比率的影响的模拟到乳腺导管的流速，使得肺腺泡树中，其中，该比值是比较大的以下跨越树结构，特征质量守恒近端世代复杂的循环不可逆的流体迹线肺泡腔内流动。随着每更深腺泡产生，肺泡到导管流量的比例逐渐降低，使得远端腺泡几代表现出更多的径向流线一样是让人联想到通货膨胀简单和气球通缩的。随着现代影像学检查，肺部影像学资料16，啮齿类动物，包括大鼠和小鼠的17，进步已经引起了一些第一CFD SIMUL的在重建肺泡解剖，重建腺泡流动ations。尽管有这样的希望的进展，这些最近的研究仍局限于终端肺泡囊只有18，19或周围的单一管道20几个肺泡解决气流的现象。其结果是，在腺泡呼吸流动现象的国家的最先进的调查仍然受到研究侧重于腺泡环境2的通用解剖风格的几何形状为主。 在实验方面，不同的设置，设有带一个或几个肺泡气道已经发展了很多年21-24。然而，存在分叉蜂窝状航空公司是能够通过在呼吸样的方式扩张和收缩模仿生理呼吸没有实验模型。由于缺乏手头有吸引力的实验平台，腺泡运输现象的研究仍然是有限的问候valida婷计算的研究和批判，仍然实验数据可用的缺乏。 近年来 ，Ma 等人 （2009）构建了由三个腺泡世代的腺泡的按比例增大的刚性壁模型;然而，在这个模型中缺乏壁运动的限制了其能力来捕获呼吸的条件下实际的肺泡流动模式。 其他规模扩大的实验，包括基于从投副本解剖数据移动壁模型，最近推出的25;然而，由于该模型仅捕获的最后两个腺泡世代（ 即 ，终端囊），它未能捕获表征更近侧腺泡世代复杂再循环流动。的规模扩大的实验后面的这些例子进一步强调这种做法的持续限制。具体而言，没有现成的试验迄今已证实从循环的假设过渡到沿径向流腺泡从而确认假设流拓扑结构的数值预测在现实肺腺泡树木7，15最关键的存在。也许，规模扩大的实验调查可吸入颗粒搬运和沉积动力学26极其有限的，由于匹配所有相关的非困难维参数（ 例如 ，粒子扩散，临界传输机制亚微米颗粒，是完全可以忽略不计）。 与正在进行的实验挑战，允许在复杂移动壁的呼吸气流和粒子动力学研究新的实验平台腺泡网络追捧。在这里， 体外模型腺泡解剖学风格的介绍。该微流体平台模拟肺腺泡直接代表腺泡规模流动，拓宽和范围日益扩大肺微流体模型27，包括支气管液塞-FLOWS 28-30和肺泡-毛细血管屏障31。 即，本设计采用了简化的五代蜂窝状气道树周期性膨胀和收缩壁，其中，环状运动是通过控制压力围绕薄的PDMS侧壁并且其中所述顶壁是由一个额外的水而变形的水室内部实现的室直接坐在腺泡结构之上。不像普通的多层微流体装置，该室被简单地通过嵌入的PDMS装置内的注射器的筒部形成的，并且不需要制备额外的PDMS模具。 这里介绍的小型化方法提供了一种用于再现复杂腺泡结构与相比，规模扩大的模式，同时捕捉腺泡流环境的基本特征移动壁的简单和灵活的手段。此平台可用于FLOW¯¯可视化使用航空（见下文代表性的成果）内的流体悬浮颗粒。在不久的将来，该模型将与空气中的颗粒可用于研究吸入腺泡粒子动力学。

<table><tbody><tr><td>Polydimethylsiloxane (PDMS) and curing agent</td><td>Dow Corning</td><td>(240)4019862</td><td>Sylgard&amp;reg; 184 Silicone Elastomer Kit</td></tr><tr><td>Plastipak 2 ml syringe</td><td>BD</td><td>300185</td><td></td></tr><tr><td>Norm-Ject Luer slip 1 ml syringe</td><td>Henke Sass Wolf </td><td>4010-200V0</td><td></td></tr><tr><td>1 mm Biopsy punch</td><td>Kai Medical</td><td>BP-10F</td><td></td></tr><tr><td>Laboratory Corona Treater</td><td>Electro-Technic Products</td><td>BD-20AC</td><td></td></tr><tr><td>PHD Ultra Syringe pump</td><td>Harvard apparatus</td><td>703006</td><td></td></tr><tr><td>Dyed red rqueous fluorescent particles</td><td>Thermo-Scientific</td><td></td><td>Uncatalloged 0.86 &amp;micro;m beads were used</td></tr><tr><td>Glycerin AR</td><td>Gadot</td><td>830131320</td><td></td></tr><tr><td>FlowMaster MITAS micro-particle image velocimetry (&amp;micro;PIV) system </td><td>LaVision</td><td>1108630</td><td></td></tr></tbody></table>

a microfluidic model of biomimetically breathing pulmonary acinar airways

在肺腺泡深度量化呼吸流动特性，以及它们如何影响吸入烟雾传输是针对优化药物吸入技术以及预测肺泡潜在有毒大气颗粒的沉积图案的关键。在这里，软光刻技术用于在在光学系统访问重现生理腺泡流动现象的真实解剖长尺度制造复杂腺泡样气道结构。微流体器件具有5代与周期性扩张和收缩的墙壁分岔蜂窝状的管道。壁的致动是通过改变内部周围都从侧面和该装置的顶部的薄的PDMS腺泡通道壁充满水的腔室中的压力来实现的。在对比普通多层微流体装置，其中需要的几个的PDMS模具堆叠，提出以制造顶部的简单方法通过嵌入注射器的筒段成PDMS模具腔。这种新颖的微流控的设置提供了生理呼吸运动而这又引起特点腺泡空气流动。在目前的研究中，微粒子图像测速（μPIV）与液体悬浮颗粒来定量这种空气流根据流体力学相似性匹配。 μPIV结果和预期腺泡流现象之间的良好的一致性表明微流体平台可能在不久的将来作为体外工具一个有吸引力的，调查在肺部的腺泡区域直接空降代表粒子输运和沉积。

在体外腺泡平台的计算机辅助设计（CAD）和显微镜图像示于图。 1，仿生腺泡模式的特点分支肺泡状圆柱形腔内衬长方形通道（ 图1）的五代。在这里，几代机型的编号，从1代（大部分近端代）代5（为最远端代）。请注意，只有通道入口通向代1是通过在PDMS中的开口的装置开放到外部环境。 16管道从第5代客场领先留给封闭的空气（ 图1a）。通过周期性地调节所述腔室中的水的压力，构成肺泡腔和管道薄壁周期性变形。同时，气道的天花板由位于导管之上的额外的水室的装置垂直变形;建立在此之上室简单的方式没有准备额外的微流体层的注射器的针筒被淹没在里面PDMS交联之前。这导致约1mm分离蜂窝状导管和顶部水室的PDMS层（参照图2）。 该水腔被连接到编程以重复一系列直线倾斜的流速以模仿的法线的平均人类成人的重潮式呼吸方案用4秒的循环时间（T）的注射泵。这将导致一个周期性下降与气道体积的增加;因为出口被密封，仅在入口是开放的环境中，管道内的流体被吸入并通过入口从设备呼出，类似于一个自然呼吸过程。这里，气道导管充满了用荧光颗粒（见协议）和微粒子图像测速（μPIV）接种甘油溶液用于映射resulti<html>整个气道树33纳克流场。 归一化的速度大小（U X /ù 的x，max）存储在流向（ 即 ，轴向）穿过通道的宽度方向示于图。 3。结果在每个5器件世代的峰吸入速度呈现，并且邻近所述管道中平面的薄板坯内代表的流动的二维投影。为了比较，稳态层流为无限长的通道36的解析解也出现在图3。 图4示出在气道中的峰吸入中平面精简肺泡腔内模式和速度幅度。 图4a，b和c描绘腺泡世代分别为1，3和5。 古尔1"SRC ="/文件/ ftp_upload / 53588 / 53588fig1.jpg"/&gt; 图1: 腺泡树网络的微流控模型 （一）全部设备的CAD图纸。 （二）腺泡树结构的特写快照显示的渠道，商会，以及薄壁隔开。紫色箭头表示图中呈现的流程配置文件的相应位置，并正Y -directions。 3，适应与裁判的许可。 33。 <img alt="图2" src="/files/ftp_upload/53588/53588fig2.jpg" /> 图 2: 微流器件的CAD设计 （一个）虚线表示从横向和顶部腔室经由T形连接器将注射器泵主导管。通过示出了PDMS铸内针筒的位置的设备的中央切（B）侧。一个dapted与REF许可。 33。 <img alt="图3" src="/files/ftp_upload/53588/53588fig3.jpg" /> 图3: 腺泡流速从PIV沿世代1信道的宽度到5在图中所示的位置中提取归一导管速度分布（U X /ù 的x，最大值 ）。 1，Y = 0恰逢整个通道的中点位置和u X，最大 =0.0104米/秒相当于这里的设备代1 PIV测试测得的峰值流向速度都在这里显示在高峰吸入（T = 0.6秒）而黑色线对应的分析速度曲线用W D = 345微米，匍匐矩形通道内流动&lt;EM&gt; H = 92微米。改编与裁判的许可。 33。 <img alt="图4" src="/files/ftp_upload/53588/53588fig4.jpg" /> 图4: 速度的大小和相应的简化模式 。数据从微PIV在位于装置世代的肺泡的平面提取的流的突起1，3和5流场示在约峰吸入（T = 0.6秒）获得。速度大小显示在对数尺度。改编与裁判的许可。 33。

Watch this Scientific Journal Video about A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways at JoVE.com

A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways

Soft-lithography was utilized to produce a representative true-scale model of pulmonary alveolated airways that expand and contract periodically, mimicking physiological breathing motion. This platform recreates respiratory acinar flows on a chip, and is anticipated to facilitate experimental investigation of inhaled aerosol dynamics and deposition in the pulmonary acinus.

仿生的微流体模型肺呼吸腺泡航空公司

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards ...

a-microfluidic-model-biomimetically-breathing-pulmonary-acinar

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.9K 次观看.  Technion - Israel Institute of Technology. Soft-lithography was utilized to produce a representative true-scale model of pulmonary alveolated airways that expand and contract periodically, mimicking physiological breathing motion. This platform recreates respiratory acinar flows on a chip, and is anticipated to facilitate experimental investigation of inhaled aerosol dynamics and deposition in the pulmonary acinus.

视频: A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

科研

免疫与感染

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient&#39;s immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

闭环惯性微流体在患者源气道分泌物中无标签中性粒细胞富集的应用

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict regional aerosol delivery. Leveraging 3D printing to generate patient-specific lung models, we outline the design of a high-throughput, in vitro experimental setup for quantifying lobular pulmonary deposition. This system is made with a combination of commercially available and 3D printed components and allows the flow rate through each lobe of the lung to be independently controlled. Delivery of fluorescent aerosols to each lobe is measured using fluorescence microscopy. This protocol has the potential to promote the growth of personalized medicine for respiratory diseases through its ability to model a wide range of patient demographics and disease states. Both the geometry of the 3D printed lung model and the air flow profile setting can be easily modulated to reflect clinical data for patients with varying age, race, and gender. Clinically relevant drug delivery devices, such as the endotracheal tube shown here, can be incorporated into the testing setup to more accurately predict a device&#8217;s capacity to target therapeutic delivery to a diseased region of the lung. The versatility of this experimental setup allows it to be customized to reflect a multitude of inhalation conditions, enhancing the rigor of preclinical therapeutic testing.

We present a high-throughput, in vitro method for quantifying regional pulmonary deposition at the lobe level using CT scan-derived, 3D printed lung models with tunable air flow profiles.

使用患者专用的 3D 打印肺模型评估区域肺沉积

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict ...

生物工程

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

模仿神经血管化初始事件的微流体模型

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Imaging-Guided Bioreactor for Generating Bioengineered Airway Tissue

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in regenerative medicine and bioreactor technologies offer opportunities to produce lab-grown functional tissue and organ constructs that can be used to screen drugs, model disease, and engineer tissue replacements. Here, a miniaturized bioreactor coupled with an imaging modality that allows in situ visualization of the inner lumen of explanted rat trachea during in vitro tissue manipulation and&#160;culture is described. Using this bioreactor, the protocol demonstrates imaging-guided selective removal of endogenous cellular components while preserving the intrinsic biochemical features and ultrastructure of the airway tissue matrix. Furthermore, the delivery, uniform distribution, and subsequent prolonged culture of exogenous cells on the decellularized airway lumen with optical monitoring in situ are shown. The results highlight that the imaging-guided bioreactor can potentially be used to facilitate the generation of functional in vitro airway tissues.

The protocol describes an imaging-enabled bioreactor that allows the selective removal of the endogenous epithelium from the rat trachea and homogenous distribution of exogenous cells on the lumen surface, followed by long-term in vitro culture of the cell-tissue construct.

成像引导的生物反应器，用于生成生物工程气道组织

Repeated injury to airway tissue can impair lung function and cause chronic lung disease, such as chronic obstructive pulmonary disease. Advances in ...

Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.

The presented protocol describes the development and use of a phalloidin-based filamentous-actin staining technique with confocal laser scanning microscopy (CLSM) to visualize adherent cell layer structure in microfluidic dynamic-culture channels and traditional fixed-well static-culture chambers. This approach aids in evaluating cell layer confluency, monolayer formation, and layer-thickness uniformity.

生理尺寸微流控培养装置中上皮细胞单层的制备和结构评价

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as ...

建立用于呼吸道感染研究的 Lung-on-chip 模型

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

作者聚焦：开发微流体 Lung-on-Chip 模型，用于深入研究人类免疫反应和感染机制

Lung-on-chip models surpass traditional 2D cultures by mimicking the air-liquid interface and endothelial cell perfusion, simulating blood flow and nutrient exchange crucial for lung physiology studies. This enhances lung research relevance, offering a dynamic, physiologically accurate environment to advance the understanding and treatment of respiratory infections.

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

由上皮细胞和原发免疫细胞组成的多细胞人类Alveal模型，用于危险评估

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

仿生的微流体模型肺呼吸腺泡航空公司

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该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

闭环惯性微流体在患者源气道分泌物中无标签中性粒细胞富集的应用

使用患者专用的 3D 打印肺模型评估区域肺沉积

模仿神经血管化初始事件的微流体模型

成像引导的生物反应器，用于生成生物工程气道组织

生理尺寸微流控培养装置中上皮细胞单层的制备和结构评价

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

用于研究肺泡粘膜免疫反应的免疫活性 Alphyolus-on-Chip 模型

由上皮细胞和原发免疫细胞组成的多细胞人类Alveal模型，用于危险评估