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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K., Sznitman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acinus-on-a-chip:+A+microfluidic+platform+for+pulmonary+acinar+flows.">Acinus-on-a-chip: A microfluidic platform for pulmonary acinar flows.</a> J. Biomech. 46 (16), 2817-2823 (2013).</li><li>Lindken, R., Rossi, M., Grosse, S., Westerweel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-Particle+Image+Velocimetry+(microPIV):+recent+developments,+applications,+and+guidelines.">Micro-Particle Image Velocimetry (microPIV): recent developments, applications, and guidelines.</a> Lab. Chip. 9 (17), 2551-2567 (2009).</li><li>Wereley, S. T., Meinhart, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+in+Micro-Particle+Image+Velocimetry.">Recent Advances in Micro-Particle Image Velocimetry.</a> Annu. Rev. Fluid Mech. 42 (1), 557-576 (2010).</li><li>Bruus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+Microfluidics.">Theoretical Microfluidics.</a> Oxford Master Series in Condensed Matter Physics. , (2008).</li><li>Haefeli-Bleuer, B., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometry+of+the+human+pulmonary+acinus.">Morphometry of the human pulmonary acinus.</a> Anat. Rec. 220 (4), 401-414 (1988).</li></ol>

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?...

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&#174; 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>1mm 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 &#181;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 (&#181;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）。</stron...

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

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

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

研究蛋白质折叠的微流体混合器

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

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 ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

微流体芯片ICPMS进样

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

影像引导下，血管源性微流控网络的基于激光的研制

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...