将YG支持IDB的奖学金。 IN是一种的阿龙研究员和教员的爱德蒙Ĵ‭。 ‬萨夫拉在特拉维夫大学生物信息学中心‭‬这项研究是支持的通过ISF授予1499年至1410年。

1。创建苏格兰带硕士5 <ol><li>绘制或打印出所需的微布局，扩大规模，在纸面上。在我们的例子中，设计是由两个Y形通道，每个通道3毫米宽（ 图2）。 </li><li>盖一个玻璃滑了一层层的透明胶带。层的数目，将确定的信道（每一层约60微米）的高度。我们用透明胶带层。 </li><li>放置在平坦表面上的布局设计。将滑过的设计模式。小心地切割胶带在载玻片上，用解剖刀，根据该布局来。 </li><li>从各地区的载玻片，但在布局的微取出透明胶带。 </li><li>滑动，在65℃的加热烘箱中放置2-3分钟。与乙醇，轻轻地擦拭。 </li></ol> 2。 PDMS微流控芯片制造<ol><li>混合基料和固化组件的PDMS，如制造商推荐的。我们用一个10:1的碱与固化剂的比率。倒入约30毫升的PDMS混合物倒入一个Petri培养皿〜0.5厘米的高度。脱气PDMS在真空中，如果需要的话。被淹没的载玻片上，在PDMS与图案的透明胶带朝上。名次后的图案的PDMS，防止形成气泡之间的滑动和培养皿底部。在65°C固化48小时，确保菜是使用气泡水平仪水平。 </li><li>在一个新的90毫米陪替氏培养皿中倒入3毫升的PDMS混合，从而导致在约0.5mm深的层。 （如果旋涂机是可用的，它可以被用于这个阶段）。 </li><li>德加和治疗为2.1。 </li><li>轻轻切出的微流体装置，用手术刀到所需的大小。该层将被称为"流层"。 </li><li>切出一个同样大小的一块薄薄的一层PDMS从第二培养皿。这将被称为"井层"。 </li><li>使用适当大小的活检打孔器（我们使用1.2毫米ID）打孔流层的入口和出口的。 </李&gt; <li>将井层很厚的玻璃滑动的设计布局和切出井，使用2毫米的ID活检冲床，对准与微通道的布局。 </li><li>清洁都PDMS层以及24毫米×60毫米盖玻片乙醇。空气干燥，保持清洁。 </li><li>等离子体治疗盖玻片和势阱层的PDMS可逆结合使用标准的等离子蚀刻机（非可逆结合）或手持式电晕6。小心地置于盖玻片上的势阱层，导致粘合性（ 图3）。轻轻揉搓出任何气泡（如果需要的话）。 </li></ol> 3。细胞成像实验<ol><li>请确保您有所需的媒体准备在适当灵活的塑料管连接到内部直径0.02"（聚乙烯），注射泵的注射器。 </li><li>成长S.酵母菌株在液体YPD所需的相位。涡彻底和300μl的每种菌株到一个离心管中。 </li><li>轻轻放置1μl的刀豆蛋白A（σ）2毫克/毫升到各孔中。洗出多余的伴刀豆球蛋白A每口井的两倍，，轻轻吹打水。 </li><li>伴刀豆球蛋白A的干燥，洗株300μL的SC中缺乏葡萄糖的两倍。洗衣机从细胞壁中的残糖有助于正确地坚持刀豆A. </li><li>涡细胞彻底。移液器0.75μl以下到它自己的井（ 图3）的各细胞悬浮液。轻轻地洗出残留的细胞与营养丰富的培养基。接下来的两个步骤需要迅速地进行，以防止干燥的细胞。 </li><li>等离子处理芯片和彻底的井使用的手持式电晕，小心不要直接打的井。小心地放置在芯片上井，密切关注两者之间的对齐方式（这一步可以做到立体镜）。轻轻按下坚持的两层PDMS。慢慢补装置完全与约50微升丰富的介质，确保没有气泡留在车内的通道。 </li><li>将设备连接到适当的进气口，插入管，并开始媒体流。护理，没有气泡被引入到该装置，因为这些可能会非常棘手中删除，这可以通过断开入口或出口，并轻轻拍打在设备上的气泡，小心，不要破坏盖玻片。 </li><li>将在显微镜下找到合适的成像点在每口井。 </li><li>丰富的介质流动下保持细胞为1小时或更长的时间，以允许恢复，如果需要的话。 </li><li>开始实验:我们使用从每个的两个注射器泵的恒定流量，以防止逆流的媒体，作为所需的改变相对流速。设置泵A到0.8毫升/小时，泵B，以0.2毫升/小时的查询结果的一种可流动的通道（ 图4）的宽度的80％以上的培养基中。这可以是动态Ç忌用改变流速。在新的条件稳定在几秒钟内，沿其整个长度的信道2。 </li></ol>

<ol>
	<li>Dertinger, S. K., Jiang, X., Li, Z., Murthy, V. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradients+of+substrate-bound+laminin+orient+axonal+specification+of+neurons.">Gradients of substrate-bound laminin orient axonal specification of neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 12542-12547 (2002).</li><li>Hersen, P., McClean, M. N., Mahadevan, L., Ramanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+processing+by+the+HOG+MAP+kinase+pathway.">Signal processing by the HOG MAP kinase pathway.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 7165-7170 (2008).</li><li>Paliwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPK-mediated+bimodal+gene+expression+and+adaptive+gradient+sensing+in+yeast.">MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.</a> Nature. 446, 46-51 (2007).</li><li>Bennett, M. R., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+measuring+gene+network+dynamics+in+single+cells.">Microfluidic devices for measuring gene network dynamics in single cells.</a> Nat. Rev. Genet. 10, 628-638 (2009).</li><li>Shrirao, A. B., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Fabrication+of+Microfluidic+Devices+by+Replicating+Scotch-tape+Masters.">Simple Fabrication of Microfluidic Devices by Replicating Scotch-tape Masters.</a> Chips &#38; Tips. , (2010).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab Chip. 6, 1548-1549 (2006).</li><li>Gerber, D., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+microfluidic+approach+to+generating+protein-interaction+networks.">An in vitro microfluidic approach to generating protein-interaction networks.</a> Nat. Methods. 6, 71-74 (2009).</li></ol>

没有利益冲突的声明。

在本文中，我们提出了一个简单的台式的方法创建一个微流体装置，同时允许以下几个酵母菌株，在动态条件下。经过数株在一个通道中，提供了一个可靠的工具比较多株的单细胞反应的动力学。我们的方法的一个优点是能够用简单的技术来制造的移动设备，而不需要一个干净的房间。事实上，我们也进行了协议跳绳等离子处理操作（步骤2.9和3.6），并取得了良好的效果，所以没有任何等离子设?...

细胞不断动态变化的环境的反应，通过改变他们的新陈代谢，基因表达谱和细胞功能。为了研究这些现象，可以量化这种变化的方法是必要的。一种类型的读出这样的回答是本地化的压力相关的转录因子中的营养应激反应的变化。 微流体装置1已被用来动态操作环境条件的细胞2-4。他们提出活细胞成像的几个优点:细胞的环境，可以精确地控制和动态地改变，细胞可以是活成像在所有时间，包括操纵外部条件期间;及需要最小的试剂量。一个非常简单的微流体设备的设计，允许两个条件之间的交替，是一个Y形的装置2。我们经常使用Y形微流体装置，让在通道中的细胞的动态变化的应用。我们使用该设备遵循荧光标记的压力相关的转录因子在酵母细胞中的定位变化。我们遵循这些变化在不断变化的营养条件下。例如，我们可以提供一个脉冲（或一系列脉冲）的期限内没有葡萄糖培养基含葡萄糖的介质，在一个非常良好的时间分辨率。通常在这样的实验中，一个是兴趣比较不同的细胞株（例如，不同的突变体，或不同的野生分离株）的响应。 Y形通道被限制在每个通道中的一个应变 - 如果使用不止一个应变，有没有简单的方法来区分菌株。为了克服这个问题，可以使用不同的渠道。如果我们要采用相同的动力条件下多株，我们希望与多渠道结合的Y-通道的概念。在实施这一解决方案有两个挑战:这是很难申请吨他相同的条件的所有信道在同一时间，并且有一个几何的，可以安装在一台设备中的Y通道的数目的限制。因此，我们在寻找一种方式来查看动态条件下几株在一个通道中。 在这里，我们描述的两层用于与多株成像动态条件下的相同的在一个通道中的微流体装置。底层由薄的PDMS与一排孔。这层附着的玻璃盖玻片创建的井到其中的将放置在不同的菌株，附着与伴刀豆球蛋白A至玻璃。第二层是一个Y形的微流体装置，使用透明胶带方法5。第二层的孔中放置的菌株后快速地对准并连接到第一个，建立一个信道与包含不同菌株的多口井。这最后的设备允许以下几个品系在一个通道中，所有的菌株被精确地在同一时间进行的相同的条件。在台式仪器上可以完成整个设计和生产过程中，使用简单的方法，没有软光刻技术。

<table border="1"><tbody><tr><td>试剂/设备</td><td>公司</td><td>目录编号</td></tr><tr><td> PDMS-SYLGARD 184 </td><td>美国道康宁</td><td></td></tr><tr><td>真空干燥器</td><td>的Nalgene </td><td> 5310-0250 </td></tr><tr><td>活检打孔器</td><td>特德·佩拉公司</td><td>哈里斯Uni-CORE核心15076（2毫米），15074（1.2毫米） </td></tr><tr><td>注射泵</td><td> Chemyx </td><td>融合200 </td></tr><tr><td>电晕处理机</td><td>电工艺产品</td><td> BD-20 </td></tr><tr><td>聚乙烯管</td><td>聚乙烯</td><td> S-54-HL </td></tr><tr><td>伴刀豆球蛋白-A </td><td>西格玛</td><td> C7275 </td></tr><tr><td>透明胶带</td><td> 3M思高</td><td>透明胶带二分之一" </td></tr></tbody></table>

a microfluidic device for studying multiple distinct strains

细胞的反应，环境变化的研究，提出了许多实验的挑战:细胞需要将被成像在变化的条件下，往往在一个比较的方式。多孔板经常使用的比较多的不同株或细胞系，但允许有限的环境动态控制。微流体装置，在另一方面，在周围条件允许精致的动态控制，但它是具有挑战性的图像和区分超过在其中少数菌​​株。在这里，我们描述了一种方法，容易且迅速地制造的微流体装置，其能够应用动态地改变的条件下，在一个通道中的多个不同的酵母菌株。通过简单的装置，该装置的设计和制造，而不需要为软光刻技术。它是由一个Y形的流路连接到的第二层窝藏微孔。该菌株中被放置在单独的微孔，并且确切的相同的动态条件下成像。我们试玩nstrate测量蛋白定位的反应在不同的酵母菌株的养分的变化的脉冲的移动设备的使用。

为了说明不同菌株之间的分离，我们拍摄了两个区分的酵母菌株在备用井。成像的完整井没有显示出细胞的孔中（ 图5a，b）之间的泄漏。这两种菌株有的转录因子MSN2标记与YFP。要测试的同时动态地改变的条件的影响，我们在两个输入信道切换的流 ​​率，创建无葡萄糖培养基，导致本地化MSN2-YFP的核（ 图5c，四 ）的步骤。在所有的孔测量的响应发生在相同的时间，示出...

Watch this Scientific Journal Video about A Microfluidic Device for Studying Multiple Distinct Strains at JoVE.com

A Microfluidic Device for Studying Multiple Distinct Strains

我们提出了一种简单的方法来施加类似的动态条件下，以多个不同的菌株，能够产生的微流体装置，而不需要一个干净的房间或软光刻。

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

a-microfluidic-device-for-studying-multiple-distinct-strains

Research

JoVE Journal

Bioengineering

8.7K 次观看.  Tel Aviv University. 我们提出了一种简单的方法来施加类似的动态条件下，以多个不同的菌株，能够产生的微流体装置，而不需要一个干净的房间或软光刻。

视频: A Microfluidic Device for Studying Multiple Distinct Strains

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

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 ...

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

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

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

协议生物膜形成的流光在微流体器件与微柱

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

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 ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

由微型条纹分析技术在微流控设备混合不均匀

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

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 ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

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.