This work was supported by grants from the National Institutes of Health, including a microphysiological systems consortium grant from the National Center for Advancing Translational Sciences (UH2TR000503), a Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship (F32DK098905 for WJM) and pathway to independence award (DK095984 for AB) from the National Institute of Diabetes and Digestive and Kidney Diseases.

1.准备本机可溶性胶原溶液中<ol><li>制备或购买200mg的酸化，可溶的，I型大鼠尾胶原在1-3毫克/毫升使用标准的分离协议，诸如报告Piez 等人 15 </li><li>比例基于改性胶原溶液的所需的最终体积上起始原料的量。大约使25-30毫升甲基化和25-30毫升琥珀酰化的胶原蛋白溶液中，每个在3毫克/毫升，从200毫克的可溶性天然胶原。 </li></ol> 2.胶原蛋白甲基化<ol><li>稀释100毫克天然，酸化（pH 2-3）的胶原溶液的为0.5毫克/毫升用冰冷无菌水的浓度，并保持该溶液在冰上，以防止凝胶化。 </li><li>调节胶原溶液以9-10的PH值用几滴1N NaOH和在RT搅拌30分钟。观察胶原沉淀，使溶液变浑浊。 </l我&gt; <li>降速在3000×g下沉淀的胶原溶液25分钟。一种透明，凝胶状沉淀物应当是在管的底部可见。吸并妥善处理上清液的。 </li><li>重悬沉淀的胶原在200ml甲醇中，用0.1N HCl和使甲基化反应，以发生在室温下搅拌4天。胶原蛋白不会解散，而应该分裂成非常小的可见部分，这将使解决方案浑浊。 </li><li>甲基化后，离心该溶液在3000×g的25分钟以沉淀甲基化胶原。吸和处置酸化甲醇上清液。 </li><li>溶解甲基化胶原在25毫升无菌PBS，得到浓度大约为3毫克/毫升，反复移液，并过滤通过60微米的细胞过滤的溶液。调节溶液的使用1N的NaOH 20μl递增pH至7.3-7.4。 </li><li>评估concentratioN使用商业鼠胶原ELISA试剂盒或羟脯氨酸检测试剂盒的解决方案。稀释至3毫克/毫升，用无菌PBS洗涤。 </li><li>通过将其与一螺丝帽转移到玻璃瓶中，小心地分层3毫升氯仿在瓶底消毒甲基化胶原溶液，使瓶子以设置O / N在4℃，然后在无菌条件下取出并存储顶层，它是甲基化的胶原蛋白。 </li><li>存储1个月内溶液在4℃下使用。 </li></ol> 3.胶原蛋白琥珀酰化<ol><li>稀释其他100毫克天然，酸化（pH 2-3）的胶原溶液的为0.5毫克/毫升用冰冷无菌水的浓度，并保持该溶液在冰上，以防止凝胶化。 </li><li>调节胶原溶液以9-10的PH值用几滴1N NaOH和在RT搅拌30分钟。胶原应沉淀，使溶液变浑浊。 </li><li>溶解SUC 40毫克互联网络信息中心酸酐（每胶原毫克0.4毫克）的10毫升丙酮的（1/20的胶原溶液的体积）。缓慢（在大约0.5 ml的增量）加该混合物的胶原溶液在搅拌下，同时连续监测pH。通过加入1或2滴的1N NaOH作为pH值接近9.0维持大于9.0的pH值。 </li><li>继续在RT下加入所有的琥珀酸酐的丙酮后搅拌120分钟。观察溶液变得清澈如琥珀酰化的胶原蛋白溶解。定期检查pH值，以确保它仍然高于9.0。 </li><li>调节溶液至4.0将pH用1N的盐酸20μl递增。再观察溶液混浊，如琥珀酰化的胶原蛋白沉淀。 </li><li>琥珀酰化后，离心该溶液在3000×g的25分钟以沉淀的琥珀酰化的胶原蛋白。吸并丢弃上清液酸化与未反应的琥珀酸酐。 </li><li>化解琥珀胶原蛋白在25ml无菌PBS，得到约3毫克/ ml的浓度，反复移液，并过滤通过60微米的细胞过滤的溶液。调节溶液的使用1N的NaOH 20μl递增pH至7.3-7.4。 </li><li>评估使用市售的大鼠胶原ELISA试剂盒或羟脯氨酸测定试剂盒的溶液的浓度。稀释至3毫克/毫升，用无菌PBS洗涤。 </li><li>通过将其与一螺丝帽转移到玻璃瓶中，小心地分层3毫升氯仿在瓶底消毒琥珀酰化的胶原蛋白溶液，使瓶子以设置O / N在4℃，然后在无菌条件下取出并存储顶层，这是琥珀酰化的胶原蛋白。 </li><li>存储1个月内溶液在4℃下使用。 </li></ol> 4.验证了胶原蛋白修饰的<ol><li>制备1 ml的样品从每个本地，甲基化，以及琥珀酰化的胶原蛋白溶液稀释到浓entration 0.1毫克/毫升，用无菌纯水的氢离子滴定。进一步稀释缓冲至少1000倍通过用水透析通过10kDa的截止membraneusing 3溶液的变化对于每个至少4小时。 </li><li>调节溶液的pH至7.3与少量的NaOH和HCl的。用pH 7.3作为任意的基准，由16 Tanford如所述创建滴定曲线用于母体，甲基化，以及琥珀酰化胶原溶液在每个样品进行氢离子滴定。 </li><li>绘制在pH变化每酸的体积加入与结合的H +的每个分子的离子数。高pH值“氨基”的范围应显示在琥珀酰化的胶原蛋白的移位朝中性点（胺基团的损失），并且低pH值“羧基”的范围应显示在甲基化胶原左移（羧基基团的损失），并用琥珀酰化的胶原右移（在羧基的增益多个），相比于天然胶原。 </li><li>通过测定氨基的％在天然胶原使用2,4,6-三硝基苯磺酸（TNBA）比色法由琥珀酰化取代，下列标准协议17,18评估琥珀酰化反应的功效。 </li></ol> 5.制造微流体装置和细胞接种<ol><li>编造使用标准方法9的微流体装置。使用PDMS从SU-8大师副本成型硅使用光刻制造微流体细胞培养室，100微米高，0.4-1.5毫米宽，1〜10毫米长的通道，细胞生长定义。 </li><li>使用等离子体清洁器以氧化装置和载玻片的表面上，然后按共同键。通过暴露杀菌设备在UV光下至少30分钟后，填充腔室50微克/毫升纤连蛋白在无菌PBS孵育在37℃下45分钟。 </li&gt; <li>种子设备用的细胞，如原代大鼠或人类肝细胞，这需要对表型或分化状态的稳定胶原凝胶。我们使用20微升新鲜分离原代大鼠肝7,19或市售冷冻保存的原代人肝细胞以14×10 6个细胞/每毫升设备。 </li><li>允许细胞附着4-6小时，然后洗出独立的细胞和生长介质取代电镀媒体。孵育O / N，以确保充分细胞扩散，创造细胞的融合单层。 </li></ol> 6.层 - 层胶原沉积<ol><li>在一个层流组织培养罩，准备足够的卷对每个器件的各溶液10应用甲基化和琥珀酰化的胶原蛋白溶液，以及媒体几毫升。置于冰上的解决方案。我们用20微升胶原蛋白（10〜15倍，总装置容量）每一层的每个设备。 </li><li>是轧花与甲基化（聚阳离子）溶液，交替与20μl的甲基化，然后冲洗设备琥珀酰化的胶原蛋白溶液，等待每个应用之间1分钟。冲洗设备总共每溶液10次，这大约需要20分钟。加速工作，以最大限度地减少时间的细胞是没有介质的数量。 </li><li>观察胶原慢慢聚集在入口/出口取决于其大小。如果流体流的阻力增加，冲洗装置一次或两次的介质，然后继续分层。 </li><li>施加所有的层之后，冲洗装置两次用新鲜培养基并返回到培养箱中。取决于细胞类型，所述细胞外基质诱导形态学变化，如在肝细胞中增强的偏振，应是在几个小时内可见。 </li><li>使用标准方法9来验证胶原基质的存在下制备用于透射电子显微镜代表性装置装配上培养的细胞的顶端。 </li></ol> 7.稳定细胞表型和功能的<ol><li>图像的细胞形态，活力，和极性使用标准方法的细胞类型。对于肝细胞，采集图像上使用相差显微镜，LIVE / DEAD染色的可行性，并CMFDA染料心尖极化以证明胶原沉积的影响14天。 </li><li>对于细胞形态，用移液管通过入口冲洗装置与20μl的PBS漂洗，注入20微升PBS，并使用图像相差显微镜设备。 </li><li>对活/死染色，通过入口用移液管与20μl的PBS漂洗，注入20微升用DAPI LIVE / DEAD染色溶液制备下列制造商的说明冲洗装置中，温育30分钟，在37℃，冲洗设备再次用20微升PBS，和图像用荧光显微镜的装置。 </li><li>对于胆汁CANALIculi染色，用移液管通过入口冲洗装置与20μl的PBS漂洗，注入20微升2用DAPIμMCMFDA染色溶液制备按照生产商的说明，孵育30分钟，在37℃，再次冲洗装置20微升PBS，和图像用荧光显微镜的装置。 </li><li>收集用过的培养基和测量细胞代谢产物在适当的时间点在培养持续时间，以确定细胞的功能。对于肝细胞，测量在用过的培养基白蛋白和尿素的量。 </li><li>上的细胞本身，例如评估酶活性的水平，抗体染色，或RNA分析执行对端点功能分析。对于肝细胞，诱导和测量细胞色素P450酶的活性或II期缀合的酶谷胱甘肽S-转移酶。 </li></ol>

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The authors have nothing to disclose.

超薄纯胶原组件可以沉积在使用改性胶原的层 - 层淀积带电的细胞或材料的表面。这项研究的结果表明，甲基化和天然胶原的琥珀酰化创建聚阳离子和聚阴离子胶原溶液（图1）可与层-层技术用于沉积超薄胶原基质组件上的细胞（ 图2）或其它带电材料表面。这种超薄基质层可以稳定形态，存活率，和偏振细胞如肝细胞（图3），播种在微流体装置，以及它们?...

Although microfluidics allows for the exquisite control of the cellular microenvironment, culturing cells, especially primary cells, within microfluidic devices can be challenging. Many traditional cell culture techniques have been developed to sustain and stabilize cell function when cultured in tissue culture plates, but translating those techniques to microfluidic devices is often difficult. 
One such technique is the culture of cells on or sandwiched between collagen gels as a model of the physiological 3D cell environment.1 Type I collagen is one of the most frequently used proteins for biomaterials applications because of its ubiquity in extracellular matrix, natural abundance, robust cell attachment sites, and biocompatibility.2 Many cells benefit from 3D culture with collagen, including cancer cells3,45, microvascular endothelial cells6, and hepatocytes7, among others. While the use of collagen gels is easy in open formats, such as tissue culture plates, the limited channel dimensions and enclosed nature of microfluidic devices makes the use of liquids that gel impractical without blocking the entire channel.
To overcome this problem, we combined the layer-by-layer deposition technique8 with chemical modifications of native collagen solutions to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These layers can stabilize cell morphology and function similar to collagen gels and can be deposited on cells in microfluidic devices without blocking the channels with polymerized matrix. The goal of this method is to modify native collagen to create polycationic and polyanionic collagen solutions and to stabilize cells in microfluidic culture by depositing thin collagen matrix assemblies onto the cells. This technique has been used to stabilize the morphology and function of primary hepatocytes in microfluidic devices.9
Although layer-by-layer deposition has previously been reported with natural and synthetic polyelectrolytes10 to cover hepatocytes in plate culture11,12 and as a seeding layer for hepatocytes in microfluidic devices13,14, this method describes the deposition of a pure collagen layer on top of hepatocytes, mimicking the 3D collagen culture techniques. In this protocol, we use hepatocytes as example cells that can be maintained using 3D collagen layers. The many other types of cells that benefit from 3D culture in collagen may similarly benefit from culture after layer-by-layer deposition of an ultrathin collagen matrix assembly.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>Life Technologies</td>
			<td>A1048301</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>Sigma-Aldrich</td>
			<td>C3867-1VL</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>EMD Millipore</td>
			<td>08-115</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>collagen type I, rat tail</td>
			<td>R%D Systems</td>
			<td>3440-100-01</td>
			<td>option for concentrated rat tail collagen</td>
		</tr>
		<tr>
			<td>succinic anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>239690-50G</td>
			<td>succinylation reagent</td>
		</tr>
		<tr>
			<td>anhydrous methanol</td>
			<td>Sigma-Aldrich</td>
			<td>322415-100ML</td>
			<td>methylation reagent</td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881-500G</td>
			<td>pH precipitation reagent</td>
		</tr>
		<tr>
			<td>hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320331-500ML</td>
			<td>pH precipitation reagent</td>
		</tr>
		<tr>
			<td>rat collagen type I ELISA</td>
			<td>Chondrex</td>
			<td>6013</td>
			<td>option for detecting collagen content</td>
		</tr>
		<tr>
			<td>hydroxyproline assay kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK008-1KT</td>
			<td>option for detecting collagen content</td>
		</tr>
		<tr>
			<td>hydroxyproline assay kit</td>
			<td>Quickzyme Biosciences</td>
			<td>QZBtotcol1</td>
			<td>option for detecting collagen content</td>
		</tr>
	</tbody>
</table>

layer-by-layer collagen deposition in microfluidic devices for microtissue stabilization

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

天然胶原可以使用甲基化和琥珀酰化创建用于在层 - 层沉积用聚阳离子和聚阴离子胶原溶液进行修改。琥珀酰化修饰天然胶原的ε-氨基与琥珀基团，和甲基化修饰天然胶原的羧基与甲基（图1A）。这些修改到胶原蛋白的氨基酸侧链改变pH滴定曲线的解决方案。琥珀酰化降低氨基团的数目，增加羧基基团的数目，而甲基化对氨基没有影响，并减少羧基基团的数目，相比于天然胶原?...

Watch this Scientific Journal Video about Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization at JoVE.com

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

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

层与层胶原沉积在微流体装置的Microtissue稳定

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

layer-layer-collagen-deposition-microfluidic-devices-for-microtissue

Research

JoVE Journal

Bioengineering

9.3K 次观看.  Massachusetts General Hospital, Harvard Medical School. The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

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

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

使用介电泳分离珠和多通道微流控芯片细胞和层流

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

科研

生物工程

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

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

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

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

微京尼平沉积技术的微模式化血管肌肉薄膜的扩展文化

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

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

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 ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

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

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...