这项工作得到了科学与台湾技术下的授权号101-2221-E-002-064-MY3部的支持。

1.制造微流体装置<ol><li>使用图形排版软件（ 如 AutoCAD的）画一个T-微通道的轮廓。为T形微通道中，两个进料通道90微米宽和2500微米长，合流通道为180μm宽和3000微米长。每个通道的一端连接到一个单独的圆的直径为1100微米。 </li><li>马克“清除”和“黑暗”的曝光和覆盖的区域，分别。对于负光致抗蚀剂（ 例如，SU-8）中，T-微通道的形状是“清除”和周围是“暗”。 </li><li>使用激光图案发生器具有442纳米的波长为2微米的最小特征尺寸，以T型微通道的图案转印到铬在玻璃上的光掩模。 </li><li>使用的光掩模，基板（ 例如，单面抛光的硅晶片）和永久环氧光致抗蚀剂（ 例如，SU-8），以通过一个标准的光刻工艺使一个模具。光致抗蚀剂层是55.2微米厚。在一般情况下，光致抗蚀剂厚度应比物镜17-19相关的深度薄。 </li><li>使用的模具和透明材料，例如聚二甲基硅氧烷（PDMS）来制造T型微通道20。 </li><li>用于流体连接的，使用2毫米的不锈钢管外径穿通孔对准PDMS上的圆形图案。 </li><li>治疗的PDMS和用氧等离子体于60W下30秒的玻璃载片的表面上。附加的PDMS到载玻片上。两种材料的氧化表面形成强大的粘合。将粘结的PDMS结构的热板上进行5分钟，在120℃。 </li><li>插入特氟隆管放置在冲压孔对流体连接。 </li></ol> 2.实验装置<ol><li>从Hoffma构建微型纹影系统n个调制对比度显微镜通过在冷凝器的前焦平面除去狭缝板和带刀口在5X物镜3的后焦平面替换调制器。相关的深度，这取决于目标17-19的数值孔径，应足以覆盖所述微流体器件的整个深度。刀口的表面通过阳极氧化铝变黑来降低其反射率。 </li><li>通过C型安装适配器安装高速摄像机显微镜的三目镜筒。有相机通过一个分束器朝向显微镜的光路中。通过以太网电缆将相机连接至一台台式电脑。相机设置的伽马校正为1，即它的灰度读数是正比于输入亮度。 </li><li>打开光源。为了避免过多的热量，使用LED（发光二极管）照明。 </li><li>使用图像处理软件（ 如&lt;/ em&gt;的在MATLAB函数imread），以获得所获取的图像的灰度级值。除去刀口，调整照明，光圈和曝光时间，以使图像的平均灰度读数比最大值低约10％。这表示背景强度为0％的截止和我们使用230的值对于8位图像。 </li><li>插入刀口完全阻断入射光。记录的图像的平均灰度读数。这表示本底密度为100％的截止和值约为15 8位图像。 </li><li>调整刀口的位置，使得所获取的图像的平均灰度读数在于为0％和100％的截止值的中间。现在截止的程度被设置为50％。 </li><li>准备两个透明流体与已知折射率21彼此作为组分完全混溶。为了评价屈光IND的依赖恩对混合物浓度，检查文献21或使用格莱斯顿，戴尔方程22。如果曲线是非线性的，在整个范围内，挑其它流体组分。然后，选择一个指定的组合物，低于该溶液的折射率与浓度线性变化。例如，使用稀的含水乙醇为0.05质量分数和水作为工作流体。 </li><li>把T型微通道上的试样台。安排T形微通道因此与平行于刀口合流通道（ 图1）。 </li><li>准备两个相同的注射器：注射器A被填充有工作流体在作为参考流体（水），和注射器B被填充有其它工作流体（稀乙醇水溶液）。注射器的大小取决于所需的流量Q和注射泵的规格：Q =πD2 V / 4，其中d是SYR的内径英格和V是柱塞的速度。流量脉动通常可以通过选择一个小的注射器，以增加V 23来防止。 </li><li>收集从T-微通道中的烧杯中的出口流体。确保特氟隆出口管固定到烧杯的壁和其端部是在液面以下，在烧杯中，以避免这将是由液滴片断离引起的振动。 </li></ol> 3.校准<ol><li>获得两种流体混合和参考图像的图像。 <ol><li>在给定的雷诺数 Re，设置注射泵的流速，Q。Q选自Q =μ（W + D）的重 /4ρ，其中，μ和ρ是粘度和工作流体的密度，和W计算和 D是T型微通道的合流通道的宽度和深度，分别。 </li><li>装入一张泵用注射器A和其他泵SYB. RINGE连接T-微通道的两个入口通过聚四氟乙烯管注射器和注射器湾启动注射泵输​​出工作流体到T-微通道以相同体积流量。 </li><li>等到源源不断建立。稳流状态是由一个固定的图案纹影的出现定义。 </li><li>使用相机控制软件来记录流体混合的20个帧在30帧每秒的帧率。 </li><li>停止其装入注射器B中的泵只有通过一个入口泵送参考流体（水）到T-微通道以恒定速率合流通道。 </li><li>等到稳流状态达到无条纹图案是观察。 </li><li>使用相机控制软件作为参考图像，当没有光学不均匀性存在于T形微通道。记录20个帧在30帧每秒的帧率。 </li><li> 3.1.1重复3.1.7到在不同的雷诺数： 重新 = 1，5，10，20和50，以便没有复杂流动结构出现在T-微通道24的合流区域。 </li></ol></li><li>使用图像处理软件由参考图像I 0（I，J）25，其中i和j是该像素指数来划分所获取的图像I（i，j）的 。 </li><li>采用CFD（计算流体动力学）封装，以模拟在T-微通道中的指定的流体的混合。 <ol><li>构造为T型微通道的几何形状的三维模型。离散流域划分为结构化网格。提高精度，采用更细的网眼的合流和T-微通道的中心区域。 </li><li>分配的流体的物理性质，并建立边界条件到流域。在求解过程中，确定从在最后获得的浓度的浓度依赖性的扩散系数迭代26，以更新的局部浓度。 </li><li>通过执行电网研究27检查的计算结果的敏感性。 </li></ol></li><li>对于每一个节点（X I，Y I）在xy -平面，应用CFD后处理工具，采取跨航道水深浓度场的平均值由梯形规则：W（X I，Y j）条 = {ΣK表 [ 瓦特（X I，YĴ和z k）的 + W（X I，YĴ和zķ1）]∙（Zķ1 - z k）的 / 2} / D 28，其中D是通道深度。使用中心差分格式来计算deriva集中略去相对于交叉流方向：（∂W /∂Y）I，J = [W（X I，YĴ+1） - W（X I，YĴ-1）] /（YĴ +1 - ÿĴ-1）。 </li><li>对于正的和负的梯度，提取灰度值I / I 0（在3.2中获得）和质量分数的梯度∂瓦特/∂ý在指定的地点，例如y = 0 的 （中心线（在3.4获得），流向的比率方向）或各种给定的x（横流方向）。 </li><li>画出的结果，并确定我之间的关系/ I 0和∂W /∂Y，I / I 0 </sub&gt;为C 1〜∂重量/∂Y + C 2（C 1和C 2是常数），用线性回归。 </li></ol> 4.定量<ol><li>重复步骤3.1至3.2中的目标微流体装置中混合。目标微流体装置的深度应该是相同或接近的T-微通道。如果不稳定现象有望，获取视频剪辑（图像序列）的步骤，而不是3.1.4。帧速率应该足够高，以解决瞬态流动明确的动态，而曝光时间应该是相同的，以在2.4，2.5，3.1.4和3.1.7中使用的值。 </li><li>使用在步骤3.6中得到的关系为灰度值的比率转换为质量分数的目标微流体装置的梯度。 </li></ol>

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作者什么都没有透露。

用于在微流体装置的流体的混合，所述微尺度纹影技术能够通过在光强度量化变化来测量浓度梯度的大小。因为这种技术的原理依赖于探测光的传播的轮换时，工作流体和微流体装置必须是透明的入射光。此外，该协议要求的溶液，其组成，以使工作流体的初步评估是必不可少的折射率之间的线性关系。除了 ​​乙醇水溶液在此证明，微型纹影技术成功地应用于测量盐度梯度29和solutocapil...

流体混合]是在许多工业过程和生物系统中发现的一个重要问题。随着微流体的出现，在微型混合带来了太多的关注，因为大众运输机制之间的扩散统治的挑战。由于设计一个有效所需的定量微混合器验证，几种测量方法被开发5-7。然而，该三维结构中，在有效的微混合器5通常发现，要求的共同测量技术不能提供浓度场的更精确的表示。由于观看角度8或反应动力学6的限制，上述方法可产生不正确地占混合物的均匀误导的结果。 对于光学透明的液体光学透明的微观混合，微型纹影技术3,9-14 </SUP&gt;提供了一个有吸引力的替代分析混合不均匀。在过去，微型纹影技术已主要用于可视可压缩流动9-13，15或相位梯度16。从两个简单的光学布局和高灵敏度的微型纹影技术的好处，能够实现特定的流动特性，使得光学干扰而非常适合于在评估混合使用不仅非侵入性的调查。在本文中，我们通过将刀口在显微镜的物镜的后焦平面构建微尺度纹影系统，描述的校准步骤，以实现定量分析，并在微流体振荡器4报告验证测量。为了实现测量时，工作流体被适当地选择，使得混合的流体的折射率与该组合物线性地变化，并且目标的微流体装置的厚度是相同的上ê用于校准。此外物种浓度，微量纹影技术可以扩展到测量其它标量是线性相关的折射率，如温度或盐度的梯度。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Permanent Epoxy Negative Photoresist</td>
			<td>MicroChem</td>
			<td>SU-8 2150</td>
			<td></td>
		</tr>
		<tr>
			<td>single side polished silicon wafer</td>
			<td>Light Technology</td>
			<td>S4W1PP5SABUP1</td>
			<td>p-type, diameter: 100&#177;0.5 mm, thickness: 525 &#177;25 mm, orientation: (1 0 0)</td>
		</tr>
		<tr>
			<td>syringe pump</td>
			<td>kdScientific</td>
			<td>kds210</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe, i.e. 10 ml</td>
			<td>Terumo</td>
			<td>SS-10L2138</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoffman modulation contrast microscope</td>
			<td>Leica Microsystems</td>
			<td>DM IL LED</td>
			<td></td>
		</tr>
		<tr>
			<td>5X objective lens</td>
			<td>Leica Microsystems</td>
			<td>N PLAN</td>
			<td>NA = 0.12</td>
		</tr>
		<tr>
			<td>knife-edge</td>
			<td></td>
			<td></td>
			<td>custom made part</td>
		</tr>
		<tr>
			<td>camera, i.e. high speed</td>
			<td>Integrated Design Tools</td>
			<td>NX7-S1</td>
			<td></td>
		</tr>
		<tr>
			<td>C-mount adapter HC 0.63x</td>
			<td>Leica Microsystems</td>
			<td>541537</td>
			<td></td>
		</tr>
		<tr>
			<td>camera operating software</td>
			<td>Integrated Design Tools</td>
			<td>MotionPro X Studio 2.02.01</td>
			<td></td>
		</tr>
		<tr>
			<td>polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>Sylgard-184 silicone elastomer kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon tubing, i.e. O.D. x I.D. 1/16 in. x 0.031 in.</td>
			<td>Supelco</td>
			<td>58700-U</td>
			<td></td>
		</tr>
		<tr>
			<td>micro glass slide</td>
			<td>Matsunami Glass</td>
			<td>S2215</td>
			<td></td>
		</tr>
		<tr>
			<td>hot plate</td>
			<td>Yeong-Shin</td>
			<td>HP-303DN</td>
			<td></td>
		</tr>
		<tr>
			<td>distilled water, i.e. HPLC grade</td>
			<td>Alps Chemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethyl alcohol, i.e. reagent grade</td>
			<td>Nihon Shiyaku Reagent</td>
			<td>EA448652</td>
			<td></td>
		</tr>
		<tr>
			<td>image processing software</td>
			<td>Mathworks</td>
			<td>MATLAB R2009a</td>
			<td></td>
		</tr>
		<tr>
			<td>computational fluid dynamics package</td>
			<td>ESI group</td>
			<td>CFD-ACE+ 2008</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing mixing inhomogeneity in a microfluidic device by microscale schlieren technique

在本文中，我们介绍了利用微型纹影技术来衡量一个微流体装置的混合不均匀。微尺度纹影系统从霍夫曼调节对比度显微镜，它提供了容易获得的物镜的后焦平面构造，通过除去狭缝板和带刀口替换调制器。微尺度纹影技术的工作原理依赖于检测由折射率1-3的变化的光偏转。偏转光要么逃逸或被刀口受阻，产生明亮或暗带，分别为。如果该混合物的折射率随其组成而线性变化，在光强度在图像平面中的局部变化是正比于浓度梯度垂直于光轴。微纹影图像为按三维不均匀性产生的干扰光的二维投影。 为了完成定量分析，我们描述了在T形微通道混合两种流体的校准步骤。我们进行了数值模拟，得到在T-微通道，与相应的微纹影图像相关密切的浓度梯度。通过比较，微纹影图像的灰度读数和在微流体设备呈现的浓度梯度之间的关系被建立。使用这种关系，我们能够分析来自准微纹影图像的混合不均匀性，并证明微尺度纹影技术的能力与测量微流体振荡器4。用于光学透明流体，微尺度纹影技术是一个有吸引力的诊断工具，以提供其保留的混合过程的三维特征的瞬时满场信息。

灰度比I / I 0 的下不同雷诺数为正的和负的质量分数的梯度示出（ 图2）具有对称带出现在T型微通道的中间。在低雷诺数，纹影带的尾部被扩展并模糊由于横跨混合界面的分散。雷诺数增加时，扩散长度缩短导致更窄的频带。在不同的下游位置，强度变化ΔI的变化量/ I 0沿着横流方向定量描绘（ 图3）。从校准过程的结果?...

Watch this Scientific Journal Video about Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique at JoVE.com

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

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 ...

analyzing-mixing-inhomogeneity-microfluidic-device-microscale

Research

JoVE Journal

Bioengineering

8.9K 次观看.  National Taiwan University. 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. 

视频: Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

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

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

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 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 ...

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 ...

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.

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 ...

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 ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

使用高通量微流体设备生成动态环境条件

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

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

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

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

生理尺寸微流控培养装置中上皮细胞单层的制备和结构评价

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