部分支持这项研究是由美国国家科学基金会（NSF 1227359），西弗吉尼亚的EPSCoR程序由美国国家科学基金会（EPS-1003907），西弗吉尼亚ADVANCE办公室主办，由美国国家科学基金会（1007978），和WVU PSCoR的分别。 WVU共享研究设施（无尘室设施）和微流控结合蜂窝片上实验室（芯片实验室）在西弗吉尼亚大学的研究工作是在微细。聚焦成像做在WVU显微镜成像设备。

1。光致抗蚀剂光刻制作模具师傅以下协议显示制程制作的微通道直径在30-60微米之间。为了得到一个更小的直径（小于30微米），一个单一的光致抗蚀剂的旋涂的微通道是必要的。 <ol><li>转移从冰箱中在4℃下回流的光致抗蚀剂的洁净室在使用前24小时，并允许其升温至室温。 </li><li>清洁硅片上并烘烤1小时，在150℃下使其脱水。脱水将协助光致抗蚀剂的硅衬底的粘附性。 </li><li>旋涂第一层的光致抗蚀剂中使用下述配方: </li></ol><table border="1"><tbody><tr><td> 步骤 </td><td> 时间（秒） </td><td> 转速（rpm） </td><td> 促进 </td></tr><tr><td> 1 </td><td> 2 &lt;/ TD&gt; <td> 300 </td><td>最高</td></tr><tr><td> 2 </td><td> 10 </td><td> 0 </td><td></td></tr><tr><td> 3 </td><td> 3 </td><td> 300 </td><td>最高</td></tr><tr><td> 4 </td><td> 60 </td><td> 600 </td><td>最高</td></tr><tr><td> 5 </td><td> 10 </td><td> 500 </td><td>最高</td></tr><tr><td> 6 </td><td> 10 </td><td> 600 </td><td>最高</td></tr></tbody></table><ol start="4"><li>然后，在加热板上的温度为110℃，持续90秒烘烤晶片。软后烘烤的光致抗蚀剂的厚度是20-30微米。 </li><li>旋涂光致抗蚀剂后，使用相同的配方，用于第一层的第二层。 </li><li>软烤晶片，再通过将其放置在加热板上，在110℃的温度下，持续90秒。在此之后软烘焙光致抗蚀剂的厚度是40-60微米。 </li><li>产生正面主图案露出photorESIST 14500兆焦耳/厘米2的曝光量，通过UV光的膜掩模。 </li><li>稀释用的去离子（DI）水（1:2（体积/体积））的显影剂。重复冲洗晶片，在溶液中，直至图案被充分发展。然后用去离子水清洗晶圆，使用氮气和干燥。 </li><li>回流焊:晶片放置在加热板上，用温度为120℃，4分钟和盖的玻璃培养皿中，以防止溶剂蒸发。从加热板上取下晶片，并使其冷却至室温。回流焊后的光致抗蚀剂的厚度为50-60微米。 </li></ol> 2。软光刻技术制作PDMS微通道网络<ol><li>准备按重量比为10:1（基料:固化剂）和聚二甲基硅氧烷（PDMS）的解决方案，彻底混合使用行星离心混合器。 </li><li>到回流的光致抗蚀剂母模铸造的PDMS解决方案。在干燥器中15分钟进行脱气，将浇铸的硅橡胶。使用氮气，以去除任何剩余的气泡，如果必要的。 </li><li>烤的PDMS在烘箱中在60℃的温度下3小时，以允许其固化。从主模，然后取出固化硅橡胶层。 </li><li>使用削尖的打孔器，创建入口/出口孔打孔渠道网络。使用氮气清洁的PDMS表面。 </li><li> 30秒内的等离子清洗机在操作压力4.5×10 -2乇，氧流量为3.5英尺3 /分氧等离子体处理将两个PDMS层。然后，对准的PDMS表面，在光学显微镜下手动。使用一滴水，如果需要更好地控制对齐。 </li><li>烘烤设备，在烘箱中在60℃下30分钟，以实现永久性粘接。 </li></ol> 3。脐静脉内皮细胞的培养和幼苗在芯片<ol><li>培养的人脐静脉内皮细胞与培养液中补充10％胎牛血清（FBS），L-谷氨酰胺的。日ê实验2和5之间的通道被使用。 </li><li>一旦融合的人脐静脉内皮细胞，计数细胞，收获他们首先用HEPES缓冲盐溶液（HEPES-BSS）冲洗细胞，然后把细胞用胰蛋白酶/ EDTA和孵育在37℃下2-6分钟胰蛋白酶消化完成后，用胰蛋白酶中和溶液中的胰蛋白酶/ EDTA。离心，悬浮细胞培养基中的8％右旋糖酐收集它们。葡聚糖用于增加介质的粘度，以帮助更好的细胞播种和附件。 </li><li>治疗设备5分钟，操作压力为4.5×10 -2乇和氧流速为3.5英尺3 /分钟的氧等离子体。然后加载装置与去离子水，并用UV光8小时在层的生物安全罩进行杀菌处理。 </li><li>前一天细胞都准备好了，洗设备1X磷酸盐缓冲液（1X PBS），然后大衣，纤维连接蛋白（100微克/毫升，DILuted用1×PBS中），并在冰箱中在4℃过夜孵育。 </li><li>纤连蛋白涂层后，用1×PBS中，然后用培养液加载洗设备。 15分钟，温度为37°C孵育设备。 </li><li>负载HUVEC细胞在8％葡聚糖培养基中，细胞浓度为3-4×10 6细胞/ ml。细胞20μl的液滴放置在该设备的一个入口，并倾斜到微流体通道中引入细胞。 15-20分钟后，细胞就会开始附加到通道的侧壁。旋转的移动设备创建一个更均匀的分布，细胞每15分钟。如果有必要，可以进行额外的负荷。 </li></ol> 4。长期灌注设定的所附的人脐静脉内皮细胞静态培养5-6小时后，将开始完全摊开。设置使用远程控制的注射泵系统10微升/小时源源不断的灌注。灌注可以调整的F或更高的流速，并且可以持续4天至2周之间的时间周期。 5。细胞染色和显微镜表征<ol><li>当细胞达到汇合里面的设备中，首先，洗涤装置用1x PBS彻底清除介质。稀释剂（4微升1毫升）稀释，用红色的染料，然后加载设备。加载染料的细胞装载过程相似。孵育的移动设备在黑暗中，在室温下5分钟，然后用培养液中洗设备停止染色。潜伏期长的染料可以导致细胞毒性和disadhesion。 </li><li>蓝色染料用1×PBS稀释（每毫升含2滴），载入装置。在室温下在黑暗中孵育5分钟，然后彻底清洗装置，用1×PBS。 </li><li>一个倒置的光学显微镜下检查细胞染色。如果染色还是不错的，与固定介质加载的微（3.5％多聚甲醛与1X PBS稀释），然后淹没固定介质的设备中，完全覆盖铝箔。将装置存放在冰箱中，在温度为4°C，以防止设备从干燥和漂白。是固定的移动设备现在已准备好用于共焦成像，这可以通过激光扫描共聚焦显微镜。 </li></ol>

作者宣称，他们有没有竞争经济利益。

<html> 1。主模具制造血管形态计量学的设计和指导原则之一是被称为穆雷的第16条，其中规定，受整个网络分布的血管直径最小能量代价。报告还指出，在分叉的父容器的直径的立方等于总和的女儿血管直径的立方（ <img alt="公式1" fo:content-width="0.9in" fo:src="/files/ftp_upload/50771/50771eq1.jpg" src="/files/ftp_upload/50771/50771eq1.jpg" width="100px" /> 19）此外，泊肃叶定律已被用于估?...

微血管，作为流通体系的一部分，调解血液和组织之间的相互作用，支持新陈代谢活动，定义组织微环境，在许多健康和病理条件下发挥了关键作用。再演在体外的功能微血管提供了一个平台，为研究复杂的血管现象。然而，传统的在体外微血管检测，如内皮细胞迁移测定，内皮细胞管腔形成的测定中，大鼠和小鼠的主动脉环测定，无法重新创建的三维（3D）的几何形状和连续的流量控制相对于体内微血管1-8。微血管的研究使用的动物模型中和在体内测定，如角膜血管生成法，鸡胚尿囊膜血管生成实验，Matrigel栓实验，是更费时，成本高，相对于观察和量化挑战，并引发伦理问题1，9-13。 显微制造和微流控芯片技术的进展已启用了各种各样的见解，到生物医学科学在削减高的实验与动物体内研究，如容易和严格控制的生物条件和动态流体环境中，不会有相关的成本和复杂性的同时一直未能与传统的大容量的技术。 在这里，我们提出了一种方法来构建内皮化微通道上的单芯片模拟3D几何体内微血管和控制的连续灌流采用回流焊的光致抗蚀剂光刻技术，软光刻技术和微流体的结合下运行。

<table border="1">
	<tbody>
		<tr>
			<td>Reagent/Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflow Photoresist</td>
			<td>AZ Electronic Materials</td>
			<td>AZP4620</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>AZ Electronic Materials</td>
			<td>AZ 400K</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB 131 Culture Medium</td>
			<td>Invitrogen</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr>
			<td>NacBlue Nuclei Staining</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH Red Stain</td>
			<td>Sigma</td>
			<td>MINI26 and PKH26GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Gibco</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffered Saline Solution</td>
			<td>Lonza</td>
			<td>CC-5024</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Invitrogen</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution</td>
			<td>Lonza</td>
			<td>CC-5002</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS Curing Agent</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Human Umbilical Vein Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-501F</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma</td>
			<td>CGLDIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Invitrogen, Molecular Probes</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma</td>
			<td>95771</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5% Paraformaldehyde</td>
			<td>Electron Microscopy Science</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner</td>
			<td>Laurell Technologies Corporation</td>
			<td>WS-400BZ-6NPP/LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>BelArt Products</td>
			<td>999320237</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump System</td>
			<td>Harvard Apparatus</td>
			<td>PHD Ultra</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Biosafety Hood</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Planetary Centrifugal Mixer</td>
			<td>Thinky</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp Oven</td>
			<td>Fisher Scientific</td>
			<td>13-246-516GAQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Zeiss</td>
			<td>Invertoskop 40C</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Barnstead/Thermolyne Cimarec</td>
			<td>SP131635</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
	</tbody>
</table>

procedure for the development of multi-depth circular cross-sectional endothelialized microchannels-on-a-chip

努力一直专注于发 ​​展在体外实验研究微血管，因为动物体内研究更费时，昂贵，观察和量化是非常具有挑战性的。然而，传统的在体外微血管检测体内微血管代表相对于三维（3D）的几何形状，并提供连续的流体流动的限制。使用回流焊的光致抗蚀剂光刻技术，软光刻技术，以及微流控芯片的组合，我们已经制定了多深的圆形横截面内皮化微的芯片，它模仿3D几何体内微血管和运行控制连续灌注下流程。使用了一个正的回流的光致抗蚀剂，制作母模的半圆形横截面的微通道网络。由对准和粘接的聚二甲基硅氧烷（PDMS）的微通道REPL从主模具icated，的圆柱形微通道网络已创建。微通道的直径可以得到很好的控制。此外，原代人脐静脉内皮细胞（HUVECs）表明，在芯片内接种细胞排列的微通道的内表面上为4天至2周之间的时间周期根据控制灌注持久。

我们的方法来制造微通道网络的多深度模仿体内微血管的复杂的三维几何形状，其中的微通道具有圆形横截面15。此外，父分支通道的女儿通道的直径近似服从默里定律保持在所需的水平的流体流动，使整体的流路阻力变低，流动速度更均匀的整个网络16-18。 电影1，图2，和电影 ，分别为一个半圆形的光致抗蚀剂制造模具师傅和圆形的横截面PDMS微?...

Watch this Scientific Journal Video about Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip at JoVE.com

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Â微通道上的一个芯片平台开发回流焊的光致抗蚀剂光刻技术，软光刻技术和微流体的结合。模拟的三维（3D）的几何形状在体内微血管内皮化的微通道平台，控制连续灌注流量下运行，允许高品质和实时成像，并可以应用对微血管研究。

发展多深的圆形横截面微内皮化的一个芯片的程序

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

procedure-for-development-multi-depth-circular-cross-sectional

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

13.8K 次观看.  West Virginia University.  Â微通道上的一个芯片平台开发回流焊的光致抗蚀剂光刻技术，软光刻技术和微流体的结合。模拟的三维（3D）的几何形状在体内微血管内皮化的微通道平台，控制连续灌注流量下运行，允许高品质和实时成像，并可以应用对微血管研究。 

视频: Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.

方法Nanoformulated的抗逆转录病毒药物的血源性巨噬细胞运输的发展

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

科研

生物工程

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

龙工程的程序

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

内皮化微流体研究在血液病的微血管相互作用

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

脱细胞猪心逆行灌注的程序

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

从藻类水热液化使用二维气相色谱时间飞行质谱法水提取部分的定性鉴定

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used techniques for the development of in vivo cancer models have considerable limitations. Therefore, in this study, we aim to implement cell sheet technology to develop an in vivo cancer model. Hepatocellular carcinoma (HCC) is successfully developed in nude rats using cell sheets created from HCC cell line cells. The cancer cell sheets are generated through intracellular adhesion and the formation of a stratified structure, controlled by the extracellular matrix. This allows for the HCC sheet transplantation into the liver and the creation of a tumor-bearing animal model within a month. In addition, the role of mesenchymal stem cells (MSC) in the development of this cancer model is investigated. In addition to the HCC cell line sheet, another two cell sheets are created: a sheet of HCC cells and bone marrow MSCs (BMMSCs) and a sheet of HCC cells and umbilical cord MSCs (UCMSCs). Sheets that have a combination of both HCC cells and MSCs are also capable of producing a tumor-bearing animal. However, the addition of MSCs reduces the size of the formed tumor, and this adverse effect on tumor development varies depending on the used MSCs' source. This indicates that a cell sheet made of certain MSC subtypes could be utilized in tumor management and control.