这项工作是支持部分由美国国家科学基金会（CBET 0846453）和美国国立卫生研究院（1R01GM089999）。

1。制备硅的主人，使用标准的SU - 8光刻1（在这个视频没有显示）。 <ol><li>使用标准的SU - 8的光刻方法来创建一个SU - 8的“主人”（SU - 8月到2050年，MicroChem，牛顿，MA）在洁净室中制造不同的组件（不同厚度的PDMS膜，合作文化室）。这种掌握模具，可以制作任何微细加工设施（例如，斯坦福大学的微流体铸造<a href="http://thebigone.stanford.edu/foundry/">;</a> http://thebigone.stanford.edu/foundry/） ​​。 </li><li>的PDMS复制之前，公开的SU - 8的主人fluorosilane蒸汽，在连接到真空源，以方便从主PDMS容易释放干燥器 。添加fluorosilane下降到纸巾，并申请一分钟的真空。拆下真空，并允许30分钟fluorosilane沉积。存放在密闭容器中以备将来使用SU - 8的主。 </li></ol> 2。 PDMS 2主从SU - 8的副本成型<ol><li>流体层和控制层是由硅橡胶成型的SU - 8主副本。 </li><li> 10:1 WT混合的PDMS预聚物和交联剂。比和德加在1小时（或直到气泡从PDMS的混合物中删除）的干燥器。 </li><li>放置在培养皿SU - 8的主，并仔细的SU - 8的掌握上的PDMS混合物倒入。德加再次在PDMS的。 </li><li>在1200转每分钟一分钟的旋转模具，模具厚度约100μm的一个统一的PDMS。 </li><li>热培养皿中包含一个小时的PDMS和SU - 8母模，在80 ° C固化的PDMS。 </li><li>取出固化硅橡胶覆盖的电磁炉SU - 8的主。 </li><li>去皮，从SU - 8的主的PDMS模具。 </li><li>在PDMS模具钻四个孔，使用20号钝针两种细菌的​​入口，一个出口和一个气动接口。 </li></ol> 3。粘接多层PDMS设备：该设备是组装三个层次的顶层，其中包含的主渠道，中间气动层，和自下而上的细菌层，包含细菌的渠道。三层组装如下所述。 <ol><li>湿的PDMS膜用甲醇和仔细剥离他们从SU - 8的主使用一个干净的镊子模具。将膜在聚四氟乙烯薄片。 </li><li>将气动薄膜氧离子室和流体层。打开血浆为40秒（氧压10 SCCM，射频功率100W）。 </li><li>曝光后的血浆中，对齐（10分钟内）使用镊子在底部的PDMS通道的膜。膜与硅橡胶层上添加甲醇，有利于自由流动层，使校准更容易。在这种情况下，设备被放置在一块纱布，使甲醇蒸气逃脱。 </li><li>对齐的PDMS层固化在80℃的烤盘上8小时</li><li>到底层的气动薄膜粘合后，重复步骤3.2 - 3.4债券到组装复合硅橡胶设备顶级渠道。 </li></ol> 4。组装的PDMS设备3,4 <ol><li>孔钻在播种真核细胞和细菌所使用的进口和出口。 </li><li>聚乙烯管（0.01英寸外径）连接的端口和操作气动层，用20ml注射器拉。 </li><li>广场的PDMS模型和预清洁的玻片上一个成一个氧离子室。血浆转20秒（氧气压力为20 SCCM，射频功率300W）不应用真空。 </li><li>为了防止岛内墙（PDMS）粘接到玻璃幻灯片之间的接触，立即注射器连接到设备，适用于真空拉动注射器和到位。 </li><li>轻轻地按在玻璃上的PDMS模型（在2分钟内）。 </li><li>固化的PDMS设备在80 ° C为10分钟。 </li></ol> 5。玻璃表面与纤维连接蛋白治疗<ol><li>在紫外光下消毒15分钟后，聚乙烯管连接到每一个端口的设备。 </li><li>填充和流入的渠道灭菌的PBS的通道，以消除任何碎片。应用流量为10分钟，以去除气泡。气泡可以很容易地逃脱的PDMS由于其气体渗透。 </li><li>气动通道，以防止气泡形成的通道进入一个填充色染料溶液。 </li><li>为了方便在玻璃上的真核细胞播种，引入设备的纤维连接蛋白1μg/ mL的溶液，孵育40分钟，在37 ° C。 </li><li> 1毫升培养基洗净多余的纤维连接蛋白。 </li></ol> 6。共生的大肠杆菌的形成和测量大肠杆菌生物膜IN群岛<ol><li>淡化M9基本媒体的共生细菌（ 如大肠杆菌BW25113/pCM18） 外径 600〜1形成一个封闭商会/离岛的共生生物膜。 </li><li>关闭岛内墙应用正液体压力。一个自制的空气，液体压力转换器是用来防止空气泡沫形成的通道。 </li><li>引进共生的大肠杆菌与阀岛的大肠杆菌封闭，并且使细菌附加1小时32 ° C 。 </li><li>洗净独立的细菌和灌注在12小时0.25mL/hr稀释的菌悬液（外径600〜0.05）。 </li><li>冲洗用新鲜的DMEM培养液生长介质的生物膜去除松散附着的细菌和真核细胞附着的准备。 </li><li>图片生物膜，使用了Leica TCS SP5共聚焦激光扫描显微镜（班诺克本，IL），和可视化生物膜结构，使用IMARIS软件5。 </li></ol> 7。播种真核细胞周围的细菌群岛6-8 <ol><li> Trypsinize接种密度为5 × 10 5细胞/ mL HeLa细胞组织培养瓶和媒体的DMEM悬浮。 </li><li>在流速2 mL / min的使用Picoplus注射泵（Apparattus哈佛大学，马萨诸塞）引进设备的HeLa细胞。确保岛内墙降低，使细菌生物膜从HeLa细胞的区域是分开的。这一步应该是执行上的光学显微镜的阶段，使播种均匀，可确定。 </li><li>夹紧单元插座，以减少渠道和孵育6 h后，在37 ° C，5％二氧化碳培养箱运动的细胞悬液。 </li><li>灌注0.5毫升/小时的设备删除独立的细胞生长介质。 </li><li>刷新HeLa和共生的大肠杆菌的DMEM媒体大肠杆菌每6小时。 </li></ol> 8。进岛和接触到真核细胞的致病菌简介<ol><li>准备E。大肠杆菌 O157：H7型（肠出血性大肠杆菌）感染HeLa细胞的感染复数（MOI）为100:1 200:1（每HeLa细胞的肠出血性大肠杆菌的数量） 。 </li><li>冲洗和洗出松散连接的共生细菌。 </li><li>引入岛屿稀肠出血性大肠杆菌悬液（外径600〜0.1）。 </li><li>在37 ° C至5％的CO 2培养箱中培养6小时无流使肠出血性大肠杆菌暴露于共生生物膜中存在的信号的设备。 </li><li>电梯的PDMS墙暴露在共生岛屿6个小时的HeLa细胞的肠出血性大肠杆菌。 </li><li>用PBS冲洗通道，并使用活/死的试剂盒（L3224，Invitrogen公司，加利福尼亚州）的活的和死的细胞染色。 </li><li>在ZEISS AXIOVERT 200M荧光显微镜和图像的多个地点，估计活的和死细胞的数量。 </li></ol> 9。代表性的成果9 消化道感染的事件的顺序是模仿开发的HeLa细胞单层和共生的大肠杆菌大肠杆菌生物膜和揭露肠出血性大肠杆菌的共生生物膜感染的HeLa细胞之前。制造微流体的共培养模型中所涉及的步骤如图1所示，图2a显示了一个共同的文化设备的三维渲染。在这两个地区共同培养装置的真核细胞培养室和细菌岛屿如图2b所示，用不同颜色的染料（真核细菌岛地区和绿色紫色）。显示在图2C使用彩色染料的细菌培养地区隔离开来的真核周围地区的可行性。图3显示了有代表性的结果，从上皮细胞和细菌共同培养，图3a所示的共生生物膜（绿色）岛的殖民统治肠出血性大肠杆菌（红色） 图3b显示并列的上皮细胞和共生的大肠杆菌与肠出血性大肠杆菌大肠杆菌的细菌岛。肠出血性大肠杆菌和绿色荧光蛋白表达的共生细菌的表达RFP的本地化中的PDMS墙降低时的岛屿，说明上皮细胞的分离细菌岛可行性。 <img src="/files/ftp_upload/1749/1749fig1.jpg" alt="图1" /> 图1：细胞共培养模型的播种计划。 （1）降低的PDMS墙形成一个岛屿，共生细菌引入岛内，和纤维连接蛋白是环岛流。 （2）HeLa细胞接种在岛屿四周的地区。 （3）在HeLa细胞达到汇合和已开发的共生生物膜，肠出血性大肠杆菌引入岛内。 （4）取消的PDMS墙揭露HeLa细胞周围的岛屿，以肠出血性大肠杆菌。插图显示阀门操作细节。 <img src="/files/ftp_upload/1749/1749fig2a.jpg" alt="图2a" /> <img src="/files/ftp_upload/1749/1749fig2bc.jpg" alt="图2BC" /> 图2：微流控模型的上皮细胞和细菌共同培养 。 （一）立体移交逃犯的合作文化诱捕地区的气动驱动装置上皮细胞之间形成的细菌群岛。每个细菌的岛屿（1200微米直径，除了1000微米）提供生长介质，并消除浪费从岛上有一个单独的入口和出口。 （二）显微设备的颜色显示不同地区的染料（区上皮细胞，细菌群岛）共同培养。 （三）气动诱捕系统的保真度降低墙上的PDMS（左侧面板）使用气动激活通道（蓝色），紫色染料引入封闭岛群岛，和它周围的黄染48小时流动当PDMS的墙（右图）提出，岛屿地区是暴露在周围的黄色染料。比例尺代表500微米。 <img src="/files/ftp_upload/1749/1749fig3.jpg" alt="图3" /> 图3：HeLa细胞和细菌共培养 。 （一）荧光图像RFP的表达肠出血性大肠杆菌和GFP表达大肠杆菌在岛的大肠杆菌BW25113 。 （二）的传输覆盖，绿色，和红色荧光图像设备。比例尺代表200微米。

<ol>
	<li>McDonald, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prototyping+of+microfluidic+devices+in+poly(dimethylsiloxane)+using+solid-object+printing.">Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.</a> Anal Chem. 74, 1537-1545 (2002).</li><li>Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+integrated+passive+valves+and+pumps+for+flexible+polymer+3-dimensional+microfluidic+systems.">Design and fabrication of integrated passive valves and pumps for flexible polymer 3-dimensional microfluidic systems.</a> Biomed Microdevices. 4, 117-121 (2002).</li><li>Baek, J. Y., Park, J. Y., Ju, J. I., Lee, T. S., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumatically+controllable+flexible+and+polymeric+microfluidic+valve+fabricated+via+in+situ+development.">A pneumatically controllable flexible and polymeric microfluidic valve fabricated via in situ development.</a> J Micromech Microeng. 15, 1015-1020 (2005).</li><li>Grover, W. H., Ivester, R. H., Jensen, E. C., Mathies, R. A., A, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+multiplexed+control+of+latching+pneumatic+valves+using+microfluidic+logical+structures.">Development and multiplexed control of latching pneumatic valves using microfluidic logical structures.</a> Lab Chip. 6, 623-631 (2006).</li><li>Lee, J., Jayaraman, A., Wood, T. K., K, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indole+is+an+inter-species+biofilm+signal+mediated+by+SdiA.">Indole is an inter-species biofilm signal mediated by SdiA.</a> BMC Microbiol. 7, 42-42 (2007).</li><li>Hsu, C. H., Folch, A. <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+with+tunable+topographies.">Microfluidic devices with tunable topographies.</a> Appl Phys Lett. 86, 023508-023508 (2005).</li><li>Hui, E. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+control+of+cell-cell+interactions.">Micromechanical control of cell-cell interactions.</a> Proc Natl Acad Sci USA. 104, 5722-5726 (2007).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+sensing+hydrogel+microstructures+into+micropatterned+hepatocellular+cocultures.">Integrating sensing hydrogel microstructures into micropatterned hepatocellular cocultures.</a> Langmuir. 25, 3880-3886 (2009).</li><li>Kim, J., Hegde, M., Jayaraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+epithelial+cells+and+bacteria+for+investigating+host-pathogen+interactions.">Co-culture of epithelial cells and bacteria for investigating host-pathogen interactions.</a> Lab-on-Chip. , (2009).</li></ol>

病原体附着和殖民化的常规检测，利用单层培养板中的真核细胞添加到其中的病原体。这些模型不是生理有关，因为他们没有纳入一个真核细胞共生细菌生物膜开发。预先生长的细菌培养的简单相加，真核细胞是不大可能导致这种构象，作为生物膜是高度的组织结构，随着时间的推移发展，是非常困难的，如果没有几乎是不可能的的，在细菌的存在真核细胞中的文化长时间没有生存能力的重大损?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>SU-8 2050</td>
<td>&nbsp;</td>
<td>Microchem Corp, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>high-resolution (16,256 dpi) photolithography mask</td>
<td>&nbsp;</td>
<td>Fineline-Imaging Inc, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>77279</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning, WI</td>
<td>184 SIL ELAST KIT 0.5KG</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH30002.02</td>
<td>&nbsp;</td>
<tr>
<td>Programmable spin coater</td>
<td>&nbsp;</td>
<td>Laurell Tech Corp</td>
<td>WS0650S</td>
<td>&nbsp;</td>
<tr>
<td>Mask aligner</td>
<td>&nbsp;</td>
<td>Neutronix-Quintel, PA</td>
<td>Q4000</td>
<td>&nbsp;</td>
<tr>
<td>Oxygen plasma etcher</td>
<td>&nbsp;</td>
<td>March Plasma System, CA</td>
<td>CS-1701</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Live/Dead Viability/Cytotoxicity Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>L-3224</td>
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microfluidic co-culture of epithelial cells and bacteria for investigating soluble signal-mediated interactions

人体胃肠道（GI）是一个独特的环境中肠上皮细胞和非致病性细菌（共生）共存。有人曾提出，微环境中的病原体在共生层遇到，在确定殖民化的程度是很重要的。调查病原菌定植目前文化特性的方法是不适合负责调查这一假说，因为他们不模仿胃肠道微环境的方式，使细菌细胞和上皮细胞的共培养。在这里，我们描述了一个微流体的共培养模型，使真核细胞和细菌的独立的文化和测试病原菌定植共生的微环境的影响。共培养模型，证明了开发一个共生<em&gt;大肠埃希氏菌</em&gt;生物膜在HeLa细胞中，随后推出的肠<em&gt; E。大肠杆菌</em&gt;（肠出血性大肠杆菌）的共生岛，在一个序列中，模仿的消化道感染的事件序列。

Watch this Scientific Journal Video about Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions at JoVE.com

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

这个协议描述为上皮细胞和细菌的同时，本地化的文化的微流体的共培养模型。该模型可用于调查发病机制不同的可溶性的分子信号的作用，以及屏幕上公认的益生菌株细菌的效力。

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

microfluidic-co-culture-epithelial-cells-bacteria-for-investigating

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Biology

14.0K 次观看.  Texas A&M University. 这个协议描述为上皮细胞和细菌的同时，本地化的文化的微流体的共培养模型。该模型可用于调查发病机制不同的可溶性的分子信号的作用，以及屏幕上公认的益生菌株细菌的效力。

视频: Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Cryosectioning方法小鼠结肠黏膜显微切割

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

由上皮细胞和原发免疫细胞组成的多细胞人类Alveal模型，用于危险评估

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...