我们要感谢G. Ø图勒在开发这些模型的指导和建议。囊性纤维化基金会（ANDERS06F0 GGA，STANTO07RO和STANTO08GA到BAS），美国国立卫生研究院（T32A107363 GGA和R01 - HL074175到BAS），这项工作是支持国家研究资源中心的生物医学研究的卓越中心（COBRE P20 - RR018787到BAS）。

1。静态共培养生物膜模型<ol><li>静态共培养生物膜模型1使用CFBE41o细胞（CFBE细胞），这是永生细胞最初是从个人发展与ΔF508- CFTR的突变 2,3,4 CF合子。 CFBE细胞应接种/ 6孔组织培养板在24孔组织培养板在最短的基本培养基辅以10％胎牛血清（MEM）或2 × 10 5 10 6个细胞的浓度， 2mm的L -谷氨酰胺，50 U / mL青霉素，50μg/ mL链霉素。我们使用1.5毫升培养基，每孔6孔板和24孔板，每孔0.5 mL于中等。 </li><li>细胞增长应在37 ° C和5％CO 2 -95％为7-10天的空气，形成与细菌接种前的汇合单层。介质必须每2-3天。这些条件已被证明导致形成一个融合的单层和紧密连接。 </li><li> 绿脓杆菌增长在5毫升LB培养基，18小时在37 ° C在200转的孵化器振动筛。在这些条件下， 体育假单胞菌的文化通常会达到5X10 9 CFU / mL的密度。 </li><li>细菌接种，CFBE细胞中删除，添加等体积的无酚红的MEM，辅以2毫米L -谷氨酰胺（显微镜中等）。聚合CFBE单层接种与体育假单胞菌的感染多重约30:1相对于原来种子CFBE细胞的数量。这相当于2 × 10 7 CFU / ml为1.5毫升的MEM / 6孔板和1.2 × 10 7 CFU / ml的0.5毫升的MEM / 24孔板。 </li><li>孵育1小时板块在37 ° C和5％CO 2 -95％的空气。 </li><li> 1小时的潜伏期后，应删除上清，而代之以新鲜显微镜0.4％，精氨酸培养基。 </li><li>在37 ° C和5％CO 2 -95％的空气和分析不同时间点（约8小时）CFBE使用相衬显微镜的单层的完整性。如果气道细胞非融合，P。假单胞菌在几分钟之内将迅速获得基底细胞表面，破坏细胞的完整性。接种与体育假单胞菌株，组成性表达GFP的荧光生物膜microcolonies可epifluorescence或共聚焦显微镜观察的结果。 </li><li>可以由共培养与磷酸盐缓冲液（PBS）洗涤2-3次，以消除浮游细菌生物膜中的细菌菌落形成单位。洗净后，用0.1％Triton在PBS或MEM X - 100的10-15分钟的裂解上皮细胞和分散的生物膜。 </li><li>涡3分钟，并准备系列稀释裂解液。这些稀释到LB琼脂板孵育过夜，于37 ° C。第二天的菌落计数来确定的CFU。 </li></ol> 2。流动细胞共培养生物膜模型<ol><li>流动细胞共培养生物膜模型（流量分析）涉及标准生物膜流动单元设备的修改，以适应CFBE 细胞 5 。 </li><li>几个直径为40毫米的盖玻片放入一个干净的100 mL烧杯。紧盖2层铝箔和高压灭菌干燥周期为20分钟。 </li><li>在细胞培养罩工作，抢一个使用乙醇洗涤钳和地点到无菌的60毫米直径的塑料盘的烧杯中无菌盖玻片。 </li><li>预热细胞生长的培养基中加入3毫升，按吸管尖盖玻片上删除被困于车底的泡沫，迫使盖玻片的塑料盘底部。 </li><li>种子2X10 6细胞每道菜和动摇的菜来回轻轻。避免纷飞，以防止离心对双方的菜细胞。 </li><li>放置在5％的CO 2 -95％空气培养箱在37 ° C的菜，饲料用3 mL新鲜生长培养基为8至10天的细胞，隔日。这些细胞会形成融合单层玻璃盖玻片上。 </li><li>拓展体育假单胞菌在5毫升LB培养基，18小时在37 °的C（孵化器振动筛在200转）。在这些条件下， 体育假单胞菌的文化通常会达到5X10 9 CFU / mL的密度。我们用P。假单胞菌株PAO1进行组成型表达GFP的6 pSMC21质粒。 </li><li>添加到1毫升细菌培养无菌的离心管。在3分钟6000转离心，洗菌体两次在1毫升显微镜介质。 </li><li>实现〜5X10 8 CFU / mL的浓度稀释成4.5毫升显微镜液0.5毫升的洗涤和悬浮细菌。 </li><li>观察活细胞需要实时成像室，再加上蠕动泵（时间的延伸长度，以确保营养物质流）和温度控制器。我们使用Bioptechs FCS2（Focht活细胞）连接到一个低流量灌注微泵的1 / 16的C - flex公司管材切成适当的长度和不育蒸压室，并通过FCS2室控制器的温度调节。我们保持在37℃的热水，位于旁边的显微镜浴介质的输入源。 </li><li>组装流室。 FCS2室包括自锁基地（坐在舞台上的适配器在成像）和灌注管和室内控制器相连的上半部分。会议厅内组装前翻转倒放在搬上舞台适配器。室的上半部分保持倒挂使灌注管是可见的，并灌注管到0.75毫米厚的橡胶垫片的通孔对齐。堆栈室顶部的橡胶垫片，确保沟槽一边是microaqueduct幻灯片，并将其放在另一个幻灯片上的橡胶垫片。这第二个垫片的厚度和内部几何确定腔的体积。我们通常用0.75毫米厚30毫米的圆形内部几何的橡胶垫片。 </li><li>加入1 ml预热（37℃）显微镜在幻灯片的中心媒介。 </li><li>检索从细胞培养孵化器的60毫米的菜，除去花中等，一次用3毫升的预热显微镜介质的细胞。此步骤可确保消除细胞生长介质中的酚红和抗生素。酚红轻度荧光灯和干涉成像，而抗生素可以消灭体育铜绿菌，才可以建立生物膜。 </li><li>使用乙醇洗镊子，从盘检索的盖玻片，降低到磁珠上腔放在显微镜媒体倒挂。现在休息盖玻片上的第二个橡胶垫片和呼吸道细胞的单层朝下。 </li><li>部件组装，一方面将堆栈的顶部室的基础，所以这一切都是右侧翻身迅速室。转动环，锁定到位的基地。 </li><li>低流量微型灌注泵入口管连接，并开始在20 mL / h的速度流，这个流量是在体育游泳的速度能力假单胞菌 。第二件显微镜介质放置在37瓶泵的油管链接℃水浴位于旁边的显微镜。 </li><li>无菌预切1 / 16的C - Flex的油管连接到会议厅的入口和出口灌注管，连接温度控制器，然后放置到一个倒置荧光显微镜显微镜阶段的组装室。 </li><li>使用1毫升的一次性注射器，注入室先前准备的菌悬液，使用两个单向阀置于行之间的泵室。在我们的特别设置的，它需要的菌悬液0.6 mL体积达到室。根据您的特定的油管长度调整本卷。我们还发现它有用的地方行的一次性0.22微米过滤器之间的泵和两个单向阀，以防止细菌从上游游泳和污染输入瓶。 </li><li>为了使细菌附着于呼吸道细胞，停止泵为2h。流，然后可以重新开始，并维持在20毫升/小时，其余实验。 </li><li>监视器微分干涉对比（DIC）的整个实验的显微镜呼吸道细胞的完整性检查单层损坏的迹象。同时，按照发展绿色荧光蛋白标记的体育通过收购一个倒置的共聚焦或宽视场荧光显微镜图像的心尖在气道细胞表面的铜绿假单胞菌生物膜。 </li></ol> 3。代表性的成果我们用一个P 的应变假单胞菌含有pSMC21质粒可组成型表达GFP的6。出于这个原因，绿色荧光蛋白标记的生物膜上CFBE单层的可epifluorescence显微镜观察。另外，可视化的生物膜可以通过染色未标注体育假单胞菌与1小时37℃1 1％calcofluor白色的解决方案。 CFBE细胞成像上形成生物膜的流动检测，我们使用一个特制的阶段适配器，但现在有几家公司可以提供专门安装阶段适配器。我们的工作与奥林巴斯IX70倒置ORCA - AG深冷CCD相机和X60油浸物镜（数值孔径1.40）配备显微镜。滤波器W配备了一个四十〇分之四百八十零纳米带通激发滤光片和一个30分之535纳米带通发射滤光片脚跟。数字图像被收购的OpenLAB 4.0.3软件包（即兴），卷迭代恢复使用Volocity 3.5.1软件（即兴）deconvolved。实现COMSTAT图像分析软件包7,8 3D生物膜结构定量分析。 对于任何合作文化模型认为，必须特别注意​​的细胞毒性模型的组件之间发展。在静态和流动细胞检测，我们发现CFBE单层，可以承受体育存在假单胞菌为接种后8小时，没有任何改动的迹象。上皮细胞单层的完整性进行评估相衬显微镜倒置显微镜 1（图1A）或微分干涉对比（DIC）的整个实验的显微镜5。随着时间的推移， 体育绿脓杆菌会产生毒素和积累，并能损害的上皮细胞单层完全或部分（图1B - 1C）的致病因素。细胞毒性，可定量评估，使用各种工具包来衡量的上皮细胞释放乳酸脱氢酶（LDH）。 LDH是一个稳定的胞内酶，裂解细胞或细胞死亡后的细胞外环境释放。 当呼吸道单层的完整性不受损害（通常为〜8 h后接种）， P. 假单胞菌生物膜可以成功地形成和发展，双方合作的文化模型（图2A - 2B）在呼吸道细胞的顶端表面。继三维重建（图2C）和量化，生物量的积累，可以准确地确定。我们也使用这些共培养生物膜，在几个表型检测。例如，如上所述，生物膜CFU可以很容易地由上皮细胞裂解，连续平板稀释裂解，电镀。我们发现这种技术优势，确定不同菌株耐抗生素治疗。此外，生物膜对上皮细胞的毒性，可使用市售的LDH检测试剂盒。在这种方式下，生物膜的毒性致病因素的作用进行评估。这些模型系统还支持一些基因表达的应用，包括启动子融合的研究，RT - PCR和基因芯片分析 1,5,9， 。 <img src="/files/ftp_upload/2186/2186fig1.jpg" alt="图1" /> 图1。 CFBE细胞和代表图像的单层损害和损害呼吸道细胞单层由于体育假单胞菌生物膜的生长。（一）代表形象，一个生长在组织培养板的CFBE细胞融合单层相衬显微镜评估。比例尺，120微米。 （二）折衷CFBE单层的例子。单层即使不显示没有明显的损伤明显的迹象，P。假单胞菌细菌之间的紧密连接的上皮细胞和基底膜获得的蔓延。生物膜的形成通常是没有实现在这些条件下，由于单层恶化的。比例尺，20微米。 （三）范例杂草丛生体育假单胞菌生物膜接种后观察24小时。成功后支持生物膜的形成，CFBE单层是无法修复的损坏，现在几乎不存在。残留生物膜，为细菌的平层增长，显示附加玻璃盖玻片。比例尺，20微米。 <img src="/files/ftp_upload/2186/2186fig2.jpg" alt="图2" /> 图2。 体育 （一） 在根尖表面使用静态共培养生物膜模型和流动细胞共培养生物膜模型的融合CFBE细胞生长的假单胞菌生物膜 。的表达GFP的体育形象代表在使用静态共培养生物膜模型，epifluorescence显微镜评估CFBE细胞融合单层假单胞菌生物膜生长。形象是相衬渠道的覆盖和荧光通道。比例尺，35微米。 （二）一个GFP标记P.的形象代表假单胞菌生物膜增长为6 h的使用流动细胞共培养生物膜模型CFBE细胞融合单层。为了方便呼吸道单层的可视化，核染色与体育接种前30分钟为10微克/毫升赫希斯特33342（分子探针） 假单胞菌 。被看作微分干涉对比（DIC）的合并，pseudocolored图像，以及相应的荧光图像显示。生物膜，呈现根尖CFBE细胞表面附着的绿色团块，分布在呼吸道的细胞。比例尺，20微米。 （三）三维重构TION的Z系列图像显示典型的蘑菇状结构的H - 6岁的体育栈假单胞菌生物膜的形成上CFBE细胞单层。比例尺，10微米。

<ol>
	<li>Anderson, G. G., Moreau-Marquis, S., Stanton, B. A., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+analysis+of+tobramycin-treated+Pseudomonas+aeruginosa+biofilms+on+cystic+fibrosis-derived+airway+epithelial+cells.">In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells.</a> Infect Immun. 76, 1423-1433 (2008).</li><li>Bruscia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CF+cell+lines+corrected+at+DeltaF508-CFTR+locus+by+SFHR-mediated+targeting.">Isolation of CF cell lines corrected at DeltaF508-CFTR locus by SFHR-mediated targeting.</a> Gene Ther. 9, 683-685 (2002).</li><li>Cozens, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+expression+and+chloride+secretion+in+polarized+immortal+human+bronchial+epithelial+cells.">CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.</a> Am J Respir Cell Mol Biol. 10, 38-47 (1994).</li><li>Hentchel-Franks, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+airway+cl-+secretion+in+human+subjects+by+adenosine.">Activation of airway cl- secretion in human subjects by adenosine.</a> Am J Respir Cell Mol Biol. 31, 140-146 (2004).</li><li>Moreau-Marquis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DeltaF508-CFTR+mutation+results+in+increased+biofilm+formation+by+Pseudomonas+aeruginosa+by+increasing+iron+availability.">The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability.</a> Am J Physiol Lung Cell Mol Physiol. 295, 25-37 (2008).</li><li>Kuchma, S. L., Connolly, J. P., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-component+regulatory+system+regulates+biofilm+maturation+and+type+III+secretion+in+Pseudomonas+aeruginosa.">A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa.</a> J Bacteriol. 187, (2005).</li><li>Heydorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+reproducibility+in+flow-chamber+biofilms.">Experimental reproducibility in flow-chamber biofilms.</a> Microbiology. 146 (Pt 10), 2409-2415 (2000).</li><li>Heydorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (Pt 10), 2395-2407 (2000).</li><li>Anderson, G. G., Yahr, T. L., Lovewell, R. R., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Pseudomonas+aeruginosa+magnesium+transporter+MgtE+inhibits+transcription+of+the+type+III+secretion+system.">The Pseudomonas aeruginosa magnesium transporter MgtE inhibits transcription of the type III secretion system.</a> Infect Immun. 78, (2010).</li><li>Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+microbial+biofilms.">Overview of microbial biofilms.</a> J Ind Microbiol. 15, 137-140 (1995).</li><li>Hoiby, N., Frederiksen, B., Pressler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+early+Pseudomonas+aeruginosa+infection.">Eradication of early Pseudomonas aeruginosa infection.</a> J Cyst Fibros. 4, 49-54 (2005).</li><li>Ko, Y. H., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis:+a+brief+look+at+some+highlights+of+a+decade+of+research+focused+on+elucidating+and+correcting+the+molecular+basis+of+the+disease.">Cystic fibrosis: a brief look at some highlights of a decade of research focused on elucidating and correcting the molecular basis of the disease.</a> J Bioenerg Biomembr. 33, 513-521 (2001).</li><li>Gibson, R. L., Burns, J. L., Ramsey, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+and+management+of+pulmonary+infections+in+cystic+fibrosis.">Pathophysiology and management of pulmonary infections in cystic fibrosis.</a> Am J Respir Crit Care Med. 168, 918-951 (2003).</li><li>Furukawa, S., Kuchma, S. L., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+their+options+open:+acute+versus+persistent+infections.">Keeping their options open: acute versus persistent infections.</a> J Bacteriol. 188, 1211-1217 (2006).</li><li>Merritt, J. H., Kadouri, D. E., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+and+analyzing+static+biofilms.">Growing and analyzing static biofilms.</a> Curr Protoc Microbiol. Chapter 1, Unit 1B 1-Unit 1B 1 (2005).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple,+convergent+signalling+pathways:+a+genetic+analysis.">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis.</a> Mol Microbiol. 28, 449-461 (1998).</li><li>Gomez, M. I., Prince, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunistic+infections+in+lung+disease:+Pseudomonas+infections+in+cystic+fibrosis.">Opportunistic infections in lung disease: Pseudomonas infections in cystic fibrosis.</a> Curr Opin Pharmacol. 7, 244-251 (2007).</li></ol>

生物膜是细菌的社区环境刺激的反应形式。这些环境的信号导致全球监管的变化，在每个细菌，导致绑定到表面，聚集，胞外多糖生产，和其他表型，如抗生素耐药性增加10。在过去几十年里，大量的证据支持的假说，即生物膜在慢性感染的发病机制中发挥了很大的作用。举例来说，它是可接受的， 体育绿脓杆菌是能够建立在囊性纤维化（CF）患者肺部慢性感染，形成生物膜</su...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FCS2 (Focht Live-Cell) chamber</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>060319131616</td>
<td>&nbsp;</td>
<tr>
<td>FCS2 chamber controller</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>060319-2-0303</td>
<td>&nbsp;</td>
<tr>
<td>40 mm glass coverslips</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>PH 40-1313-0319</td>
<td>&nbsp;</td>
<tr>
<td>MEM</td>
<td>&nbsp;</td>
<td>Mediatech, Manassass, VA</td>
<td>#10-010-CV</td>
<td>&nbsp;</td>
<tr>
<td>MEM without phenol red</td>
<td>&nbsp;</td>
<td>Mediatech, Manassass, VA</td>
<td>Mediatech, Manassass, VA</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

co-culture models of pseudomonas aeruginosa biofilms grown on live human airway cells

细菌生物膜与多种人类疾病的数量，但生物膜的发展已普遍在非生物表面研究。在本文中，我们描述了人类呼吸道上皮细胞培养（CFBE细胞）形成铜绿假单胞菌生物膜的协议。在第一种方法（称为静态共培养生物膜模型），P.绿脓杆菌是与标准组织培养板汇合单层生长的CFBE细胞培养。虽然细菌是上皮细胞的毒性，除了单层的破坏精氨酸延误时间足够长的生物膜形成上的CFBE细胞。第二种方法（称为流细胞共培养生物膜模型），涉及适应生物膜的流动池装置，这是在生物膜研究中，经常使用，以适应支持CFBE细胞融合单层玻璃盖玻片。这是接种单层与体育假单胞菌和蠕动泵，然后流经细胞的新鲜培养基。在两个系统中，细菌生物膜的形成，接种后6-8小时内。生物膜的可视化是提高使用的 P. 假单胞菌菌株表达绿色荧光蛋白（GFP）。静态和流动细胞共培养生物膜检测早期体育的模型系统绿脓杆菌感染的囊性纤维化（CF）的肺，这些技术允许不同方面的P.假单胞菌生物膜的形成和毒性研究，包括生物膜的细胞毒作用，生物膜CFU测量，染色和可视化生物膜。

Watch this Scientific Journal Video about Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells at JoVE.com

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

本文介绍了成长的不同方法<em&gt;绿脓杆菌</em&gt;对培养的人类呼吸道上皮细胞的生物膜。这些协议可以适应研究生物膜形成的不同方面，包括可视化的生物膜，生物膜染色，测量菌落形成单位（CFU）的生物膜，研究生物膜的细胞毒性。

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

co-culture-models-pseudomonas-aeruginosa-biofilms-grown-on-live-human

Research

JoVE Journal

Immunology and Infection

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

22.1K 次观看.  Dartmouth College. 本文介绍了成长的不同方法绿脓杆菌对培养的人类呼吸道上皮细胞的生物膜。这些协议可以适应研究生物膜形成的不同方面，包括可视化的生物膜，生物膜染色，测量菌落形成单位（CFU）的生物膜，研究生物膜的细胞毒性。

视频: Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

科研

免疫与感染

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

慢性假单位病伤口感染的延迟接种模型

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.