Name: p. aeruginosa infected 3d co-culture of bronchial epithelial cells and macrophages at air-liquid interface for preclinical evaluation of anti-infectives
Uploaded: 2020-06-15T13:00:00
Description: Watch this Scientific Journal Video about P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives at Jo

根据第642028号H2020-MSCA-ITN-2014、NABBA -设计和开发先进的纳米药物，用于研究、技术开发和示范项目，这项工作得到了欧盟HORIZON 2020计划的资助，以克服生物障碍并治疗严重疾病。我们感谢安娜·科斯塔博士和珍妮·容特克博士对共同文化的发展给予的大力支持，感谢阿尔加·哈特维格为ELISA测定、佩特拉·科尼格、贾娜·韦斯特胡斯博士和基亚拉·德罗西博士提供的科学插图，感谢他们对细胞培养、分析和显微镜的支持。我们也感谢切尔西·索恩校对我们的手稿。

1. 渗透支持插入中细胞的生长和分化
<ol><li>培养 CFBE41o- 在 含有 10% 胎儿小牛血清 （FCS）、1% 非必需氨基酸和 600 mg/L 葡萄糖的 T75 烧瓶中，其最低基本介质 （MEM） 含有 5% 的 CO2 气氛。每2~3天向细胞中加入新鲜介质。<ol>
<li>在37°C下用3 mPin-乙二胺四聚氰胺四聚氰胺酸（EDTA）在烧瓶中达到70%汇合后分离细胞，15分钟。加入 7 mL 的新鲜 MEM，在室温 （RT） 下以 300 x g 离心 4 分钟?...

<ol>
	<li>Cutting, G. R. <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+genetics:+from+molecular+understanding+to+clinical+application.">Cystic fibrosis genetics: from molecular understanding to clinical application.</a> Nature Reviews Genetics. 16 (1), 45-56 (2015).</li><li>Lyczak, J. B., Cannon, C. L., Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+Pseudomonas+aeruginosa+infection:+Lessons+from+a+versatile+opportunist.">Establishment of Pseudomonas aeruginosa infection: Lessons from a versatile opportunist.</a> Microbes and Infection. 2 (9), 1051-1060 (2000).</li><li>Wilke, M., Buijs-Offerman, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+cystic+fibrosis:+Phenotypic+analysis+and+research+applications.">Mouse models of cystic fibrosis: Phenotypic analysis and research applications.</a> Journal of Cystic Fibrosis. 10, 152-171 (2011).</li><li>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=Pseudomonas+aeruginosa+biofilm+formation+in+the+cystic+fibrosis+airway.">Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway.</a> Pulmonary Pharmacology and Therapeutics. 21 (4), 595-599 (2008).</li><li>Yu, Q., et al. <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+evaluation+of+tobramycin+and+aztreonam+versus+Pseudomonas+aeruginosa+biofilms+on+cystic+fibrosis-derived+human+airway+epithelial+cells.">In vitro evaluation of tobramycin and aztreonam versus Pseudomonas aeruginosa biofilms on cystic fibrosis-derived human airway epithelial cells.</a> Journal of Antimicrobial Chemotherapy. 67 (11), 2673-2681 (2012).</li><li>Moreau-Marquis, S., Redelman, C. V., Stanton, B. a., Anderson, G. G. <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+models+of+Pseudomonas+aeruginosa+biofilms+grown+on+live+human+airway+cells.">Co-culture models of Pseudomonas aeruginosa biofilms grown on live human airway cells.</a> Journal of visualized experiments : JoVE. (44), e2186 (2010).</li><li>Stanton, B. A., Coutermarsh, B., Barnaby, R., Hogan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+reduces+VX-809+stimulated+F508del-CFTR+chloride+secretion+by+airway+epithelial+cells.">Pseudomonas aeruginosa reduces VX-809 stimulated F508del-CFTR chloride secretion by airway epithelial cells.</a> PLoS ONE. 10 (5), 1-13 (2015).</li><li>Lambrecht, B. N., Prins, J., Hoogsteden, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+dendritic+cells+and+host+immunity+to+infection.">Lung dendritic cells and host immunity to infection.</a> European Respiratory Journal. (18), 692-704 (2001).</li><li>Murgia, X., De Souza Carvalho, C., Lehr, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+the+pulmonary+barrier:+New+insights+to+improve+the+efficiency+of+inhaled+therapeutics.">Overcoming the pulmonary barrier: New insights to improve the efficiency of inhaled therapeutics.</a> European Journal of Nanomedicine. 6 (3), 157-169 (2014).</li><li>Ding, P., Wu, H., Fang, L., Wu, M., Liu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmigration+and+phagocytosis+of+macrophages+in+an+airway+infection+model+using+four-dimensional+techniques.">Transmigration and phagocytosis of macrophages in an airway infection model using four-dimensional techniques.</a> American Journal of Respiratory Cell and Molecular Biology. 51 (1), 1-10 (2014).</li><li>Hartl, D., Tirouvanziam, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+Immunity+of+the+Lung:+From+Basic+Mechanisms+to+Translational+Medicine.">Innate Immunity of the Lung: From Basic Mechanisms to Translational Medicine.</a> Journal of Innate Immunity. 10 (5-6), 487-501 (2018).</li><li>Esposito, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+proteostasis+to+repair+the+F508del-CFTR+defect+in+cystic+fibrosis.">Manipulating proteostasis to repair the F508del-CFTR defect in cystic fibrosis.</a> Molecular and cellular pediatrics. 3 (1), 13 (2016).</li><li>Hein, S., Bur, M., Schaefer, U. F., Lehr, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+Pharmaceutical+Aerosol+Deposition+Device+on+Cell+Cultures+(PADDOCC)+to+evaluate+pulmonary+drug+absorption+for+metered+dose+dry+powder+formulations.">A new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to evaluate pulmonary drug absorption for metered dose dry powder formulations.</a> European Journal of Pharmaceutics and Biopharmaceutics. 77 (1), 132-138 (2011).</li><li>Kletting, S., Barthold, S., et al. <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+human+alveolar+epithelial+(hAELVi)+and+macrophage+(THP-1)+cell+lines.">Co-culture of human alveolar epithelial (hAELVi) and macrophage (THP-1) cell lines.</a> Altex. 35 (2), 211-222 (2018).</li><li>Schwende, H., Fitzke, E., Ambs, P., Dieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+state+of+differentiation+of+THP-1+cells+induced+by+phorbol+ester+and+1,25-dihydroxyvitamin+D3.">Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3.</a> Journal of leukocyte biology. 59 (4), 555-561 (1996).</li><li>Castoldi, A., Empting, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspherical+and+Spherical+InvA497-Functionalized+Nanocarriers+for+Intracellular+Delivery+of+Anti-Infective+Agents.">Aspherical and Spherical InvA497-Functionalized Nanocarriers for Intracellular Delivery of Anti-Infective Agents.</a> Pharmaceutical Research. 36 (1), 1-13 (2019).</li><li>Ebensen, T., Delandre, S., Prochnow, B., Guzm&#225;n, C. A., Schulze, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Combination+Vaccine+Adjuvant+System+Alum/c-di-AMP+Results+in+Quantitative+and+Qualitative+Enhanced+Immune+Responses+Post+Immunization.">The Combination Vaccine Adjuvant System Alum/c-di-AMP Results in Quantitative and Qualitative Enhanced Immune Responses Post Immunization.</a> Frontiers in cellular and infection microbiology. 9, 31 (2019).</li><li>Brockman, S. M., Bodas, M., Silverberg, D., Sharma, A., Vij, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer-based+selective+autophagy-induction+rescues+&#948;F508-CFTR+and+inhibits+Pseudomonas+aeruginosa+infection+in+cystic+fibrosis.">Dendrimer-based selective autophagy-induction rescues &#948;F508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis.</a> PLoS ONE. 12 (9), 1-17 (2017).</li><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> Infection and Immunity. 76 (4), 1423-1433 (2008).</li><li>Moreau-Marquis, S., Bomberger, J. M., et al. <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> American journal of physiology. Lung cellular and molecular physiology. 295, 25-37 (2008).</li><li>Braakhuis, H. M., Kloet, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+future+of+in+vitro+models+to+study+translocation+of+nanoparticles.">Progress and future of in vitro models to study translocation of nanoparticles.</a> Archives of Toxicology. 89 (9), 1469-1495 (2015).</li><li>Bosshart, H., Heinzelmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=THP-1+cells+as+a+model+for+human+monocytes.">THP-1 cells as a model for human monocytes.</a> Annals of Translational Medicine. 4 (21), 4-7 (2016).</li><li>Daigneault, M., Preston, J. a., Marriott, H. M., Whyte, M. K. B., Dockrell, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+of+markers+of+macrophage+differentiation+in+PMA-stimulated+THP-1+cells+and+monocyte-derived+macrophages.">The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages.</a> PLoS ONE. 5 (1), (2010).</li><li>Bismarck, P. V., Schneppenheim, R., Schumacher, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+treatment+of+pseudomonas+aeruginosa+respiratory+tract+infection+with+a+sugar+solution+-+A+case+report+on+a+lectin+based+therapeutic+principle.">Successful treatment of pseudomonas aeruginosa respiratory tract infection with a sugar solution - A case report on a lectin based therapeutic principle.</a> Klinische Padiatrie. 213 (5), 285-287 (2001).</li><li>Klinger-Strobel, M., Lautenschl&#228;ger, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspects+of+pulmonary+drug+delivery+strategies+for+infections+in+cystic+fibrosis+-+where+do+we+stand.">Aspects of pulmonary drug delivery strategies for infections in cystic fibrosis - where do we stand.</a> Expert Opinion on Drug Delivery. 5247, 1-24 (2015).</li><li>Ehrhardt, C., Collnot, E. -. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+an+in+vitro+model+of+cystic+fibrosis+small+airway+epithelium:+characterisation+of+the+human+bronchial+epithelial+cell+line+CFBE41o-.">Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line CFBE41o-.</a> Cell and tissue research. 323 (3), 405-415 (2006).</li><li>Anderson, G. G., Kenney, T. F., Macleod, D. L., Henig, N. 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=Eradication+of+Pseudomonas+aeruginosa+biofilms+on+cultured+airway+cells+by+a+fosfomycin/tobramycin+antibiotic+combination.">Eradication of Pseudomonas aeruginosa biofilms on cultured airway cells by a fosfomycin/tobramycin antibiotic combination.</a> Pathogens and Disease. 67 (1), 39-45 (2013).</li><li>Cavalieri, F., Tortora, M., Stringaro, A., Colone, M., Baldassarri, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomedicines+for+antimicrobial+interventions.">Nanomedicines for antimicrobial interventions.</a> Journal of Hospital Infection. 88 (4), 183-190 (2014).</li><li>Savla, R., Minko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+approaches+for+inhalation+treatment+of+fibrosis.">Nanotechnology approaches for inhalation treatment of fibrosis.</a> Journal of Drug Targeting. 21 (10), 914-925 (2013).</li><li>Ho, D. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+strategies+in+drug+delivery+systems+for+treatment+of+pulmonary+infections.">Challenges and strategies in drug delivery systems for treatment of pulmonary infections.</a> European Journal of Pharmaceutics and Biopharmaceutics. 144, 110-124 (2019).</li></ol>

本文描述了受感染气道的3D共培养方案，由人类囊性纤维化支气管上皮细胞系CFBE41o和人类单细胞衍生巨噬细胞系THP-1组成。该协议允许评估上皮屏障的完整性，巨噬细胞迁移，细菌生存和炎症，这是测试药物疗效和宿主反应时同时的重要参数。模型中的新颖性在于将上皮细胞（即人类CF细胞系和巨噬细胞）与急性细菌感染（即P.aeruginosa）结合在一起。上皮细胞的急性感染证明是由抗生素（即...

伪多多多酸菌是 囊性纤维化（CF）中的相关病原体，有助于肺组织损伤1。多糖的生产，如藻酸盐和其他粘液外聚糖，协调疾病的进展，这导致顽强的细菌坚持，限制抗生素向细菌的输送，并保护细菌免受宿主免疫系统2。在这种情况下 ，P. aeruginosa从 浮游生物期过渡到生物膜形成是一个关键问题，也促进了抗生素耐受性的发生。
在 CF 的上下文中，鼠标主要用作模型。然而，小鼠不会自发地发展这种疾病与CF突变3的引入。大多数细菌生物膜开发和药物易感性研究是在非生物表面进行的，如培养皿。但是，此方法并不表示体内的复杂性。例如，重要的生物屏障是不存在的，包括免疫细胞以及粘膜上皮。虽然P.aeruginosa对上皮细胞相当有毒，但一些团体已经成功地与人类支气管细胞共同培育了早期的P.aeruginosa生物膜。这些细胞起源于囊性纤维化患者与CFTR突变（CFBE41o-细胞）4，并允许评估抗生素疗效5或评估感染期间CFTR蛋白的纠正6。这种模式被证明可以提高药物疗效的可预测性，此外还能够对药物在药物开发后期阶段失败的问题进行定性。
然而，在肺，粘膜上皮暴露在空气中。此外，存在于气道中的免疫细胞，如组织巨噬细胞，对吸入的病原体或颗粒8起起至关重要的作用。巨噬细胞通过不同的细胞层迁移，到达支气管流明并对抗感染。此外，吸入的药物还必须应付粘液的存在作为肺气血屏障9的附加非细胞元素。事实上，已经开发了几个复杂的三 维 （3D）体外模型，旨在提高 体内的相关性 。联合培养系统不仅增加了药物发现 体外 系统的复杂性，而且能够研究细胞-细胞相互作用。这种复杂性在有关巨噬细胞迁移10、中性粒细胞释放11、粘液在感染中的作用9、上皮细胞对过度损伤的反应等研究中已经得到解决。然而，一个可靠的CF感染 体外 模型，功能在CF的基因突变，暴露在空气中（增加的生理条件），并整合免疫细胞仍然缺乏。
为了弥补这一差距，我们描述了一个协议，用于稳定受感染气道的人类3D共培养。该模型由人类CF支气管上皮细胞和巨噬细胞组成，感染了 P.aeruginosa， 能够代表扩散和免疫屏障。以在相当高的通量下测试抗感染物为目的，利用两种人类细胞系：CFBE41o和 THP-1单细胞衍生巨噬细胞，在井板插入物的渗透滤膜上建立了这种共培养物。此外，为了最终研究气溶胶抗感染剂13的沉积，该模型是在空气-液体界面（ALI）而不是液体覆盖条件（LCC）建立的。
正如我们在这里报告，这个模型不仅允许评估抗生素治疗的细菌生存，而且允许评估细胞毒性，上皮屏障完整性，巨噬细胞转运和炎症反应，这些都是药物开发的基本参数。
该协议结合了两种相关的细胞类型，用于肺气道的吸入治疗：巨噬细胞和CF支气管上皮。这些细胞在可渗透支撑刀片的对面播种，允许细胞暴露在空气中（称为空气-液体接口（ALI）条件）。这种宿主细胞的共培养随后感染了 P.aeruginosa。两个宿主细胞系都是人类起源的：上皮细胞代表囊性纤维化支气管上皮，在CF通道（CFBE41o-）上发生突变，THP-114 细胞是一个特征良好的巨噬细胞状细胞系。在将巨噬细胞样细胞添加到相反的隔间之前，首先允许在井板插入物的上侧形成汇合上皮层。一旦在ALI建立共同培养，系统就接种 了P.aeruginosa。 然后，这种受感染的联合培养系统用于评估抗生素的疗效，例如去布拉霉素。分析了以下终点：跨皮质电阻（TEER）、细胞和细胞-细菌相互作用的可视化、共生激光扫描显微镜（CLSM）、通过计数菌群形成单位（CFU）、宿主细胞生存（细胞毒性）和细胞因子释放的细菌存活率。

<table border="0" cellpadding="0" cellspacing="0" style="width:1308px;" width="1306">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Accutase</td>
			<td style="width:525px;">Accutase</td>
			<td style="width:205px;">AT104</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Carl Roth, Germany</td>
			<td>HP62.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CASY TT Cell Counter and Analyzer</td>
			<td>OLS Omni Life Sciences</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace Far Red</td>
			<td>Thermo Fischer</td>
			<td>C34564</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge Universal 320R</td>
			<td>Hettich, Germany</td>
			<td>1406</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">CFBE41o- cells</td>
			<td style="width:525px;">1. Gruenert Cell Line Distribution Program 
			2. Sigma-Aldrich</td>
			<td style="width:205px;">1. gift from Dr. Dieter C. Gruenert 
			2. SCC151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chopstick Electrode Set for EVOM2, 4mm</td>
			<td>World Precision Instruments, Sarasota, USA</td>
			<td>STX2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Laser-Scanning Microscope CLSM</td>
			<td>Leica, Mannheim, Germany</td>
			<td>TCS SP 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytokines ELISA Ready-SET-Go kits</td>
			<td>Affymetrix eBioscience, USA</td>
			<td>15541037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytokines Panel I and II</td>
			<td>LEGENDplex Immunoassay (Biolegend, USA).</td>
			<td>740102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytotoxicity Detection Kit (LDH)</td>
			<td>Roche</td>
			<td>11644793001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+) Glucose</td>
			<td>Merck</td>
			<td>47829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dako Fluorescence Mounting Medium</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI (4&#8242;,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fischer</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epithelial voltohmmeter</td>
			<td>World Precision Instruments, Sarasota, USA</td>
			<td>EVOM2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Permeable Support for 12 Well Plate with 3.0&#956;m Transparent PET Membrane, Sterile</td>
			<td>Corning, Amsterdam, Netherlands</td>
			<td>353181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum</td>
			<td>Lonza, Basel, Switzerland</td>
			<td>DE14-801F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse (H+L) Cross-adsorbed secondary Antibody, Alexa Fluor 633</td>
			<td>Invitrogen</td>
			<td>A-21050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Lactate Dehydrogenase (LDH), rabbit muscle</td>
			<td>Roche, Mannheim, Germany</td>
			<td>10127230001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB broth</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>L2897-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM (Minimum Essential Medium)</td>
			<td>Gibco Thermo Fisher Scientific Inc.</td>
			<td>11095072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco Thermo Fisher Scientific Inc.</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P. aeruginosa strain PAO1</td>
			<td>American Type Culture Collection</td>
			<td>47085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P. aeruginosa strain PAO1-GFP</td>
			<td>American Type Culture Collection</td>
			<td>10145GFP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde Aqueous Solution -16%</td>
			<td>EMS DIASUM</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffer solution buffer</td>
			<td>Thermo Fischer</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>Greiner</td>
			<td>664102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma, Germany</td>
			<td>P8139-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision Cover Glasses</td>
			<td>ThorLabs</td>
			<td>CG15KH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified Mouse anti-human ZO-1 IgG antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610966</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roswell Park Memorial Institute (RPMI) 1640 medium</td>
			<td>Gibco by Lifetechnologies, Paisley, UK</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soda-lime glass Petri dish, 50 x 200 mm (height x outside diameter)</td>
			<td>Normax, Portugal</td>
			<td>5058561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saponin</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S4521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T75 culture flasks</td>
			<td>Thermo Fischer</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">THP-1 cells</td>
			<td>Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany)</td>
			<td>No. ACC-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tobramycin sulfate salt</td>
			<td>Sigma</td>
			<td>T1783-500MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA 0.05%</td>
			<td>Thermo Fischer</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween80</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P1754</td>
			<td></td>
		</tr>
	</tbody>
</table>

p. aeruginosa infected 3d co-culture of bronchial epithelial cells and macrophages at air-liquid interface for preclinical evaluation of anti-infectives

f治疗肺部感染的Drug研究正在向高复杂性 的预测体外 模型发展。细菌在肺模型中的多方面存在可以重新适应上皮排列，而免疫细胞则协调对微环境中细菌的炎症反应。 虽然在囊性 纤维化的背景下，体内模型一直是测试新的抗感染药物的选择，但它们仍然 不能准确地模仿人类 此类疾病的体内条件和治疗结果。基于 人类细胞 （支气管上皮和巨噬细胞）和相关病原体的受感染气道的复杂体外模型可以弥补这一差距，并有助于将新的抗感染药物转化为临床。为此，建立了人类囊性纤维化支气管细胞系CFBE41o和 THP-1单细胞衍生巨噬细胞的共同培养模型，模拟 了P.aeruginosa 在空气-液体界面（ALI）条件下感染人类支气管粘膜。该模型在七天内建立，并同时评估以下参数：上皮屏障完整性、巨噬细胞迁移、细菌存活和炎症。本协议描述了一个强大且可重复的系统，用于评估药物疗效和宿主反应，该系统可能与发现新的抗感染药物和优化其气溶胶输送到肺部有关。

图1A 显示了在渗透支架的渗透支架的渗透侧和基础侧生长24小时后，人类支气管上皮细胞和巨噬细胞共同培养的形态。上皮屏障完整性由较高的TEER（834°cm2）和CLSM通过免疫固结蛋白ZO-1（图1B）显示。在未感染的CFBE41o的屏障完整性方面观察到的相同结果- 单一培养可以在未受感染的上皮-巨噬细胞共培养物中看到。
为...

Watch this Scientific Journal Video about P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives at Jo

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

我们描述了一个协议，一个三维共培养模型的感染气道，使用CFBE41o- 细胞，THP-1 巨噬细胞，和伪多莫纳斯aeruginosa，建立在空气-液体接口。该模型提供了一个新的平台，同时测试抗生素疗效，上皮屏障功能和炎症标记。

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 ...

因此，当我们评估新的抗感染药物时，我们必须解决两件事。一边是细菌。如果它们是浮游生物膜或知情生物膜，另一边是宿主细胞的存在，特别是上皮宿主细胞，它们形成紧密的生物屏障。如果这些障碍生效，怪物反应可能如何，并且有了这个新模型，我们实际上可以同时监视这两个参数。在治疗细菌感染时，我们必须意识到什么器官受到影响。例如，如果是肺，我们可以利用，并顶做药物作为气溶胶。然而，如果我们想要一个模型来研究这个问题，我们需要一个模型，也允许气溶胶的位置，这是我们的模型。另一个优点是它基于人类细胞，这样我们就可以避免由于动物和物种差异而导致的问题。该系统可以让您更深入地了解感染研究领域，在宿主细胞和细菌的相互作用过程中揭示分子和细胞事件。首次尝试此协议时，请记住检查细胞培养基的不育性和细胞形态，在处理可渗透支架时要小心。在含有10%FCS、1%非必需氨基酸和每升600毫克葡萄糖的T 75烧瓶中培养CFBE41 O减细胞，含5%二氧化碳。每两到三天向细胞中加入一次新鲜介质。细胞达到70%的汇合后，用10毫升PBS清洗，并在37摄氏度下分离3毫升的尝试性EDTA，15分钟。然后，加入7毫升新鲜MM，将细胞在300倍G下离心4分钟。丢弃上清液后，在 10 毫升的 MEM，同时用温和的移液扰乱团块。使用自动细胞计数器或血细胞计室对细胞进行计数，然后稀释到每500微升20万个细胞的浓度，用于在12个带渗透支架的井板中播种。将每井的细胞悬浮液加入 500 微升，用于渗透支撑的阴面。然后向基底侧添加1.5毫升新鲜介质。在37摄氏度和5%的二氧化碳下孵育细胞72小时。在播种后的第三天，先从基础侧侧去除介质，然后从水平侧移除介质，从而创建空气液体界面。在基底侧侧加入500微升新鲜 MEM，并每隔两天更换介质，直到细胞形成汇合单层。要评估细胞的上皮屏障特性，将500微升细胞培养基添加到细胞侧，向基底侧添加1.5毫升，然后将板返回培养箱一小时。使用 AN STX2 筷子电极和上皮伏欧姆表测量运输材料电阻或 TEER。为了培养THP1细胞，使用RMMI 1640基基在T 75烧瓶中生长，在37摄氏度和5%的二氧化碳下辅以10%FCS。每两天在一个新的T75烧瓶中播种200万个细胞，每天分裂细胞。为了区分THP1细胞，将烧瓶中300倍G的内端物离心4分钟，丢弃上一代。将颗粒重新放入新鲜介质中，放入新的 T75 烧瓶中。每毫升PMA向细胞中加入10毫微克，然后返回培养箱。要像细胞一样分离巨噬细胞，在37摄氏度下用PBS清洗一次，并在室温下用含有0.5毫摩尔EDTA的三毫升细胞分离溶液孵育10分钟。在倒置显微镜下检查细胞分离。当细胞分离后，加入7毫升新鲜介质，并在300倍G下离心4分钟。取出上清液后，在15毫升锥形管中将巨噬细胞重新在THP1培养基的三毫升中，数数细胞，并在建立共培养之前孵育最多一小时。要建立上皮巨噬细胞共培养，请从 CFBE41 O 减去单层的下腔中去除介质，并小心地反转无菌玻璃培养皿内的支撑，使用细胞刮刀去除通过膜孔隙过度生长的细胞。将 200，000 个 THP1 巨噬细胞放在每个井中倒置刀片的基底侧侧 200 微升 RPMI 中，关闭培养皿，并孵育两小时。然后将刀片放回 12 孔微板中，并将 500 微升 MEM 介质添加到可渗透刀片的基底侧侧。接种15毫升磅，每毫升氨基林补充300微克，用PAO1GFP的单一聚落P aeruginosa，在37摄氏度下孵育18小时，同时在180RPM下颤抖。孵育后，将细菌转移到50毫升锥形管中，并在3，850次G下离心机进行5分钟。丢弃上一杯，加入10毫升的无菌PBS，这些 PBS 已预热至 37 摄氏度。以600纳米测量光学密度，将细胞培养的细菌浓度调整为每毫升20万个菌落形成单位的最终浓度，这相当于每个上皮细胞感染一个细菌的多样性。将100微升的细菌悬浮液加入渗透支撑的阴膜侧，在37摄氏度和5%的二氧化碳下孵育一小时，让细菌附着在细胞上，然后用移液器小心地去除脂肪液体，以恢复ALI条件。对于药物治疗的实验，将500微升的药物溶液稀释在细胞培养的细胞侧。然后向基底侧添加1.5毫升细胞培养基。收集500微升的非典型和基底侧向介质，并汇集它们来评估非附着细菌的CFU。为了评估附着细菌或内化细菌的存活率，在渗透支撑的每个隔间中加入500微升无菌去越冷水，并在室温下孵育细胞30分钟。如果从冷冻样品中评估CFU，在37摄氏度下解冻10分钟。然后使用移液器尖端刮膜表面，注意不要给油井施加太大的压力。上下移位删除所有粘附的内容。与两个分数的细菌悬浮液，使一到10连续稀释与PBS补间和板细菌在磅加糖板。在30摄氏度下孵育的麦格板16至72小时，然后计算菌落并计算CFU。此处显示了人类支气管上皮细胞和巨噬细胞在渗透支架的渗透侧和罗勒侧产生的共培养的形态。对于紧密结点蛋白ZO1，更高的TEER测量和免疫保持，证明了上皮屏障的完整性。为了模拟细菌感染，P aeruginosa在CFBE41 o减细胞上接种。在感染后6小时，在共培养体的一侧观察到巨噬细胞。TEER 下降到每厘米 250 欧姆平方，表明上皮屏障受损，ZO1 染色也暗示了这一点。微噬细胞通过可渗透的过滤孔和细菌吸收的THP-1细胞在脂肪侧移动迁移显示在这里。在THP-1单培养中，巨噬细胞早在感染后一小时就迁移。同时，在共同培养的感染后三小时后，也可以看到这种迁移。使用托布拉霉素治疗6或20小时的感染共培养物被用共声扫描激光显微镜成像。如果不进行治疗，上皮细胞或巨噬细胞在感染20小时后死亡。尽管在显微镜图片中进行了六个小时的治疗后，CFU的测定表明细菌并没有扩散。然而，经过20小时的治疗，细菌恢复了其增殖能力。还对单一文化和共同文化的TEER进行测量。与单一培养相比，CFBE41 o减去THP-1细胞的共培养并没有引起上皮屏障完整性的任何变化。感染后，TEER 值下降。执行此过程时，确保细胞正确安装且滤膜不被破坏至关重要。因此，需要仔细执行细胞播种步骤。除了这个协议，插入物上的抗生素雾化将有可能看到细菌的生存是否变化相比，淹没的条件。

Research

JoVE Journal

Immunology and Infection

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

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

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

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 ...

科研

免疫与感染

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Application of a Mouse Ligated PeyerÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

使用人类肠道上皮细胞和BALB / C小鼠巨噬细胞的容忍脂肪酸和霍乱毒素浓度的测定

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

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

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

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 ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Pseudomonas aeruginosa Induced Lung Injury Model

Pseudomonas aeruginosa Induced Lung Injury Model

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. ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

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

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