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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Accutase | Accutase | AT104 | |
| Ampicillin | Carl Roth, Germany | HP62.1 | |
| CASY TT Cell Counter and Analyzer | OLS Omni Life Sciences | – | |
| CellTrace Far Red | Thermo Fischer | C34564 | |
| Centrifuge Universal 320R | Hettich, Germany | 1406 | |
| CFBE41o– cells | 1. Gruenert Cell Line Distribution Program 2. Sigma-Aldrich |
1. gift from Dr. Dieter C. Gruenert 2. SCC151 |
|
| Chopstick Electrode Set for EVOM2, 4mm | World Precision Instruments, Sarasota, USA | STX2 | |
| Confocal Laser-Scanning Microscope CLSM | Leica, Mannheim, Germany | TCS SP 8 | |
| Cytokines ELISA Ready-SET-Go kits | Affymetrix eBioscience, USA | 15541037 | |
| Cytokines Panel I and II | LEGENDplex Immunoassay (Biolegend, USA). | 740102 | |
| Cytotoxicity Detection Kit (LDH) | Roche | 11644793001 | |
| D-(+) Glucose | Merck | 47829 | |
| Dako Fluorescence Mounting Medium | DAKO | S3023 | |
| DAPI (4′,6-diamidino-2-phenylindole) | Thermo Fischer | D1306 | |
| Epithelial voltohmmeter | World Precision Instruments, Sarasota, USA | EVOM2 | |
| Falcon Permeable Support for 12 Well Plate with 3.0μm Transparent PET Membrane, Sterile | Corning, Amsterdam, Netherlands | 353181 | |
| Fetal calf serum | Lonza, Basel, Switzerland | DE14-801F | |
| Goat anti-mouse (H+L) Cross-adsorbed secondary Antibody, Alexa Fluor 633 | Invitrogen | A-21050 | |
| L-Lactate Dehydrogenase (LDH), rabbit muscle | Roche, Mannheim, Germany | 10127230001 | |
| LB broth | Sigma-Aldrich, Germany | L2897-1KG | |
| MEM (Minimum Essential Medium) | Gibco Thermo Fisher Scientific Inc. | 11095072 | |
| Non-Essential Amino Acids Solution (100X) | Gibco Thermo Fisher Scientific Inc. | 11140050 | |
| P. aeruginosa strain PAO1 | American Type Culture Collection | 47085 | |
| P. aeruginosa strain PAO1-GFP | American Type Culture Collection | 10145GFP | |
| Paraformaldehyde Aqueous Solution -16% | EMS DIASUM | 15710-S | |
| Phosphate buffer solution buffer | Thermo Fischer | 10010023 | |
| Petri dishes | Greiner | 664102 | |
| Phorbol 12-myristate 13-acetate (PMA) | Sigma, Germany | P8139-1MG | |
| Precision Cover Glasses | ThorLabs | CG15KH | |
| Purified Mouse anti-human ZO-1 IgG antibody | BD Transduction Laboratories | 610966 | |
| Roswell Park Memorial Institute (RPMI) 1640 medium | Gibco by Lifetechnologies, Paisley, UK | 11875093 | |
| Soda-lime glass Petri dish, 50 x 200 mm (height x outside diameter) | Normax, Portugal | 5058561 | |
| Saponin | Sigma-Aldrich, Germany | S4521 | |
| T75 culture flasks | Thermo Fischer | 156499 | |
| THP-1 cells | Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany) | No. ACC-16 | |
| Tobramycin sulfate salt | Sigma | T1783-500MG | |
| Trypsin-EDTA 0.05% | Thermo Fischer | 25300054 | |
| Tween80 | Sigma-Aldrich, Germany | P1754 |
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