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

Summary

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.

Abstract

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.

Introduction

Pseudomonas aeruginosa is a relevant pathogen in cystic fibrosis (CF) that contributes to lung tissue impairment¹. The production of polysaccharides, such as alginate and other mucoid exopolysaccharides, coordinates the progress of the disease, which leads to tenacious bacterial adherence, limits the delivery of antibiotics to bacteria and protects the bacteria against the host immune system². The transition of P. aeruginosa from the planktonic stage to biofilm formation is a critical issue in this context, also facilitating the occurrence of antibiotic tolerance.

In the cont....

Protocol

1. Growth and differentiation of cells in permeable support inserts

1. Cultivate CFBE41o^- in a T75 flask with 13 mL of minimum essential medium (MEM) containing 10% fetal calf serum (FCS), 1% non-essential amino acids and 600 mg/L glucose at 37 °C with 5 % CO₂ atmosphere. Add fresh medium to the cells every 2–3 days.
   1. Detach the cells after reaching 70% confluence in the flask with 3 mL of trypsin- Ethylenediaminetetraacetic acid (EDTA) at 37°C for 15 mi.......

Discussion

This paper describes a protocol for a 3D co-culture of the infected airways, constituted by the human cystic fibrosis bronchial epithelial cell line CFBE41o- and the human monocyte-derived macrophage cell line THP-1. The protocol allows the assessment of epithelial barrier integrity, macrophage transmigration, bacteria survival, and inflammation, which are important parameters when testing drug efficacy and host-responses simultaneously. The novelty in the model lies within the incorporation of epithelial cells (i.e........

Materials

Name	Company	Catalog Number	Comments
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