Establishing a Co-Culture Model to Study Host-Pathogen Interactions at the Air-Liquid Interface
JoVE Encyclopedia of Experiments
Microbiology
0 views • 1:50 min • July 1st, 2026
Begin with a multi-well plate containing a transwell membrane insert.
The insert contains a confluent bronchial epithelial cell layer on the upper side of the membrane.
The insert's lower side holds macrophage-like cells in medium, mimicking the human airway epithelial barrier.
Add pathogenic bacteria suspended in a buffer over the epithelial layer.
Incubate to allow bacterial attachment to the epithelial cells and their colonization.
Then, remove the buffer from the insert while retaining the medium in the well to create an air-liquid interface.
The bacteria secrete virulence factors that interact with epithelial tight junctions and disrupt them.
This increases permeability across the epithelial layer and triggers the release of pro-inflammatory cytokines from the epithelial cells.
These cytokines facilitate the movement of macrophage-like cells toward the epithelial layer, where they recognize and engulf the bacteria.
This establishes an epithelial-macrophage co-culture model at the air-liquid interface, representing pathogen exposure in the respiratory tract.
Add 100 microliters of bacterial suspension to the apical side of the permeable support and incubate the plate at 37 degrees Celsius and 5% carbon dioxide for one hour to allow bacteria to attach to the cells. Then, carefully remove apical liquid with a pipette to restore ALI conditions.
Overview
This article presents a 3D co-culture model of bronchial epithelial cells and macrophage-like cells at the air-liquid interface (ALI) to study host-pathogen interactions, specifically focusing on Pseudomonas aeruginosa infection. The model mimics the human airway epithelial barrier and enables preclinical evaluation of anti-infective strategies by observing epithelial integrity, immune cell activation, and pathogen clearance mechanisms.
Key Study Components
Area of Science
- Cell biology
- Immunology
- Microbiology
- Respiratory research
Background
- The airway epithelium serves as a primary barrier against respiratory pathogens.
- Macrophages play a crucial role in innate immune defense within the lung.
- Traditional monoculture models lack the complexity of cell-cell interactions present in vivo.
- 3D co-culture systems at ALI better replicate physiological conditions for infection studies.
Purpose of Study
- To establish a physiologically relevant in vitro model for studying P. aeruginosa infection.
- To investigate the interplay between bronchial epithelial cells and macrophage-like cells during infection.
- To assess epithelial barrier integrity and immune responses upon bacterial challenge.
Methods Used
- Culture of bronchial epithelial cells to confluence on transwell membrane inserts.
- Seeding of macrophage-like cells on the lower side of the membrane to mimic the airway barrier.
- Addition of P. aeruginosa to the apical (upper) side of the epithelial layer.
- Incubation to allow bacterial attachment and colonization.
- Establishment of air-liquid interface by removing apical liquid while maintaining medium below.
- Observation of epithelial disruption, cytokine release, and macrophage migration and phagocytosis.
Main Results
- Bacterial infection disrupts epithelial tight junctions, increasing permeability.
- Infected epithelial cells release pro-inflammatory cytokines.
- Cytokines promote migration of macrophage-like cells toward the epithelial layer.
- Macrophage-like cells recognize and engulf bacteria, modeling pathogen clearance.
Conclusions
- The 3D co-culture ALI model effectively simulates respiratory tract infection dynamics.
- It enables detailed study of epithelial barrier function and immune cell responses.
- This system is valuable for preclinical evaluation of anti-infective therapies targeting airway infections.
Frequently Asked Questions
Executive Industry Relevance
Establishing a 3D co-culture model at the air-liquid interface enables predictive evaluation of host-pathogen interactions in a physiologically relevant respiratory context. This system supports mechanistic de-risking for anti-infective discovery by modeling epithelial barrier disruption and immune cell responses. The approach enhances translational confidence for preclinical anti-infective candidate assessment and portfolio triage.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of host-pathogen interaction mechanisms in a controlled, disease-relevant system.
- Supports biological de-risking by modeling epithelial barrier integrity and immune activation.
- Facilitates functional target validation for anti-infective strategies targeting airway infections.
Screening & Assay Development
- Provides a validated co-culture platform for quantitative assessment of epithelial disruption and immune cell migration.
- Enables reproducible measurement of cytokine release and pathogen clearance as assay endpoints.
- Supports screening readiness for evaluating anti-infective compound efficacy in a complex biological context.
Translational & Preclinical Research
- Aligns with disease-relevant respiratory models for translational biomarker development.
- Ensures continuity from discovery through preclinical validation by modeling human airway responses.
- Reduces translational risk by providing predictive data on epithelial and immune cell dynamics.
Pipeline & Workflow Integration
This co-culture model integrates into the discovery-to-preclinical continuum for anti-infective R&D, bridging early mechanistic studies and preclinical candidate evaluation.
- Discovery Biology: Supports hypothesis testing on epithelial barrier function and immune response to infection.
- Screening: Delivers quantitative, reproducible outputs for compound efficacy assessment.
- Analytics: Enables measurement of membrane integrity, cytokine levels, and phagocytic activity.
- Translational Research: Provides a platform for biomarker alignment and disease-relevant validation.
- Enterprise Reuse: Offers a reusable, standardized system for cross-program anti-infective evaluation.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation and mechanistic understanding of infection.
- Operational Value: Standardizes co-culture workflows for reproducibility and scalability across projects.
- Strategic Value: Informs go/no-go decisions and reduces late-stage biological risk for anti-infective portfolios.
- Portfolio Impact: Enables risk-adjusted prioritization of candidates based on functional, disease-relevant data.
Implementation Considerations
- Requires expertise in airway epithelial and immune cell culture techniques.
- Needs access to transwell systems and analytical tools for barrier integrity and cytokine measurement.
- Demands cross-team standardization for reproducible co-culture setup and readout analysis.
- Adaptation may be needed for different pathogen or cell type combinations.
- Model complexity may limit throughput compared to simpler monoculture assays.
Biopharma FAQs
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Last updated: 22 August 2026