Establishing a Co-Culture Model to Study Host-Pathogen Interactions at the Air-Liquid Interface

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.