In vivo-comparable in vitro assay for lung epithelial barrier injury

A method of testing a test substance in an in vitro model of a human tracheobronchial respiratory tract, said method comprising:

• providing the in vitro model comprising a cell culture comprising airway epithelial cells, a basolateral compartment below the airway epithelial cells and an apical compartment above the airway epithelial cells, wherein the airway epithelial cells form a barrier between the basolateral compartment and the apical compartment;
• adding the test substance to the apical and/or basolateral compartment;
• adding a tracer to the basolateral compartment, wherein the tracer is fluorescent, has a molecular weight within 5 kDa of a molecular weight of human albumin and is added to the basolateral compartment before, during or after adding the test substance to the apical and/or basolateral compartment;
• incubating the cell culture system in a presence of the tracer;
• collecting at least one sample from the apical compartment of the cell culture;
• and
• measuring a fluorescence of the at least one sample to determine an effect of the test substance on the airway epithelial cells.