Protocol for differentiating primary human small airway epithelial cells at the air-liquid interface

Area of Science:

• Pulmonary research
• Cell culture and differentiation
• Airway epithelial biology

Background:

• Air-liquid interface (ALI) culture models the lung's physiological environment.
• Small airway epithelial cells (SAECs) are underutilized compared to bronchial cells.
• SAECs are crucial for studying bronchiolar pathologies and serve as surrogates for alveolar cells.

Purpose of the Study:

• To provide an optimized, detailed protocol for culturing and differentiating primary human SAECs at the ALI.
• To address the current knowledge gap in SAEC ALI culture methodology.
• To establish a reliable model for studying small airway diseases.

Main Methods:

• Optimization of key culture conditions: passage number, pH, CO2, seeding density, and collagen coating.
• Development of a FITC-dextran permeability assay for assessing SAEC barrier integrity.
• Exposure of differentiated SAECs to cigarette smoke, LPS, and poly(I:C) to validate the model.

Main Results:

• Identified critical culture parameters for high-quality, uniform SAEC differentiation at ALI.
• Demonstrated the utility of the FITC-dextran assay for pre- and post-experiment barrier integrity assessment.
• Showcased model responsiveness through increased cytokine production and epithelial damage upon exposure to stimuli.

Conclusions:

• The provided protocol successfully bridges the methodological gap for differentiating primary human SAECs at ALI.
• This model is essential for advancing research into small airway diseases, including COPD.
• The optimized SAEC ALI model facilitates the study of airway responses to various insults.