Basal Cell Expansion Conditions Influence Differentiation and Phenotypic Outcomes in Primary Nasal Epithelia Grown at Air–Liquid Interface

Background/Objectives: Primary airway epithelia grown at air–liquid interface (ALI) are widely used to model respiratory endophenotypes and to enable individualized functional testing. However, culture conditions used to establish ALI vary considerably and may introduce selection biases. We aimed to adapt our lung explant-based ALI protocol to nasal basal cells obtained by minimally invasive brushing and to identify expansion conditions that optimize the number of primary epithelia that can be generated from nasal brushing while preserving donor-specific features. Methods: Primary nasal basal cells from nine healthy adult donors were expanded using different protocol variations in a paired design. We systematically varied two factors during the expansion phase: (a) supplementation with Rho-associated kinase (ROCK) and SMAD pathway inhibitors and (b) a single passage prior to ALI differentiation. Outcomes included the likelihood of obtaining a well-formed, differentiated ALI epithelium and functional readouts relevant to respiratory phenotyping. Results: The probability of establishing a well-differentiated ALI epithelium in a single attempt was highest when expansion was performed without ROCK/SMAD inhibitors and without passaging. Among successfully established ALI cultures, replicate inserts from the same donor showed comparable CFTR expression and ciliary beat frequency; however, functional chloride secretion could only be robustly detected in a subset of epithelia and was associated with low epithelial apoptosis. Conclusions: Avoiding strong selection pressures during the expansion phase—by omitting ROCK/SMAD inhibitors and minimizing passaging—improves the success rate of obtaining well-differentiating primary nasal ALI cultures and likely preserves cellular diversity among founder cells of the epithelium. These findings support the use of patient-derived nasal ALI cultures as robust, reproducible platforms for individualized respiratory phenotyping and functional assessment in personalized medicine.