TransPillar‐Based Culture Platform for Reproducible and Standardized Formation of Human‐Derived Airway Organoids

This study proposes an optimized culture strategy for standardized human‐derived airway organoids using the newly developed TransPillar platform. Human airway tissue consists of basal, goblet, and ciliated cells that maintain apical–basal polarity, and airway organoids recapitulate this cellular composition to mimic native tissue. Organoid standardization is essential for preserving cellular composition, polarity, and assay reliability while reducing size variability. However, conventional FlatWell‐based platforms exhibit inherent limitations, as gravity‐driven downward redistribution of cells and extracellular matrix leads to their accumulation at the plate interface, thereby contributing to structural heterogeneity within Matrigel domes. In contrast, the TransPillar platform maintains the Matrigel dome in an inverted configuration beneath the pillar, enabling gravity‐driven redistribution of cells toward the distal end of the dome. Organoids preferentially form in this region without attachment to a rigid substrate, reducing growth heterogeneity and improving structural uniformity. Immunohistochemical analysis confirmed the presence of basal, goblet, and ciliated cells with preserved apical–basal polarity. Compared with FlatWell cultures, TransPillar exhibited higher retrieval efficiency and maintained structural integrity, facilitating reliable histological evaluation. These findings establish TransPillar as a robust platform for standardized airway organoid culture, providing a simple yet effective strategy to improve reproducibility in organoid‐based research.