Culture platform influences marker expression and morphology of human lung organoids

Abstract The cultivation platform strongly shapes the development and phenotype of human lung organoids derived from adult stem cells. We compared three systems - animal-derived (AD), animal-free (AF), and scaffold-free (SF) - under basal-out, apical-out, and proximally differentiated conditions. Our goal was to assess how each platform influences epithelial cell-type-associated marker expression and morphology. AD and AF matrices yielded smaller, more uniform organoids, whereas SF cultures produced markedly larger structures, especially in apical-out and proximally differentiated conditions. Bulk RT-qPCR analysis revealed distinct platform-associated marker-expression profiles: the basal cell marker KRT5 peaked in AD basal-out organoids. Under proximal differentiation, KRT5 expression declined across systems, reaching the lowest levels in AF, and SCGB1A1 became undetectable in all systems. In contrast, SF cultures showed the highest expression of the AT1 cell-associated marker AQP5 (basal-out and apical-out) and of the ciliated cell-associated marker FOXJ1 (proximally differentiated), whereas MUC5AC was only slightly upregulated. Notably, ACE2 and TMPRSS2 - key mediators of SARS‑CoV‑2 entry - were induced by proximal differentiation: ACE2 was strongly upregulated in SF organoids, and TMPRSS2 increased under all proximal conditions, independent of platform. Immunofluorescence corroborated mature epithelial phenotypes, revealing robust ciliation across platforms and particularly advanced cilia maturation in AF cultures. Together, these data indicate that the cultivation platform strongly influences lung organoid growth, morphology, and cell-type-associated marker expression. Our findings provide an evidence-based guide for selecting systems to model human respiratory physiology, infection, and regeneration.