Establishment and characterisation of primary human oesophageal organoids as a versatile epithelial model

Purpose Human patient-derived oesophageal epithelial models are limited, and organoid cultures often have short passaging lifespans, restricting studies of tissue homeostasis and disease. We aimed to establish and characterise primary oesophageal organoids from non-pathologic epithelium as a renewable epithelial source and adaptable modelling platform. Methods Primary oesophageal tissues were obtained from endoscopic biopsies and surgical resections. Basal epithelial cells were isolated and cultured under optimised conditions to establish long-term three-dimensional organoids. Organoid-derived cells were expanded as two-dimensional monolayers and re-seeded into 3D culture. Epithelial identity and differentiation were assessed by immunostaining and RT-qPCR (reverse transcription-quantitative polymerase chain reaction) analysis of markers. Results Primary organoids were established and maintained while preserving epithelial markers. Expression of KRT14, involucrin, loricrin and filaggrin confirmed the oesophageal phenotype. Larger, multilayered primary organoids showed increased expression of differentiation-associated markers relative to 2D monolayers and re-seeded small organoids, indicating culture configuration influences epithelial differentiation while maintaining lineage identity. Conclusions We established a robust and reproducible protocol for generating and maintaining oesophageal organoids across culture formats. These models retained epithelial features while exhibiting culture-dependent differentiation. This platform enables complementary 2D and 3D investigation of oesophageal epithelial biology and supports future comparison of healthy and disease-derived tissues for patient-specific modelling and mechanistic studies.