Constructing Salivary Gland Organoids from Human Minor Salivary Glands

This study describes a protocol for constructing human minor salivary gland (hMSG) organoids to establish a reproducible model for tissue regeneration research. Salivary gland stem/progenitor cells (hMSG-SCs) and mesenchymal stem cells (hMSG-MSCs) were isolated from pediatric minor salivary gland tissues, expanded, and co-seeded in a Matrigel-based three-dimensional (3D) system. Self-organization led to the formation of organoids with ductal-acinar-like structures, recapitulating native cellular heterogeneity more effectively than epithelial-only models. The current protocol emphasizes optimized culture conditions to preserve cell phenotypes and standardized steps for passaging, ratio mixing, and handling Matrigel (a commercial basement matrix), thereby enhancing reproducibility. Importantly, the presence of mesenchymal cells provides a supportive microenvironment that promotes epithelial survival, proliferation, and morphogenesis, overcoming limitations of low efficiency and incomplete structure. This approach enables consistent generation of salivary gland organoids and offers a robust model for studying development, functional restoration, and disease mechanisms, while also supporting potential translational applications in regenerative therapies.