Abstract
Regenerative therapies for salivary gland dysfunction remain unavailable. We establish a fully chemically defined, xeno-free three-dimensional culture system to generate functional human salivary gland organoids. These organoids recapitulate the typical glandular architecture and display multilineage cellular composition, supporting long-term expansion with high transcriptomic fidelity to the primary tissue. The organoids exhibit key salivary functions, including glycoprotein secretion, amylase expression, and calcium flux in response to cholinergic stimulation. Single-cell transcriptomic analysis reveals preserved epithelial heterogeneity within the organoids and indicates a basal-to-ductal-to-acinar bifurcated differentiation trajectory. In the orthotopic transplantation model using non-obese diabetic mice with Sjögren syndrome, the organoids significantly improve salivary secretion. In the ectopic subrenal capsule transplantation model using immunodeficient mice, the organoids achieve glandular tissue reconstruction and vascularization. This study establishes a robust, functionally mature, and clinically translatable human salivary gland organoid system as a platform for tissue regeneration therapies targeting salivary hypofunction disorders such as Sjögren syndrome.