Salivary gland organoid culture maintains distinct glandular properties of murine and human major salivary glands

Salivary glands that produce and secrete saliva, which is essential for lubrication, digestion, immunity, and oral homeostasis, consist of diverse cells. The long-term maintenance of diverse salivary gland cells in organoids remains problematic. Here, we establish long-term murine and human salivary gland organoid cultures. Murine and human salivary gland organoids express gland-specific genes and proteins of acinar, myoepithelial, and duct cells, and exhibit gland functions when stimulated with neurotransmitters. Furthermore, human salivary gland organoids are established from isolated basal or luminal cells, retaining their characteristics. Single-cell RNA sequencing also indicates that human salivary gland organoids contain heterogeneous cell types and replicate glandular diversity. Our protocol also enables the generation of tumoroid cultures from benign and malignant salivary gland tumor types, in which tumor-specific gene signatures are well-conserved. In this study, we provide an experimental platform for the exploration of precision medicine in the era of tissue regeneration and anticancer treatment.

(a) mSMG organoids were cultured in EGF-containing media and subjected to brightfield, H&E, or IF imaging for ductal (KRT5/KRT7) or acinar/myoepithelial (AQP5/ACTA2) markers. Red arrows indicate organoids with keratinized core. Scale bars indicate 500 μm for brightfield images and 50 μm for H&E and IF images. (b) Proportions of keratinized organoids (left) and expressions of Involucrin mRNA (right) were compared between the organoids cultured in EGF-or NRG1-containing media (n = 3). (c) H&E staining of mSMG organoids cultured in the GEM with or without retinoic acid (RA). Black arrow indicates the keratin core. Scale bar indicates 50 μm.
(d) The expression of WNT gene family members was assessed in the organoids cultured in media containing EGF, HB-EGF, NRG1, or TGFα. The gene expression of organoids cultured in EGF-containing media was normalized as 1 (n = 3). (e) Dose-dependent effect of A83-01 (left), Noggin (middle), and RSPO1-CM (right) on organoid growth was determined with brightfield images. Scale bar indicates 200 μm. The selected conditions used in mouse GEM are highlighted with red outlines. (f) FGF-induced growth support was observed using brightfield microscopy. FGF1, FGF2, FGF7, or FGF10 were added solely or in combination to NRG1-based media.
Scale bar indicates 500 μm. (g) Expression of the acinar-related gene (Smgc and Prol1) was measured at different compositions of FGF1 or FGF7. Expressions were not detected in the organoids cultured without FGFs (n = 3). (h) mSMG organoid were cultured in GEM supplemented with DMSO or 2 μM IWP-2 for 7 days. Organoid growth was determined with brightfield images (left) and quantified via luminescence (right, n = 3). Scale bar indicates 200 μm. Data are representative of at least three independent experiments, and presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.
Supplementary Figure 2. Gene expression and functionality of long-term cultured murine salivary gland organoids.
(a) mSMG organoids were maintained in the GEM or GEM lacking each factor and their maximum passage number (left) and proliferation capacity (right, n = 3) were evaluated. (b) mSMG organoids were maintained for longer period (passage 30). The growth of a single organoid was tracked in time-lapse of images obtained at each time point. (c) mRNA expression of ductal (Krt5, Krt7), acinar (Aqp5, Bhlha15), and myoepithelial (Krt14, Acta2) was determined in mouse SMG tissues and organoids that were cultured for different periods. Expressions in organoids cultured for 1 month were normalized as 1 (n = 3). (d-g) mSMG organoids maintained for 30 passages were used for assessment of marker expression, genetic stability, and functionality. (f) Organoids from CD49f + CD26and CD49f + CD26 + cells were maintained in the GEM for 3 weeks, followed by 3 days in the DAM, and subjected to H&E staining or IF staining for duct (KRT5/KRT7), acinar (AQP5/MIST1), and myoepithelial (ACTA2/KRT14) markers. White arrows indicate MIST1 + cells or ACTA2 + KRT14 + myoepithelial cell. Nuclei were stained with Hoechst 33342 (blue).
Scale bars indicate 50 μm. Data are representative of at least three independent experiments, and presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.
Supplementary Figure 8. Identification of cell types in salivary gland tissues and cluster-specific gene signatures in organoids.
Human PG, SMG, and SLG organoids were cultured in the GEM for 1 month and differentiated in the DAM for another 3 days. The harvested organoids were subjected to scRNA-seq. Human SMG organoids were cultured in the GEM for 1 month or 3 months, and differentiated in the DAM for another 3 days. The harvested organoids were subjected to scRNA-seq, and clusters were visualized as UMAP. (a) The different culture periods were illustrated with different colors (early for 1 month, orange; late for 3 months, blue). (b) Based on the clustering strategy used in Figure   5A, clusters from early passage were displayed with all cells from the late passage. Cluster 8 in early passage was not detected in the late passage. (c) Each cluster from early passage was illustrated with all cells from the late passage.