Constructing Salivary Gland Organoids from Human Minor Salivary Glands

Salivary glands are essential exocrine glands in the oral cavity, responsible for secreting saliva to maintain oral moisture, aid digestion, and provide antimicrobial defense¹. Dysfunction of these glands, such as in Sjögren's syndrome or post-radiation damage, can lead to severe oral health problems, and effective regenerative treatments are currently lacking². Organoid technology, an emerging method for constructing cell substitutes that mimic the structure and function of tissues and organs in a three-dimensional (3D) culture system, offers a promising tool for salivary gland regeneration research³.

In recent years, mouse and human salivary gland organoid culture systems have been successfully established^4,5. Various stem cell types, including salivary gland stem/progenitor cells^4,5, pluripotent stem cells^6,7, and epithelial-derived stem cells such as dental follicle stem cells⁸, have been used to generate these organoids. Nevertheless, several technical challenges remain. Primary human salivary gland epithelial stem/progenitor cells (hSG-epiS/PCs) exhibit limited proliferation and low survival in vitro, which restricts organoid formation efficiency and reproducibility⁹. Moreover, organoids derived solely from epithelial cells often lack multicellular heterogeneity and fail to fully recapitulate the branched architecture and functional acinar-ductal organization of native glands¹⁰.

Against this background, this study aims to establish a standardized method for constructing human minor salivary gland organoids. First, we isolated stem cells (hMSG-SCs) and mesenchymal stem cells (hMSG-MSCs) from human minor salivary gland tissues^11,12,13. Organoids were subsequently initiated via a self-assembling mechanism by co-seeding hMSG-SCs and hMSG-MSCs in Matrigel. This method provides a reproducible platform for salivary gland organoid culture and lays an important foundation for future research in salivary gland regeneration and disease modeling.

Human minor salivary gland (hMSG) samples were obtained from patients undergoing lip surgery at the Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. The patients (n > 10) were aged between 3 months and 10 years, with no history of gland-related disorders or autoimmune diseases. All tissues were obtained from redundant, histologically normal minor salivary glands excised during cleft lip repair, which provides an ethically approved and optimal source of viable tissue for stem/progenitor cell isolation. Informed consent was obtained preoperatively, and the study was approved by the Medical Ethics Committee of Shanghai Ninth People's Hospital (Approval No.2018-56). The reagents and the equipment used are listed in the Table of Materials.

1. Human minor salivary gland tissue collection

1. Immediately after excision, immerse the salivary gland tissues in Dulbecco's Modified Eagle Medium (DMEM) supplemented with antibiotics and maintain at 4 °C until further processing (within 6 h).

2. Cell isolation from human minor salivary gland tissues

1. Ensure sterile conditions by preparing an autoclaved surgical kit and performing all procedures inside a biosafety cabinet (Class II BSC).
2. Transfer the excised tissue into a culture dish containing Dulbecco's Phosphate Buffered Saline (PBS) and wash thoroughly. During the washing process, use forceps to carefully remove any connective tissue and residual blood, ensuring the salivary gland tissue is completely cleaned.
3. Add 1 mL of serum-free medium to prevent tissue dehydration. Mince the tissue into pieces no larger than 0.5 mm³ using sterile ophthalmic scissors.
4. Using sterile forceps, evenly distribute the tissue pieces onto the bottom of a T-25 culture flask.
5. Add 10 mL of culture medium to the culture flask.
   NOTE: For the culture of epithelial stem cells (hMSG-SCs), the primary culture medium used is Keratinocyte Medium (KM), containing 5 µg/mL bovine serum albumin, 50 µg/mL bovine pituitary extract, 5 µg/mL transferrin, 3.75 µg/mL insulin, 3 ng/mL fibroblast growth factor 2, 1 ng/mL epidermal growth factor, 500 ng/mL epinephrine, 0.5 µg/mL hydrocortisone, 10^-8 M prostaglandin E₂, and 30 nM T₃. For the culture of mesenchymal stem cells (hMSG-MSCs), the primary culture medium consists of DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1% Penicillin-Streptomycin (5,000 U/mL) and 1% GlutaMAX supplement.
6. Place the T-25 flask vertically in a 37 °C, 5% CO₂ incubator for 4-5 h. In the vertical position, the medium does not contact the minced tissue, allowing the explants to settle and firmly attach to the bottom surface.
7. After 4-5 h, switch the flask to a horizontal position so that the culture medium completely covers the attached tissue pieces and supports cell outgrowth.
8. Replace half of the medium on day 4 and then replace the medium every 3 days until cell colonies appear. When cell growth inhibition is observed, proceed with passaging.

3. Cell passaging

1. Passage cells when primary cell colonies show growth inhibition or when cell confluence reaches 80%-90%.
2. Aspirate the culture medium and wash the cells once with PBS.
3. Add 1 mL of 0.25% Trypsin-EDTA and incubate at 37°C, 5% CO₂ for 4 min for epithelial cells and 1.5 min for mesenchymal cells, respectively.
4. Neutralize trypsin by adding 5 mL of pre-warmed DMEM medium supplemented with 10% FBS. Gently pipette the cells to ensure proper dissociation, then transfer the cell suspension to a centrifuge tube and centrifuge at 300 × g for 5 min.
5. Discard the supernatant and resuspend the hMSG-SCs in KM medium, and resuspend the hMSG-MSCs in Mesenchymal Stem Cell Medium (MSCM).
6. Seed the resuspended cells in T-25 culture flasks at a 1:3 ratio, then incubate at 37 °C, 5% CO₂ for continued expansion.

4. Self-organization of hMSG-MSCs and hMSG-SCs for organoid construction

1. Thaw Matrigel on ice for 3-4 h.
2. Preheat a 24-well plate at 37 °C in an incubator and pre-chill pipette tips at 4 °C for precise handling.
3. Add 450 µL of pre-chilled sterile PBS to 1 mL of fully thawed Matrigel.
4. Mix gently using pre-chilled pipette tips and distribute 350 µL of the diluted Matrigel per well into the preheated 24-well plate, ensuring no bubbles are formed.
5. Tilt the plate gently to ensure even Matrigel coating.
6. Place the plate into a 37 °C, 5% CO₂ incubator for 20-30 min to allow the Matrigel to solidify. Avoid over-drying the Matrigel.
7. Culture hMSG-MSCs and hMSG-SCs at passages 3-5 separately until they reach 80%-90% confluency. Digest cells with 0.25% Trypsin-EDTA, neutralize with complete medium, and count cell numbers using an automated cell counter or haemocytometer.
8. Centrifuge at 300 × g for 5 min, discard the supernatant, and resuspend in fresh culture medium.
9. Mix hMSG-MSCs and hMSG-SCs at a 1:1 ratio.
10. Seed the cells into Matrigel-coated 24-well plates at a total cell concentration of 2 x 10⁵ cells per well.
11. Add 1 mL of organoid culture medium (MSCM:KM = 1:1) to each well.
12. Incubate at 37 °C, 5% CO₂ , and change the medium every 3 days.
13. Subculture organoids every 7 days at a ratio of 1:2 to 1:4, and resuspend in cell freezing media for storage at -80 °C.

5. Statistics and reproducibility

1. Analyze all data using statistical and graphing software and present as the mean ± standard deviation (SD). Two-tailed unpaired Student's t-tests were used for comparisons between two groups. P < 0.05 was considered statistically significant.
   NOTE: All experiments were independently repeated at least three times with similar results, and all statistical analyses were based on at least three independent experiments or samples.

Human minor salivary gland tissue explants cultured in T25 flasks showed clear cell outgrowth around the tissue blocks after approximately 5 days. In serum-rich stem cell culture medium, the cells exhibited spindle-shaped morphology characteristic of hMSG-MSCs (Figure 1A,B). Flow cytometry analysis revealed that these cells expressed high levels of mesenchymal stem cell markers, including CD29, CD90, CD105, and CD166 (Figure 1C). In contrast, when cultured in KM medium, cobblestone-like colonies appeared around day 7. These colonies could be stably expanded in vitro and were identified as hMSG-SCs (Figure 1 A,B). Flow cytometry analysis of passage 3 hMSG-SCs showed high expression of the epithelial marker CD49f, while mesenchymal stem cell markers CD90 and CD105 were negative (Figure 1C). Together, these results demonstrate that hMSG-SCs are distinct from mesenchymal stem cell populations and exhibit typical characteristics of epithelial origin.

When hMSG-MSCs and hMSG-SCs were co-seeded in Matrigel at a 1:1 ratio, the cells self-assembled into organoids, forming distinct ductal branches and gradually maturing. Acinar-like spherical structures extended into the Matrigel, while duct-like structures appeared as hollow tubular formations composed of monolayer or multilayer epithelial cells (Figure 2A,B). Immunofluorescence analysis revealed epithelial heterogeneity and proliferative activity (Figure 2C). CK5 and Ki67 co-staining identified proliferative basal epithelial cells, while strong MUC5B expression indicated secretory features. Whole-mount staining further confirmed the presence of both epithelial and mesenchymal components (Figure 2D). CK15/CK19 co-staining indicated the presence of basal epithelial progenitors (CK15⁺) together with luminal ductal cells (CK19⁺), demonstrating multiple epithelial subpopulations within the organoids. In addition, Vimentin/CK15 co-staining revealed basal epithelial cells in close association with surrounding mesenchymal cells, highlighting epithelial-mesenchymal interactions during organoid organization.

To directly evaluate the role of epithelial-mesenchymal interactions, we compared organoids generated from MSC-only, SC-only, and MSC-SC co-culture conditions (Figure 3). MSC-only cultures formed aggregates accompanied by extensive outward migration of fibroblast-like cells, with no organized epithelial structures. SC-only cultures produced small, simple spheroids with limited size and minimal morphological complexity. MSC-SC co-culture generated significantly larger 3D organoids with branching extensions and early ductal-acinar-like structures, demonstrating that cooperation between MSCs and SCs is essential for robust morphogenesis. Together, these findings indicate that co-assembly of hMSG-MSCs and hMSG-SCs promotes the formation of multicellular organoids with both structural complexity and differentiation.

Figure 1: Isolation and stable expansion of hMSG-MSCs and hMSG-SCs. (A) Outgrowth of cells from human minor salivary gland explants. Left: hMSG-MSCs with spindle-shaped morphology; Right: hMSG-SCs with cobblestone-like morphology. Scale bar = 100 µm. (B) Passage 3 cells retain their respective morphologies. Left: hMSG-MSCs; Right: hMSG-SCs. Scale bar = 100 µm. (C) Flow cytometry of passage 3 cells. hMSG-MSCs express mesenchymal markers CD29, CD90, CD105, and CD166, whereas hMSG-SCs express the epithelial marker CD49f and are negative for CD90 and CD105, confirming their distinct identities. Please click here to view a larger version of this figure.

Figure 2: hMSG-MSCs and hMSG-SCs self-organize to form salivary gland organoids. (A,B) Co-seeding of hMSG-MSCs and hMSG-SCs in Matrigel results in the formation of organoids with distinct ductal-acinar-like structures. Scale bars = 50 µm (A), 200 µm (B, left), and 50 µm (B, right). (C) Co-staining of CK5 (green) and Ki67 (red) identifies proliferative basal epithelial cells. Staining of MUC5B (green) indicates secretory features. Nuclei are counterstained with DAPI (blue). Scale bar = 25 µm. (D) Whole-mount immunofluorescence staining demonstrates epithelial and mesenchymal components within organoids. Co-staining of CK15 (green) and CK19 (red) confirms multiple epithelial subpopulations, whereas co-staining of Vimentin (green) and CK15 (red) shows mesenchymal cells surrounding CK15⁺ basal epithelial cells. Nuclei are counterstained with DAPI (blue). Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 3: Morphological comparison of organoids generated from hMSG-MSCs alone, hMSG-SCs alone, and MSC-SC co-culture. Representative bright-field images show that: MSC-only cultures formed aggregates surrounded by abundant fibroblast-like cells, without organized epithelial structures. SC-only cultures produced small, simple spheroid structures with limited size and minimal morphological complexity. MSC-SC co-culture generated larger 3D organoids with visible branching and early ductal-acinar-like architecture. Scale bars = 500 µm (top row) and 100 µm (bottom row). Please click here to view a larger version of this figure.

This protocol enables the successful construction of human minor salivary gland organoids through the isolation and culture of hMSG-SCs and hMSG-MSCs. The formation of organoids depends on careful preparation of tissue explants, maintaining distinct culture conditions for epithelial and mesenchymal cells, and accurate co-seeding of both cell types on Matrigel. Appropriate cell ratios and seeding densities are critical for supporting self-organization and reproducible formation of ductal-acinar-like structures.

During the procedure, challenges such as poor epithelial cell outgrowth, uneven organoid formation, or cell death during passaging may arise. These issues can be addressed by optimizing culture media, ensuring timely tissue adherence, and carefully monitoring enzymatic dissociation.

To promote the formation and maturation of salivary gland organoids, various strategies have been explored, including optimization of the growth environment, such as the use of bioreactors, and regulation of the microenvironment through growth factors and small molecule signaling modulators^{9,12,14,15,16}. Since MSCs and hMSG-SCs are derived from the same tissue source, their co-assembly not only provides diverse cell types and intercellular interactions but also enables paracrine functions of MSCs to support further organoid maturation. Compared with organoids generated from hMSG-SCs alone, this approach yields organoids with more complex ductal-acinar structures and enhanced expression of functional proteins. Although this protocol effectively generates structurally defined organoids in vitro, their functional maturation and long-term culture stability remain to be validated.

Importantly, the organoids generated using this co-culture protocol have broad potential for disease modeling. Their stable ductal-acinar organization and preserved epithelial-mesenchymal interactions provide a physiologically relevant platform for studying salivary gland disorders, including radiation-induced gland injury, secretory dysfunction, inflammatory responses, and fibrotic remodeling. Because the system supports controlled perturbations in a 3D microenvironment, it may further enable drug-testing applications and mechanistic studies of epithelial regeneration or glandular dysfunction.

Overall, this study provides a crucial experimental foundation for salivary gland regeneration and disease modeling research, with potential applications in the treatment of salivary gland dysfunction and drug screening.