Constructing Salivary Gland Organoids from Human Minor Salivary Glands

Summary

This protocol generates human minor salivary gland organoids through the self-organization of human minor salivary gland stem cells (hMSG-SCs) and human minor salivary gland mesenchymal stem cells (hMSG-MSCs) within a 3D Matrigel system.

Abstract

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.

Introduction

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 research

Protocol

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 ....

Results

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, wh.......

Discussion

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	C11885500BT
0.25% Trypsin-EDTA	Gibco	25200056
Dulbecco’s Modified Eagle Medium (DMEM)/F12	Gibco	C11330500BT
Fetal bovine serum (FBS)	Gibco	10270106
GlutaMAX	Gibco	35050061
Keratinocyte Medium	Sciencell	2101
Matrigel	BD	356230
Mesenchymal Stem Cell Medium (MSCM)	Sciencell	7501
Penicillin/Streptomycin	Gibco	15140122
Phosphate-Buffered Saline (PBS)	Gibco	C20012500BT