Development of a Soy Protein-Based Composite Hydrogel as a Platform for Three-Dimensional Cell Culture of Human Salivary Glands

Background: Xerostomia, or dry mouth symptoms, affects billions of people worldwide and currently has no definitive cure. Tissue engineering with three-dimensional (3D) platforms has become increasingly popular for modelling salivary gland diseases. For decades, soybeans have been used in the pharmaceutical industry owing to their abundance of proteins, peptides, and amino acids, and recently, they have emerged as a source of sustainable biomaterials. Methods: A composite hydrogel from soy protein isolate (SPI), alginate (A), and gelatin (G) was developed by the hanging-drop technique and crosslinked with CaCl2 for ionic gelation. The scaffold was characterized by scanning electron microscopy (SEM), fourier-transform infrared spectroscopy (FTIR), swelling and degradation rate, and mechanical tests. The in vitro biocompatibility of the scaffold on cellular proliferation, organoid formation, phenotype expression and functionality of two cell types: human submandibular gland cells (HuSMG) and primary salivary functional units (SFUs), was also assessed. Results: Soy-based hydrogels have a stiffness comparable to native salivary gland tissue (1–3 kPa) and controlled swelling and degradation rates after 3 weeks of incubation. Biological assessment indicated greater sustained cell viability (>90%) and betterorganized spheroid/organoid structures in the soy-based hydrogels after 1 month of culture. Robust expression of key salivary gland markers, including zonula occludens-1 (ZO-1), aquaporin 5 (AQP5), cytokeratin 7 (CK7), α-amylase (AMY1) and alpha-smooth muscle actin (αSMA), was observed in both cell types cultured in the soy-based hydrogel. Additionally, the spheroids/organoids exhibited physiological responsiveness and functional maturation through agonist-induced stimulation, including ATP, Carbachol, and Isoprenaline. Conclusions: These findings validate the soy-based hydrogel as a biocompatible, sustainable and physiologically relevant 3D culture system for salivary tissue engineering and regeneration.