Dysfunctional salivary gland (SG) is an unsolved clinical challenge, which is presented as xerostomia. Cell therapy is a promising treatment for restoring SG function. Salispheres are spheroid cellular organizations derived from SG stem cells. Benefitting from these cellular organizations, SG stem cells can be expanded to regenerate SG. During in vitro culture, the spontaneous reorganization of salispheres may change the features of residing SG stem cells. Therefore, it is imperative to explore ways to maintain the spheroid structure of salispheres during cell expansion in vitro. Herein, we explored biomaterial approaches using chitosan. Chitosan based biomaterials were fabricated in different forms to offer distinct interactive surfaces for cultured salispheres. The number and size of the salispheres increase in the chitosan-containing systems without increasing the incidence of spheroid cavitation. The effect of chitosan increases with high chitosan concentrations, which is optimum when chitosan is fabricated in a soluble form. The chitosan effect contributes to the regulation of the intercellular interactions and polarization within the spheroid structures. By retarding the process of salisphere cavitation, chitosan preserves the features of salivary gland progenitor cells in the cultured salispheres. The results suggest that the chitosan-containing system could effectively maintain the primitive structures and properties of salispheres during in vitro expansion, which demonstrates the potential application of salispheres for cell therapy of dysfunctional SG.