Diabetes is a disease that affects the patient's quality of life. Although there are several studies of therapeutic alternatives, there is still no definitive cure. Polymeric scaffolds represent a promising therapeutic strategy to preserve cell mass through 3D cultures. The aim of this study is to explore an alternative polymeric scaffold based on a mix of chitosan (Chi), gelatin (Ge), and polyvinyl alcohol (PVA) functionalized with VEGF for the culture of insulin-producing cells. The scaffolds were obtained by freeze-thaw cycles and lyophilization, followed by neutralization and, functionalization with vascular endothelial growth factor (VEGF). Physicochemical characteristics, biocompatibility and functionality were evaluated. Scaffolds obtained had interconnected heterogeneous pores. The presence of functional groups confirmed the integration of all the components without significantly losing thermal stability and mass. The functionalized and neutralized scaffolds positively impacted the biocompatibility and insulin secretion. Cell respiration was sustained, and cell morphology demonstrated the formation of cell clusters. It can be concluded that neutralization and functionalization of the scaffolds combined with VEGF are necessary to improve biocompatibility and functionality. Moreover, all these characteristics generated encouraging results on the diffusion of nutrients and cell adhesion, which could be valuable in the translational application for diabetes treatment.