The main goal of this study was the design, development and characterization of a chitosan based scaffolding substrate including three glycosaminoglycans and collagen to provide an optimal microenvironment for human mesemchymal stem cells isolated from adipose tissue (hMSCs). Chitosan scaffolds provide a moist wound environment which promotes healing and epidermal regeneration. Furthermore, the importance of extracellular molecules such as glycosaminoglycans in wound healing makes them essential ingredients in these types of formulations. The physical properties of hydrogels scaffolds and stability were investigated. The scaffolds were evaluated by structural and microscopic assays, as well as cell culture analyses. The hydrogel with best suitable properties was selected as candidate scaffold for hMSCs encapsulation. The viability of hMSCs remained above 75%, indicating good cell viability. The number of living hMSCs in the scaffold reached a steady state up to ~100% at days 5 and 7. Scanning electron microscopy showed irregular compartments with the presence of the hMSCs. These findings indicated that our hydrogel scaffold provided a suitable niche for cell viability which could be considered a promising candidate for further in vivo studies.