In this study, cross-linked hydrogels for 3D cell culture systems were made from a mixture of poly (vinyl alcohol) (PVA) and gelatin using a 60Co γ-ray irradiation technique. The physical properties of these hydrogels, including their gelatin content, water absorptivity, and gel strength, were examined to evaluate the applicability of these hydrogels as the 3D constructs for diploid cell cultures. The gel content and strength increased upon increasing the radiation dose and decreasing the concentration of gelatin. The degree of swelling was inversely proportional to the gel content and strength. The cyto-compatibility of these hydrogels, with respect to their gelatin contents, was evaluated in vitro in cultures of mouse fibroblasts. The fibroblast adhesion in the hydrogels increased upon increasing the gelatin content. In addition, the fibroblasts cultured in gelatin-containing hydrogels showed improved spreading ability and growth ratios. Therefore, these hydrogels appear to be suitable for scale-up, and they are promising scaffold materials for large-scale diploid cell cultures.