The authors have developed an allogeneic cultured dermal substitute (CDS) through cultivation of fibroblasts on a two-layered spongy matrix of hyaluronic acid (HA) and atelocollagen (Col). The Col spongy layer is essential for attachment and proliferation of fibroblasts on the two-layered spongy matrix. The HA spongy layer is necessary for maintaining the moisture environment on the wound surface. The optimal weight ratio of HA/Col is determined by considering the following characteristics: mechanical properties for handling, cell viability after thawing, potency of vascular endothelial growth factor (VEGF) release after thawing, efficacy of wound healing, and manufacturing cost. This study is designed to investigate the physical properties for handling, the growth behavior of fibroblasts on the spongy matrix, and the quantitative analysis of VEGF released from fibroblasts in the fresh or cryopreserved CDS. The results of this study suggest that a CDS composed of Col spongy matrix alone has the highest potency in regard to the release of VEGF. However, taking into account the manufacturing cost, coupled with the potency of VEGF release, a two-layered sponge of HA and Col with a weight ratio of 5/2 is very promising for commercial application.