78. Skin Reconstruction Combining Cultured Dermis Only from Human Fibroblasts and Cultured Epidermis

PURPOSE: Skin defects have significant physical and psychosocial implications for patients. For patients lacking autologous skin for grafting, dermal substitutes prepared using animal collage have become a viable alternative. However, it is still difficult to reconstruct full-thickness skin using cultured epidermal autograft (CEA) and dermal substitutes. Recently, cell therapy and regenerative medicine have emerged as promising strategies to address these limitations. We have developed a scaffold-free cultured dermis generated only from human fibroblasts using the net-mold method and showed that our cultured dermis promoted wound healing in murine full-thickness skin defects. In this study, we tried to reconstruct full-thickness skin by combining our cultured dermis and cultured epidermis (CE) by transplanting them into immunodeficient mice. METHODS: Human dermal fibroblast (HDF) spheroids were fabricated on an ultra-low attachment (ULA) surface. We established a novel culture platform that enables cultured dermis generation through spheroid fusion, without adding extracellular matrix. CE was prepared using Green’s method and transplanted CE onto the cultured dermis, which measured 6 mm in diameter and 0.5 mm in thickness.The constructs were co-cultured at the air-liquid interface (ALI) for 1 day and 7 days. We grafted the full-thickness skin after culturing for 1 day into the subcutis of nude mice and took the specimen on day 7. We observed the full-thickness skin histologically before grafting and 7 days after grafting. RESULTS: H&E staining and pan-cytokeratin (pan-CK) immunostaining were performed for histological evaluation. 7 days after ALI culture, both the dermal and the epidermal layers were observed. We also observed full-thickness skin structure 7 days after grafting by H&E staining and AZAN staining. CONCLUSION: We were able to prepare cultured dermis from human fibroblasts only without adding extracellular matrix using the net-mold method, and generate full-thickness skin by combining cultured dermis with cultured epidermis. The morphology of the full-thickness skin was maintained for 7 days in subcutaneous transplantation. In the next step, we plan to evaluate the efficacy of full-thickness skin for skin defects. In the future, we hope to produce autologous full-thickness skin in vitro and use it as a graft to cover the full-thickness defects after burn injury or trauma.