Development of a Self-Assembled Dermal Substitute from Human Fibroblasts Using Long-term Three-Dimensional Culture

Skin substitutes have emerged as an alternative to autografts for the treatment of skin defects. Among them, scaffold-based dermal substitutes have been extensively studied; however, they have certain limitations, such as delayed vascularization, limited elasticity, and the inability to achieve permanent engraftment. Self-assembled, cell-based dermal substitutes are a promising alternative that may overcome these shortcomings but have not yet been developed. In this study, we successfully developed a cell-based dermal substitute (cultured dermis) through the long-term culture of human dermal fibroblasts using the net-mold method, which enables three-dimensional cell culture without the use of a scaffold. Spheroids prepared from human dermal fibroblasts were poured into a net-shaped mold and cultured for 2, 4, or 6 months. The dry weight, tensile strength, collagen and glycosaminoglycan levels, and cell proliferation capacity were assessed and compared among the 2-, 4-, and 6-month culture periods. We found that collagen and glycosaminoglycan levels decreased over time, while the dry weight remained unchanged. Tensile strength increased at 4 months, suggesting that remodeling had progressed. In addition, the cell proliferation capacity was maintained, even after a 6-month culture period. Unexpectedly, the internal part of the cultured dermis became fragile, resulting in the division of the cultured dermis into two collagen-rich tissues, each of which had a thickness of 400 mu m and sufficient strength to be sutured during in vivo analysis. The divided 4-month cultured dermis was transplanted to skin defects of immunocompromised mice and its wound healing effects were compared to those of a clinically available collagen-based artificial dermis. The cultured dermis promoted epithelialization and angiogenesis more effectively than the collagen-based artificial dermis. Although further improvements are needed, such as the shortening of the culture period and increasing the size of the cultured dermis, we believe that the cultured dermis presented in this study has the potential to be an innovative material for permanent skin coverage. Impact statementThis is the first report of the successful development of a self-assembled, cell-based dermal substitute derived from human dermal fibroblasts through long-term three-dimensional culture. The scaffold-free cultured dermis exhibited superior properties, such as sufficient tensile strength, high biocompatibility, and efficacy in promoting wound healing when compared to the collagen-based artificial dermis.