INTRODUCTION: Vitiligo, the most common human pigmentary disorder, significantly impacts patients' quality of life through its profound psychological and social consequences. The condition's complex pathogenesis and substantial effect on patients' well-being underscore the critical need for advanced therapeutic approaches. Treatment for vitiligo by autologous cultured epidermal transplantation has been developed, and the efficacy of this treatment has been determined. However, there is concern that differences in postoperative pigmentation of affected areas may occur due to difficulty in adjusting melanocyte content. This study aimed to develop a reproducible method for fabricating cultured epidermal sheets containing melanocytes for use in clinical applications. METHODS: Human epidermal keratinocytes and melanocytes derived from skin tissues of volunteer donors were prepared under different culture conditions and the proliferation of keratinocytes and melanocytes was determined. The density and percentage of melanocytes in cultured epidermal sheets were evaluated by DOPA staining and flow cytometry. Moreover, a novel clinical-grade melanocyte culture medium with specific supplements was developed that avoids bovine pituitary extract containing unknown animal-derived unknown materials. RESULTS: The density of melanocytes was higher when melanocytes were seeded first, as opposed to the simultaneous seeding of melanocytes and keratinocytes. The best condition in the present study for preparing cultured epidermal sheets with high melanocyte density was to seed melanocytes at 1.0 × 10(4) cells/cm(2) on Day 1, followed by feeder cells on Day 2 and keratinocytes at 8.0 × 10(4) cells/cm(2) on Day 3. The optimal culture medium for melanocytes was determined to contain 10 nM aMSH, 10 nM endothelin-1, and 10 μg/mL FGF-2 as culture supplements. CONCLUSIONS: The results of this study establish a foundation for the development of cell therapy effective for vitiligo.