Objective: Human oral mucosal epithelial cell(hOMEC)sheets have been successfully used to treat epithelial defects of the cornea and esophagus.In conventional research,cell source of the epithelial cell sheets have been collected by using enzyme.The cells are then seeded on cell culture inserts,as a primary culture.However,the size of resectable tissue is limited,restricting the resultant cell numbers.Therefore,it is important to establish a subculture method for generating sufficient numbers of cells for hOMEC sheet fabrication.The aim of this study was to fabricate and examine a hOMEC sheet cultured on a temperature-responsive surface via an explant culture method.Method: Human excess oral mucosa was harvested by tonsillectomy and then minced as finely as possible.The tissue was placed in a culture dish coated with type Ⅰ collagen and subjected to primary explant culture in keratinocyte growth medium(KCM).Subsequently,the cultured cells were trypsinized and seeded onto a temperature-responsive cell-culture insert at a density of 8–60×10^4 cells/cm^2.In addition,we examined whether hOMEC sheets could be generated from frozen cells.The sheets were analyzed by histology and flow cytometry.Results: We observed approximately 80%increase in cell proliferation between days 12 and 16 of primary cell culture.The efficacy of our approach was comparable with conventional methods,yielding five times as many or more cell sheets than these methods.The higher the number of harvested cells used for seeding,the shorter the time required for cell sheet generation.It was also possible to generate cell sheets using cells that had been frozen for 2 months.Conclusion: We have demonstrated that tissue-engineered epithelial cell sheet grafts can be fabricated using temperature-responsive culture via the explant culture method.We believe that the new method would be useful to create cell sheets for clinical application.