Vitrification and rewarming methods for triple-layered rabbit chondrocyte sheets. (A-E) Coating method. A cell sheet treated with ES and VS is placed on a stainless mesh using forceps (A) and exposed to liquid nitrogen (LN) vapor for vitrification (B). (C) A cell sheet vitrified on mesh. Note the transparency of the vitrified sheet. For rewarming, the vitrified sheet on a mesh device is placed on a heating plate for thawing (D) and then transferred to RS using forceps (E). (F-J) Wrapping film method. An ES- and VS-treated cell sheet was wrapped in an envelope of polyvinylidene chloride film (F) and exposed to LN vapor for vitrification (G). (H) A cell sheet vitrified in wrapping film. Note that the cell sheet is transparent when it solidifies. For rewarming, the sheet vitrified in the wrapping film is placed on a heating plate (I), followed by transfer into RS (J). (K-O) Dish method. A cell sheet pretreated with ES and VS is placed in 2 ml VS (K) and exposed to LN vapor for vitrification (L). (M) A cell sheet solidified with VS in a dish. Note the transparency of the vitrified solution and the lack of any cracks. (N) A dish containing a cell sheet on a heating plate for rewarming. The opacity of the solution indicates that ice crystals have formed during the warming process. The thawed cell sheet is then transferred into RS using forceps (O).