Fig. 1.

The structure of the circulating vitrification bag and operation procedure. a: (Left) The structure of the circulating vitrification bag. An inflow tube and an outflow tube (polytetrafluoroethylene tube, inner diameter: 1.2 mm) are attached to the polyethylene bag (110 × 85 mm; film thickness: 0.063–0.064 mm). The inflow tube has 15 holes (hole diameter: 0.6 mm) to allow quick inflow of the solutions (indicated by arrows). The outflow tube has 30 holes (hole diameter: 0.6 mm), allowing for smooth solution discharge (indicated by arrows). (Right) A photograph of the circulating vitrification bag. b: A circulating vitrification bag attached on an orbital shaker that was operated with the following conditions; rotation number: 15 rpm, rocking angle: 20°, turning angle: 360°, under room temperature (24–27 °C). c: Operation procedure of the circulating vitrification bag. (1) Insert the cell sheet sandwiched between two nylon meshes into the circulating vitrification bag and close the opening. Then, attach the bag to the shaker. (2) While shaking the circulating vitrification bag, inject the equilibration solution-1 via the inflow tube (arrows). (3) Keep the cell sheet in solution for a given amount of time (Table 1). (4) Discharge the solution in the circulating vitrification bag from the outflow tube (arrows). Repeat steps (2) to (4) to successively inject and discharge the pre- and post-vitrification solutions shown in Table 1.