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Tobacco BY-2 cells were successfully cryopreserved by a simple slow prefreezing (equilibrium freezing) method using an encapsulation technique. After the cells were immobilized in alginate gel beads, the beads were treated with cryoprotectant solution (2 M glycerol, 0.4 M sucrose) for 45 min. The beads were then transferred to a laboratory freezer at −30°C, stored for 2 h, and then immersed in liquid nitrogen. To initiate the regrowth of cells, the beads were warmed in a water bath. Following dilution of cryoprotectant solution, the beads were suspended and cultured in normal medium. With this method, suspension cell cultures were regrown within 7 days. There were no differences in the morphology or growth profiles between cryopreserved cell cultures and the original cell cultures.

Simple cryopreservation protocol with an encapsulation technique for tobacco BY-2 suspension cell cultures