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Arabidopsis thaliana suspension cells were preserved in liquid nitrogen for over three years, using embedding of cells in calcium-alginate prior to subculture in sucrose-enriched medium, air-drying, and direct quenching in liquid nitrogen. Survival of cells reached 34%, yielding regrowth at the surface of all cryopreserved beads in less than 7 days. Following pretreatment and dehydration, the water content dropped from 2300% to 34% with respect to dry weight. Differential scanning calorimetry showed that glass transition occurred on cooling, followed by a slight crystallization event on rewarming. The survival of cells was independent of the cooling rate. The tolerance of the acute dehydration step increased progressively with sucrose pretreatment duration, indicating the requirement for adaptative cellular alterations. Ultrastructural studies revealed several changes in cells after sucrose pretreatment prolonged from 1 to 7 days: reversal of the initially plasmolyzed state, microvacuolation, numerous autophagic structures, scarcity of ribosomes, increase in number and size of starch grains. No cell division seemed to occur during this period. After air-drying and after a freeze-thaw cycle, followed by 24 h rehydration, regenerating cells had recovered a high level of ultrastructural organization and contained numerous polysomes suggesting an intense metabolic activity. Trehalose, a cryoprotective disaccharide not considered to be a metabolic substrate, yielded only 70% regrowth after freezing. Biochemical analysis showed that soluble sugars accumulated during the pretreatment, essentially sucrose or trehalose; the monosaccharide content also increased. In the light of these results, the action of sucrose in inducing freezing tolerance is discussed.

Cryopreservation of a Catharanthus cell suspension was performed after encapsulation in alginate beads. Encapsulated cells were precultured in sucrose-enriched medium for several days, dried over silica gel, and directly cooled in liquid nitrogen. After rewarming in air at room temperature, alginate beads were placed on semi-solid culture medium. Following regrowth, beads transferred to liquid medium generated a new cell suspension. Cell survival and regrowth from cryopreserved encapsulated cells depended on preculture duration and residual water content after air-drying.