Cryopreservation of cell suspension cultures of Taxus x media and Taxus floridana
Skrlep, K.,National Inst. of Biology, Ljubljana (Slovenia) | Bergant, M.,National Inst. of Biology, Ljubljana (Slovenia) | De Winter, G.M.,Leiden Univ., (The Netherlands). Inst. of Biology | Bohanec, B.,University of Ljubljana (Slovenia). Biotechnical Faculty | Zel, J.,National Inst. of Biology, Ljubljana (Slovenia) | Verpoorte, R.,Leiden Univ., (The Netherlands). Inst. of Biology | Van Iren, F.,Leiden Univ., (The Netherlands). Inst. of Biology | Camloh, M.,National Inst. of Biology, Ljubljana (Slovenia)
Different lines of cell suspension cultures of Taxus x media and Taxus floridana were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately -1 deg C per min to -80 deg C. The highest post-thaw cell viability was 90%. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.
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