Optimization of Osmotic Agents and Cryoprotectants for the Cryopreservation of Transgenic Rice Cells

초록

Recently, production of recombinant proteins has been studied using transgenic plant cell cultures. The transgenic rice cell lines (Oryza sativa L.) were utilized to produce recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in this study. General long-term preservation method for plant cells is known to be the repeated subcultures. One drawback of this traditional method is genetic instability of transformed cell lines. For that reason, a cryopreservation method was developed for the long-term storage of the transgenic plant cells that could maintain the genetic stability. Plant cells are damaged by ice formation during freezing and thawing procedures because of high water content up to 90%. To solve this problem, an optimal method has been designed by adding osmotic agents in pre-culture medium as well as various cryoprotectant mixtures before cooling step. In pre-culture, sucrose, mannitol and sorbitol were used as osmotic agents. Molar concentration of each agent was adjusted at 0.2, 0.4, and 0.6 M, respectively. When 0.4 M of osmotic agents were added, F/D ratio (fresh weight/dry weight ratio) was 6.01, 5.97, and 6.32 respectively at day 5, which was 0.34-, 0.34-, and 0.30-fold decrease compared to that of control. Compared with each agent, relative cell viability was the best in the case of 0.4 M sucrose treatment. In addition, optimization of cryoprotectant concentration was performed with range from 0 to 2 M. Cryoprotectants such as sucrose, glycerol and dimethylsulfoxide (DMSO) were applied to the pre-cultured cells. The best combination was found to be 1 M sucrose, 0.5 M glycerol and 0.5 M DMSO. The relative cell viability with the optimized condition was 57.3% after cryopreservation by two-step freezing for 7 day. In conclusion, we could optimize the osmotic agents and the cryoprotectants for the cryopreservation of transgenic rice cells.