Optimization of Osmotic Agents in the Pre-culture Medium for the Cryopreservation of Transgenic Rice Suspension Cells

초록

Recently, the production of recombinant proteins (plant-made pharmaceuticals) has been studied using transgenic plant cell cultures. The transgenic rice cell lines (Oryza saitva 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. Cryopreservation method was developed for the long-term storage of the transgenic plant cells that could maintain the genetic stability. However. additional step is essential in plant cells damaged by ice crystal formation because of high water content up to 90%. For the purpose of solving this problem, various osmotic agents (sucrose, mannitol, and sorbitol) were applied to reduce the water content of plant cells. Molar concentration of each agent was adjusted at 0.2 M, 0.4 M, and 0.6 M, respectively. F/D ratio was decreased and cell viability was rapidly decreased at 0.6 M due to high osmotic stress. When 0.4 M of osmotic agents were added, F/D ratio was 6.06, 7.92, and 7.06 respectively at day 2, which was 0.50-fold, 0.34-fold, and 0.42-fold decrease compared to that of control. Relative cell viability was the best in the case of 0.4 M sucrose treatment. Therefore, we can develope the optimal pretreatment method for transgenic plant cell cryopreservation.