Effects of Inoculation Time and Proline on Cell Viability of Transgenic Rice Cells during Cryopreservation

초록

Recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was produced using transgenic rice cell lines (Oryza sativa L.). Serial subculturing of cells is time-consuming and can often result in genetic instability. To maintain genetic stability, long-term preservation of highly valuable cells is important. Inoculation time before pre-culture was optimized for cryopreservation and the effects of various concentrations of cryoprotectant mixture was investigated. In pre-culture step, the cells in exponential phase were found to be more resistant to cryo-damage than the cells in the lag or stationary phase because these cells have small vacuoles. Cryoprotectants consisted of sucrose, glycerol, and dimethylsulfoxide (DMSO) was used. However, toxicity of cryoprotectants is a fundamental limiting factor for successful cryopreservation. To overcome this problem, proline was used for replacing DMSO as a non-toxic alternative cryoprotectant. Cryopreservation with proline led to enhanced post-thaw cell viability and stabilization of cells. In conclusion, appropriate inoculation time and utilization of proline were proved to be important factors for enhancing cell viability during cryopreservation of transgenic rice cells.