Enhanced Cell Viability for the Efficient Cryopreservation of Transgenic Rice Cells

초록

Transgenic rice cell suspension cultures (Oryza sativa L.) have been utilized to produce recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). General preservation method for plant cells has been known to be the repeated subcultures. One drawback of this method is the genetic instability of transformed cell lines. For that reason, a cryopreservation method was developed. Dimethyl sulfoxide (DMSO) is known to be an effective cryoprotectant, but the cells are damaged during thawing procedures. To solve this problem, an optimal method was designed by using various cryoprotectant mixtures and the cryoprotectants were removed by negative pressure. Optimal concentration of cryoprotectant was found to be 1 M sucrose, 0.5 M glycerol and 0.5 M DMSO, respectively. To remove toxic cryoprotectant in thawing steps, the time required for negative pressure treatment was found to be 2 min and the resulting cell viability was 84.7%. After 3 weeks, the cells could be recovered to callus efficiently and cell mass was increased. In conclusion, we could enhance the viability of the transgenic rice cells after the cryopreservation by optimized cryoprotectants-related steps.