Development of long-term cryopreservation method for the hCTLA4Ig production

초록

Recently, transgenic rice (Oryza sativa L.) cell suspension cultures have been utilized to produce recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig). General long-term preservation method for plant cells has been known to be the repeated subcultures. However, this method has several problems such as genetic instability of transformed cell lines. For that reason, a cryopreservation method was developed for the long-term storage of the transgenic plant. In plant cells, cryopreservation is difficult due to high water content up to 90%. To overcome this problem, dimethyl sulfoxide (DMSO) has been widely used as a cryoprotectant to reduce the water. In this study, optimal method was designed by using various cryoprotectant mixtures and thawing steps. The optimal concentration of cryoprotectant was found to be 1 M sucrose, 0.5 M glycerol and 0.5 M DMSO, respectively. Furthermore, removal of toxic cryoprotectant using negative pressure was investigated in thawing steps. When thawed cells were treated with mild pressure for 2 min, the cell viability was maintained up to 84.7% after cryo-banking for 30 days. Using optimized procedures, cells were cryopreserved for 5 years, thawed and regenerated to calli than suspended into liquid medium. The growth and production characteristics including growth rate, productivity of 5-year-cryopreserved cells were proved to be similar with those of control cells. In conclusion, long-term cryopreservation was possible in transgenic rice cells and these results could be applied to the transgenic plant cell cultures.