Characterization of long-term cryopreservation for hCTLA4Ig production in transgenic rice suspension cell cultures

초록

Plant-made pharmaceuticals (PMPs) are therapeutic proteins derived from transgenic plant cells. Recently, transgenic rice cell suspension cultures (Oryza sativa L.) 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, due to error control, this method has several problems such as genetic instability of transformed cell lines, high cost, and contamination possibility. For these reasons, a cryopreservation method has developed for the continuous supply and long-term storage of the transgenic plant cells that could maintain the genetic stability. Cryopreservation is a process where the cells are preserved in liquid nitrogen (-196℃) with cryoprotectant. Cryopreservation of cell lines is a crucial activity performed as a part of biopharmaceutical industry. Various strategies have been approved in the other systems such as animal cells and microbes following the current good manufacturing practices (cGMPs). However, plant-specific procedures have not yet been developed. In plant cells, the 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 crystallization but cells can be damaged by toxicity of DMSO. In this study, optimal method was designed by using various cryoprotectant mixtures and thawing steps. Optimal concentration of cryoprotectant was found to be 1 M sucrose, 0.5 M glycerol and 0.5 M DMSO, respectively. Furthermore, removal method of toxic cryoprotectant using negative pressure was investigated during thawing steps. When the thawed cells were treated with mild pressure for 2 min, viability was maintained up to 84.7% after cryo-banking for 30 days.