Cryopreservation of transgenic rice suspension cells producing recombinant hCTLA4Ig.
Transgenic suspension cells of Oryza sativa L. cv. Dongjin utilized as a host for producing recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) were preserved in liquid nitrogen (-196 degrees C) after slow prefreezing in a deep freezer (-70 degrees C). The development of an optimal procedure for long-term storage was investigated by the addition of various concentrations of cryoprotectant mixture and osmoticum in preculture media before cooling. A pre-deep-freezing time of 120 min was the most effective for maintaining cell viability. Compared with mannitol, sorbitol, trehalose, and NaCl under the same osmotic conditions, 0.5 M sucrose was found to be the best osmoticum for preculture media. The cryoprotectant comprising sucrose, glycerol, and dimethylsulfoxide (DMSO) was applied to the precultured cells, and a combination of 1 M sucrose, 1 M glycerol, and 1 M DMSO provided the best result. The viability with this optimized condition was 88% after cryocell-banking for 1 day. The expression of hCTLA4Ig in recovered callus from cryopreservation was also kept stable, and the production level was similar to that observed in noncryopreserved cultures.
Cho
Ji-Suk
JS
Department of Biological Engineering, Inha University, Incheon, 402-751, Republic of Korea.
Hong
Seok-Mi
SM
Joo
Sung-Yeon
SY
Yoo
Ji-Seon
JS
Kim
Dong-Il
DI
D000069594:Abatacept; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D006801:Humans; D018796:Immunoconjugates / Q000235:genetics / Q000378:metabolism*; D008353:Mannitol / Q000494:pharmacology; D012275:Oryza / Q000166:cytology* / Q000235:genetics; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012965:Sodium Chloride / Q000494:pharmacology; D013012:Sorbitol / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D014199:Trehalose / Q000494:pharmacology
D018796:Immunoconjugates; D011994:Recombinant Proteins; D008353:Mannitol; D012965:Sodium Chloride; D013012:Sorbitol; D013395:Sucrose; D000069594:Abatacept; D014199:Trehalose