Ice-Binding Protein Derived from Glaciozyma Can Improve the Viability of Cryopreserved Mammalian Cells.
Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1°C/min in a -80°C freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.
Department of Chemistry, Pukyong National University, Busan 48513, Republic of Korea.
Hak Jun
HJ
Kim
Next-Generation Pharmaceutical Research Center, Korea Institute of Toxicology, Daejeon 34114, Republic of Korea.
Hye Eun
HE
Shim
Division of Polar Biology, Korea Polar Research Institute, Incheon 21990, Republic of Korea.
Jun Hyuck
JH
Lee
Department of Chemistry, Pukyong National University, Busan 48513, Republic of Korea.
Yong-Cheol
YC
Kang
Southeast Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Namhae 52440, Republic of Korea.
Young Baek
YB
Hur
D000818:Animals; D001487:Basidiomycota / Q000737:chemistry*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D003412:Cricetulus; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents / Q000302:isolation & purification / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D051379:Mice
D003451:Cryoprotective Agents; D005656:Fungal Proteins
Glaciozyma sp.; antifreeze protein; cryopreservation; ice recrystallization inhibition; ice-binding protein; mammalian cells