Beneficial effect of intracellular free high-mannose oligosaccharides on cryopreservation of mammalian cells and proteins

The cryoprotective effect of intracellular free high-mannose oligosaccharides (HMOS) on mammalian cells and proteins was examined by monitoring PC-12 cell viability and assaying protein kinase C (PKC)-ε activity. 1-Deoxymannojirimycin, an inhibitor of α-mannosidase, to cause an increase in intracellular free HMOS, significantly rescued PC-12 cells with 2-h freezing insult at −15 °C in a concentration (1–50 mM)- and pretreatment time (48–72 h)-dependent manner, as compared with unpretreated cells; full rescue from freezing injury was obtained with 1-deoxymannojirimycin at more than 25 mM for 48-h pretreatment and more than 3 mM for 72- and 96-h pretreatment. For PC-12 cells pretreated with 1-deoxymannojirimycin at 1 mM for 72 h, thawed cell viability after more than 8-w cryopreservation at −80 °C in 10% (v/v) dimethyl sulfoxide was much higher than that for cells without pretreatment. PKC-ε activity was well preserved after 16-h cryopreservation at −20 °C in the presence of mannose 9-N-acetylglucosamine 2 (Man9-GlcNAc2) (1 mM), an HMOS, while the activity was reduced to 15% without Man9-GlcNAc2. Collectively, the results of the present study suggest that intracellular free HMOS is a key molecule to protect mammalian cells and proteins from freezing injury; in other words, HMOS could be a new target for cryopreservation of mammalian cells and proteins.