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)-epsilon activity. 1-Deoxymannojirimycin, an inhibitor of alpha-mannosidase, to cause an increase in intracellular free HMOS, significantly rescued PC-12 cells with 2-h freezing insult at -15 degrees C in a concentration (1-50mM)- and pretreatment time (48-72h)-dependent manner, as compared with unpretreated cells; full rescue from freezing injury was obtained with 1-deoxymannojirimycin at more than 25mM for 48-h pretreatment and more than 3mM for 72- and 96-h pretreatment. For PC-12 cells pretreated with 1-deoxymannojirimycin at 1mM for 72h, thawed cell viability after more than 8-w cryopreservation at -80 degrees C in 10% (v/v) dimethyl sulfoxide was much higher than that for cells without pretreatment. PKC-epsilon activity was well preserved after 16-h cryopreservation at -20 degrees 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.
Watanabe
Kanako
K
Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan.
Yaguchi
Takahiro
T
Yang
Dongqin
D
Kanno
Takeshi
T
Nagai
Kaoru
K
Yamamoto
Satoshi
S
Fujikawa
Hirokazu
H
Yamamoto
Hideyuki
H
Nagata
Tetsu
T
Tashiro
Chikara
C
Nishizaki
Tomoyuki
T
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D008351:Mannans / Q000378:metabolism* / Q000494:pharmacology; D008358:Mannose / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D016716:PC12 Cells; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D051381:Rats; D043323:alpha-Mannosidase / Q000037:antagonists & inhibitors
D003451:Cryoprotective Agents; D004791:Enzyme Inhibitors; D008351:Mannans; D009844:Oligosaccharides; C475461:mannosyl(9)-N-acetylglucosamine2; D017485:1-Deoxynojirimycin; D051744:Protein Kinase C-epsilon; D043323:alpha-Mannosidase; D008358:Mannose