2.Freezing Survival of the Nematode Caenorhabditis elegans in the Presence of a Cryoprotectant

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For the establishment of successful cryopreservation of Caenorhabditis elegans (C. elegans), freezing survival of the nematode particularly in the larva L4 and adult stages, was investigated in the presence of a cryoprotectant. The highest rate of freezing survival for larva L4 was obtained in the presence of 5% (v/v) dimethyl sulfoxide (Me_2SO) dissolved in M9 buffer. The survival rate increased with the dipping time in the Me_2SO solution until 13min before freezing, which corresponded with the decrease in the nematode volume. Although the volume first decreased, it recovered from shrinkage with further dipping time, indicating that Me_2SO can permeate into the nematode, which was confirmed by the use of gas chromatograph. Glycerol, on the other hand, can hardly permeate into the nematode; the volume recovery was minimal. Therefore, the remarkable increase in the rate of freezing survival when Me_2SO was added, compared with glycerol, might be caused by its permeation into the nematode body. Cooling rate, lowest temperature of cooling and initiation temperature of freezing also affected freezing survival. Slow cooling accompanied by ice seeding at a higher subzero temperature was effective in avoiding freezing of the internal nematode. Freezing avoidance inside the nematode due to osmotic dehydration in combination with the concentration of Me_2SO in the body was found to be necessary for the cryopreservation of C. elegans in both the L4 and adult stages. Taking these results into consideration, two-step freezing was conducted in which the initial cooling down to -100℃ at the rate of 0.2℃ min^<-1> with ice seeding at -3℃ was followed by plunging into liquid N_2. A survival rate above 85% was then obtained for the L4 nematode after slow warming.
2009-10-31

2.Freezing Survival of the Nematode Caenorhabditis elegans in the Presence of a Cryoprotectant

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