Methods Freezing

The methods available are those developed for sperm, oocyte, and embryo freezing or vitrification.[1-3] In prin ciple, the material is suspended in a supportive solution of ions and other additives, of the correct osmotic strength and the appropriate pH. One or more cryoprotective agents are added and the suspension is packaged into plastic straws or tubes designed to withstand cooling to —200°C and rewarming. The samples are frozen carefully by cooling in a controlled-rate freezer designed for the purpose, or in the vapour phase over the surface of liquid nitrogen. The material is stored under the surface of liquid nitrogen (—196°C) or more recently, responding to concerns about cross contamination, in the vapour phase. For recovery of the material, thawing is generally rapid (to avoid damaging recrystallization of ice) often in a water bath to increase the rate of heat transfer. Stored material has an indefinite time-span estimated at hundreds of years because metabolic processes are arrested, leaving only cosmic radiation to cause DNA damage.

However, the frozen sample is subjected to severe stresses during cooling and rewarming that result in considerable cell damage. Only about 50% of spermatozoa will survive an optimized cryopreservation protocol, and success with embryos depends on species. Oocytes have been cryopreserved with only modest success. The stresses arise from one or more of the following: 1) osmotically driven water movements across the cell membranes caused by the addition and removal of cryoprotectant; 2) the removal of water as ice during freezing and its dissolution during rewarming; and 3) cytotoxicity from high concentrations of cryoprotect-ants used.

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