To achieve slow cooling, ampoules must be heavily insulated. A block of polystyrene containing individual holes sufficiently large to take ampoules should be prepared. The polystyrene must be 1 - 2 cm deep all around the ampoule, with no air spaces. The insulation required is placed in the block, which is then placed near the middle of the freezer and left overnight (16 – 24 h) before being transferred to a liquid nitrogen storage vessel. Before using this method on a regular basis, a series of tests should be made to monitor the cell viability after freezing. If it varies significantly from the viability of the cells prior to freezing, i.e. a drop of more than 15 – 20%, the insulation will have to be modified. This method is not recommended for preparing master or reference stocks for long term storage. Two-stage freezing Ampoules are kept at a holding temperature of – 20° to – 40°C for up to 24 – 48 h and then transferred directly to – 196°C. Similar results can be achieved for a small number of ampoules by using a small device which holds them in liquid nitrogen vapour in the neck of a Dewar flask. After a holding time of 10-20 min the ampoules are plunged into liquid nitrogen and then transferred to their final storage location. Preliminary experimental work is required to determine the optimum conditions. In all cases, the cells should be examined for viability following cryopreservation. For certain specialized cell types, it may be considered necessary to include particular essential growth factors to the freeze medium in order to maintain surface receptor stability during cryopreservation. For example, hybridomas often cause problems on revival. It is essential to use freshly prepared freezing mixture every time. DMSO should be obtained from a supplier who is able to offer the most recently prepared stocks available. Once received, it should be filter sterilized (0, 2 μm) through a filter specifically designed for DMSO and stored in a glass container with an air-tight stopper or lid at – 20°C. This will ensure that no problems
Applied Veterinary Virology: The isolation and identification of viruses using cell cultures 30 | P a g e develop due to oxidation of the DMSO. In those cases where DMSO causes differentiation, glycerol should be used instead. Thawing • Achieve rapid thawing by transferring ampoules directly to a water bath containing water at 37°C. If an ampoule contains potentially hazardous material, it is advisable to add 1 - 2% (w/v) chloramine-T to the water in the water bath. • Take care not to submerge the cap of the plastic ampoule in order to prevent contaminated water from entering the ampoule. A simple method is to use a plastic rack designed to hold tubes of the same diameter as that of the ampoules, and to place it in the correct depth of water. Alternatively, a piece of foam polystyrene foam containing holes into which the ampoules are fitted; this will float on the surface of the water.
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- Fall '12
- Test, representative, Elastase, Cell culture, Earle, Applied Veterinary Virology