Remove the cryoprotective agents from the cells as quickly and gently as possible to avoid the damage that prolonged exposure to these agents can cause. How you remove the cryoprotective agents depends on the type of agent and the type of cells — cells that are sensitive to cryoprotective agents, for example, require gentle centrifugation to remove the agent. When glycerol is the cryoprotectant, the sudden addition of a large volume of fresh medium to the thawed cell suspension can damage or destroy the cells. To avoid this, take the cells through several stepwise dilutions with an equal volume of warm medium every 10 minutes before further processing to give the cells time to adjust.
In general, most cells recover normally if the cryoprotective agent is removed through a medium change within six to eight hours of thawing.
When successfully preserved, frozen cells need little maintenance and can be a lifeline if you lose cell cultures to contamination or accident. Frozen cell cultures are especially useful for long-term experiments, as their suspended animation ensures that biological variants are kept to a minimum.