Next, the cell suspension inside the vial must be thawed. A frequently used rule of thumb is that upon beginning the cell thawing process in a standard cryovial with 1 mL cell suspension, all ice needs to have disappeared within a few minutes. Rapid heating of the cell suspension prevents localized recrystallization during cell thawing, which can cause cellular damage . Sometimes thawing cells within a few minutes is not possible, e.g. due to time spent to find the exact vial or due to distance between the location of the liquid nitrogen storage and the thawing location. In this case, it is preferable to leave the cryovials at the lowest possible temperature followed by rapid thawing instead of both slow thawing and leaving the cells in thawed freezing medium for longer than necessary .
When all ice has disappeared, any further adverse effects of the cryoprotective agent on the cells need to be minimized by two possible ways. Cells can be either cultured directly after thawing, for example in a T-flask, while ensuring the freezing medium is diluted by at least a factor of ten with normal cell culture medium. Alternatively, it is possible to dilute the freezing medium with normal cell culture medium, centrifuge the tube, discard the medium, and resuspend the thawed cells in fresh culture medium [2, 3, 4]. To check that the cell thawing protocol was successful, it is recommended to determine the percentage of viable cells (e.g. with a trypan blue staining and a cell counter) .
To improve cell thawing standardization, it is good practice to monitor the cell culture over several days for any abnormalities in shape or growth rate (see image at the top for an example). Poor or inconsistent cell growth can be a sign of cell stock problems or problems within the cryopreservation process. Therefore, early detection can help to minimize experimental errors.
How to thaw cells – Advantages and disadvantages of different methods
Different methods and equipment exist to thaw cells in cryovials – water bath, hand-warming, bead bath, and dedicated devices.
Read more about the advantages and disadvantages of different cell thawing methods, as well as lab- and cell-type specific considerations in our White Paper “Cell Thawing Protocol Standardization - Guide for More Reproducible Cryopreservation Results” -