Thawing Frozen Cells

Thawing Cells

Stocks of cells we aren't currently passaging, transfecting, or working with through other means are stored frozen in the -80C freezer or in liquid nitrogen. These stocks of cells serve as a backup for replacing any cell cultures we have that become infected or unviable. However, these frozen cells must be thawed out in specific manners to ensure that the cells will be alive and usable post-thawing. Simply removing the cells from the freezer and waiting for them to thaw at room temperature will thaw the cells too slowly, and they will die. For this reason, the thawing cells protocol must be properly followed.

Ideally, cells would would be frozen in liquid nitrogen if being stored for long periods of time (multiple months or longer). If this is the case, a liquid nitrogen tank containing the cells would ideally be transported near the tissue culture hood and the frozen cells could be immediately transported into the hood from the frozen storage (or transported on ice between the liquid nitrogen and the hood). However, we currently do not have access to liquid nitrogen as a freezing method, so the cells are freezing in the -80C freezer until new means become available. This means that when thawing the cells, the cells must be transported outside of the freezer from the second floor to our lab/tissue culture hood on the fifth floor. For this reason, a VERY cold ice bath must be prepared to transport the cells.

Such an ice bath can be prepared using dry ice (if you have some available), or using normal ice and the frozen gel packs found in the bottom of Freezer B. Even with this ice bath, it is advised that the cells remain outside of the -80C freezer/warmed media for as short of a time as possible to ensure the cells do not thaw too fast. Once on the fifth floor, the cells should be immediately warmed as quickly as possible in a hot water bath and with pre-warmed growth media (see cell culture protocol and video).