Every so often we are asked if storing insect cells at -80°C is a viable option if you haven’t got access to liquid nitrogen cryogenic facilities. Our standard response is that this is not a good idea for long term storage of viable cells. This is largely based on historical dogma that says that cells do not preserve their viability for long at these temperatures. However, since one of our protocols for freezing insect cells involves an overnight cooling step in propanol in the -80°C we thought we would test the longevity of Sf21 cells left at this temperature for a little while longer.
Two vials of cells were left in the -80°C for two months and then thawed prior to viability check and dispensing to a cell culture flask. We found that their viability was reduced to between 5-10% (normally, the viability of cryogenically stored cells [in liquid nitrogen] is 80-90% after thawing). Despite the low viability of the cells in the vials, they were able to establish a culture as a monolayer, although after seven days they are still very thin.
What this rather limited tests shows is that storing insect cells at -80°C for any period longer than a month is probably not a good idea. However, it does show that if you have purchased some cells from a vendor such as OET, you don’t have to revive them immediately after they arrive. You can probably safely leave them in a -80°C freezer for at least a week and their viability shouldn’t decrease by too much. Although for this test we used Sf21 cells, we expect the same results would be obtained by storing any other cell type at this temperature.
Although many stocks of insect cells are provided at a concentration suitable for setting up as shake/suspension cultures directly from cryogenic storage, it is also good practice to set aside some of these cells as a monolayer culture. The Sf21 cells we tested for viability here were intended for revival as a spinner/suspension culture. Had we put the cells into this format it is unlikely they would have grown owing to their low viability. By seeding them into a monolayer culture so that they attach, low density cells always seem to recover better than in suspension.