Freezing and recovering Sf9 Cells

Sf9 cells can be frozen in a manner quite similar to methods used to preserve mammalian cells. This involves putting them in a suitable medium, including 7.5-10% DMSO, and a controlled freezing rate.

Freezing Procedure:

The procedure that I use is to grow cells in a suspension culture into log phase growth with doubling times on the order of 24 hours or better and up to a density of 2-2.5E6 cells/mL. Of course all of the following operations must be done with particular care for sterility. Count the cells and determine the volume required to resuspend the cells to 2E7 to 1E8 cells/mL, I usually stay in the 3-5E7 zone. Prepare fresh at least 1/2 this resuspension volume of fresh medium containing 15% DMSO, sterile filter this DMSO/Medium through a 0.2 um membrane filter that is resistant to 15% DMSO; nylon membranes work well for this.

Gently pellet the cells, for example in a table top centrifuge at 1000-2000 rpm for 5-10 minutes. Remove the conditioned medium, but save a sufficient amount to gently resuspend the cell pellet at 1/2 the final target volume. For example, grow 500 mL of cells in a 1 L or larger spinner or shake flask to a density of 2.5E6 (this assumes the conditions you're using will reach a density of 4E6 or greater, if not, back off on the density to 50 or 60% of the max final density). This would be about 1.2E9 cells. To suspend those cells at 4E7 cells/mL would require about 30 mL. So save 15 mL of conditioned medium and resuspend the cell pellet in this medium.

Once the cells are well suspended, add the fresh DMSO/Medium and mix thoroughly. Immediately dispense into 1 mL aliquots in prelabeled 1.8 mL cryovials and place in an appropriate freezing container (e.g. "Mr. Frosty," Nalgene#5100-0001, VWR# 55710-200). Work quickly and place the freezing container in a <-70C freezer as soon as practicable. Leave the cells at -70C for at least overnight, and then it is best to transfer to liquid nitrogen storage, though they will do OK at -70C for long periods.

Thawing Procedure

To thaw the cells, prepare a flask with sufficient medium in it to suspend the cells at 6E5-1E6, in our example above with 4E7 cells/vial, that would be 50 mL at 8E5 cells/mL. Thaw the cells quickly at room temperature or slightly above. Avoid water baths as they are a frequent source of contamination. Use a clean dry block at 32C, or frequently they are simply warmed in sterile-gloved hands sprayed with with 70% Isopropanol.

As soon as the cells are thawed, add them to the flask containing the fresh medium. Usually the cell viability will be close to 90% immediately upon thaw, however, over the first 24 hours it is not unusual to lose half the cells. In the next 24 hours you should see substantial growth. so by 48 to 72 hours you should be back to the seeded density, and doubling should resume fairly regularly after that.

A good Sf9 line I have is very reliable in this manner, and will resume a 22-24 hour doubling time by 72 hours post-thaw. A less vigorous Sf21 line I have will take significantly longer, with 30 hour doubling times at first, and requiring 4 to 6 passages before the doubling time gets back down to less than 24 hours.

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