Sf9 Cells in Grace's

The following protocol describes a general procedure for thawing cryopreserved cells. For detailed protocols, always refer to the cell-specific product insert.

1. Remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37°C water bath.
2. Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial.
3. Transfer the vial into a laminar flow hood. Before opening, wipe the outside of the vial with 70% ethanol.
4. Transfer the desired amount of pre-warmed complete growth medium appropriate for your cell line dropwise into the centrifuge tube containing the thawed cells.
5. Centrifuge the cell suspension at approximately 200 x g for 5-10 minutes. The actual centrifugation speed and duration varies depending on the cell type.
6. After the centrifugation, check the clarity of supernatant and visibility of a complete pellet. Aseptically decant the supernatant without disturbing the cell pellet.
7. Gently resuspend the cells in complete growth medium, and transfer them into the appropriate culture vessel and into the recommended culture environment.

Note: The appropriate flask size depends on the number of cells frozen in the cryovial, and the culture environment varies based on the cell and media type.

Insect cells do not require CO2 or high humidity to grow, they can grow in a lab drawer at room temperature. We recommend this so people don't waste CO2 and other resources necessary for maintaining a tissue culture incubator. It should be noted, however, that the cells will grow in a humidified incubator.

Yes, you can grow Sf9 cells in glass vessels. The only concern would be if your glass vessels are not clean enough and there may be residual detergent left which will hurt your cells.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

These cells appear after cultures have been grown for several weeks. These do not seem to be detrimental to plating of high titer stocks or expression. However, if they are in high numbers, it may indicate that the cells are becoming old and that the culture should be re-started with a new stock of frozen cells.

Yellow particles could be cell organelles, aggregates, or debris. We see this when we first thaw frozen cells. To avoid this, you can let the shaker culture sit for 5 minutes, then transfer top 1/3 to a new flask, making sure to count cells first. You can also use heparin at up to 200 U/mL to decrease aggregation. Pluronic solution at a final concentration of 0.2% can also be used to decrease shearing, and increase shake speed to 100-120 rpm. Recently thawed cells seem to be breaking up and releasing small vesicles, as observed under high magnification. To reduce the amount of those small particles, cells need to be rapidly but completely defrosted for successful thawing to take place. Also:

1. Place vial on ice during transfer from water bath to sterile hood.
2. Pipette as gently as possible because cells shear easily due to larger surface area.
3. Cells may not have been placed in cold media after removal from defrosted vial into flask.
4. Media may not have been changed after 30-45 minutes once a majority of cells had attached. Media change should be with pre-warmed media (27degrees C). 10% DMSO in freezing medium will kill the cells if left on them for long periods of time (1 hour seems to be a maximum).
5. Lastly, cells should be checked for contamination. To do so, plate a small portion of culture in a T-25 flask and incubate for 3 days, checking for cloudiness.