Sf9 cells in Sf-900™ III SFM

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.

It should be okay to thaw the cells into Sf-900 II SFM. This is a richer media compared to the Sf-900 III SFM so the cells would have an easy time adapting. We would recommend taking the cells through 3 passages in the new medium before using them for any experiments as that they have enough time to adapt.

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

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.