High Five™ Cells in Express Five™ Medium

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.

To get High Five cells that have been adapted for suspension culture to adhere to a flask for selection of stable clones, it is recommended to check for heparin in the media (heparin helps keep the cells in suspension) and add 0.1% FBS to the culture for 10-20 minutes, then change the media. Keep in mind that cells which have adapted to suspension culture often take time to revert to adherent culture. Maintain the cells in the same flask over a few passages under selection, each time passaging at 1:4 or 1:5, without spinning the cells down. This should select for the adherent cells.

We recommend a density of 3.0x10E6 cells/vial for adherent cultures and 10-15x10E6 cells/vial for suspension cultures.

L-Glutamine is an essential nutrient in cell cultures for energy production as well as protein and nucleic acid synthesis. In general, insect cell media contain higher L-Glutamine levels than serum-free media for mammalian cells, primary cells, stem cells, etc. This is to support the fast metabolism of insect cells, especially when used for virus production and protein expression applications.

Note that removal of L-Glutamine from the media formulation aids in product shelf-life by preventing glutamine degradation during storage.