Gibco™ Express Five™ SFM

Cells can be adapted by Sequential or Direct Adaptation. Suggested protocols for each are below, and you can also find more information by searching "Adaptation of Cell Cultures to a Serum-Free Medium" from our website home page.

SEQUENTIAL ADAPTATION
1) Subculture the cells growing in serum-supplemented medium into a 25%:75% mixture of SFM and serum supplemented medium.
2) When the cell density is 5 x 10E5 cells/ml, subculture the cells into a 50%:50% mixture of SFM and serum supplemented medium at a cell density 2.5 x 10E5 to 3 x 10E5 cells/ml.
3) Continue to subculture after the cell density 5 x 10E5 cells/ml in gradually increasing proportions of SFM until the serum is ~0.1% with about 85% cell viability.
4) Subculture the cells into SFM with an innoculum of 2.5 x 10E5 to 3 x 10E5 cells/ml.
5) When the cell density is 1 x 10E6 to 3 x 10E6 cells/ml (4 to 6 days post planting) subculture the cells again.
6) Stock cultures of SFM adapted cells should be subcultured in SFM every 3 to 5 days when the cell density is 1 x 10E6 to 3 x 10E6 cells/ml with 90% viability.

DIRECT ADAPTATION
Some cells can be directly adapted from serum-containing medium to SFM. For direct adaptation, the cell innoculum should be 1.5 x 10E5 to 3 x 10E5 cells/ml.
Cells should be subcultured when the cell density is 1 x 10E6 to 3 x 10E6 cells/ml. Cells are fully adapted to SFM when the cell density is 2 x 10E6 to 4 x 10E6 cells/ml after 4 to 7 days in culture.
Stock cultures of cells adapted to SFM should be subcultured in SFM every 3 to 5 days when the cell density is 1 x 10E6 to 3 x 10E6 cells/ml with 90% viability.

The glutamine concentration is correct. Insect media have historically been 2 to 10 fold higher in glutamine concentration vs. classical mammalian media. Express Five SFM was optimized for High Five cells which have especially high requirements for glutamine and asparagine. The liquid basal media is actually 1.09x in concentration and was designed to be diluted to the correct 1x composition with 90 ml/L of the 200 ml L-glutamine. Because the L-glutamine concentration is so high, we left it out of the basal formulation to avoid problems associated with glutamine degradation and the resulting ammonia build-up which can be toxic.

Some cells, such as insect cells, are sensitive to changes in their medium. By sequentially adapting cells, the medium is changed with minimal effects on cell growth.

Many antibiotics are suitable for use with insect cells. The table on page 11 of the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Insect_Cell_Lines_UG.pdf) summarizes some of the most commonly used antibiotics, their working concentrations, and their methods of action. Please see page 38 for ordering information.

We recommend that you use L-Glutamine instead of GlutaMAX, as insect cells lack the ability to sufficiently cleave the dipeptide bond in the GlutaMax, making it a poor source of glutamine for these cells.