All procedures should be carried out using sterile techniques in a biosafety cabinet.
HEK TF is formulated without L-glutamine. For applications requiring this amino acid, supplement with 6-8 mM L-glutamine prior to use. Supplementation of L-glutamine directly to the culture is recommended.
2 Culture conditions
Cultures should be maintained at 37 °C. For cultivation in an incubator, a 5% CO2 atmosphere is necessary.
3 Instructions for use
3.1 Thawing of cells
1) Quickly thaw a vial of frozen cells in a 37 °C water bath.
2) Transfer the cells aseptically to a centrifugation tube containing 10 mL of HEK TF.
3) Centrifuge cell suspension at 115×g for 5 minutes.
3.3 Stepwise adaptation from serum-containing cultures
1) Expand the culture in serum-containing standard medium.
2) Centrifuge a sufficient number of cells for inoculation of suspension culture with 4–6·105 cells/mL at 115×g for 5 minutes.
3) Resuspend cells in Xell medium (if necessary include 6-8 mM L-glutamine) and 2% fetal bovine serum (FBS).
3.4 Bioreactor cultivation
For best performance the inoculation density in bioreactor should be in the range of 4–6·105 cells/mL in Xell medium. Suggested starting parameters for bioreactor cultivations of HEK cells using Xell medium are pH 7.0–7.2, 40% DO, and a temperature of 37 °C.
3.5 Freezing of cells
Cells can be frozen in HEK TF without the use of serum.
1) Choose a well-growing culture with viabilities above 90%.
2) Prepare a freezing medium consisting of 90% HEK TF Medium and 10 % dimethyl sulfoxide (DMSO; cell culture grade).
3) Cool down the freezing medium to 2–8 °C.
3.6 Transfection of cells
HEK TF has been developed to especially support transfection applications and allows high transient gene expression.
The setup for transfection can vary depending on the application and cell line used. It is advisable to use established protocols or test different protocols to reach optimum performance.
1) One day before transfection, seed cells with an appropriate inoculum to reach 3∙106 cells/mL on the day of transfection.
2) Before transfection, spin down cells and resuspend in fresh HEK TF. The cell culture should have high viability.