HEK293 System FAQ
Q. What is the glucose concentration in the HEK 293 medium and feed?
Q. How should adherent HEK 293 cells (banked in a FCS containing medium) be adapted into suspension in BalanCD HEK 293 medium?
A. We recommend starting with adapting adherent HEK cells to suspension culture in BalanCD HEK 293 medium with serum (the same concentration the cells have been adapted to). Follow the standard protocol we suggest in our product insert (125 mL shake flask). Using the suggested protocol, customers can add additional 0.1% of poloxamer to protect cells in suspension against possible damage during stirring in shake flask. After the cells are adapted to suspension culture, take the cells in HEK 293 medium with X% FBS and gradually taper off to 0% FBS. The adaptation to 0% FBS should be quick but growth in suspension may be difficult and take a while. We highly recommend banking the cells after each adaption.
Q. Can BalanCD HEK 293 medium be used for HEK 293 transfected cells to make stable suspension cells?
A. Yes. BalanCD HEK 293 can be used for HEK 293 transfected cells to make stable suspension cells. The customer may need to add Anti-Clumping Supplement to prevent aggregation.
Q. What is the preferred transfection method to be used with BalanCD HEK 293?
A. Our recommended method is PEI-Mediated Transfection, please refer to the product insert for the general guideline. BalanCD HEK 293 medium is also compatible with other transfection methods using cationic liposomes or electroporation. Optimization of transfection parameters is highly encouraged, since optimal transfection parameters may vary depending on the application.
Q. What is recommended feeding guide for BalanCD HEK 293?
A. BalanCD HEK293 Feed can be evaluated with the suggested standard feed method shown in the product insert. For transient transfection, we advise to test 12-20% of the initial culture volume in total, at a maximum of 5% daily for 4 feeding events. For stable transfection, we advise to test 10-25% of the initial culture volume in total, at a maximum for 5 feeding events. Nutrient depletion analysis in the media can be used to optimize the feeding schedule if necessary. If feeds need to be used at more than 20-25% of the initial culture volume, monitor osmolality to ensure that is it not above 400 mOsm/kg.
Q. Does BalanCD HEK293 Feed inhibit the cell doubling time but still maintain the VCD? Is the addition of valporic acid or sodium butyrate needed to inhibit cell growth?
A. BalanCD HEK 293 Feed is used to improve cell growth and protein production and will not inhibit cell growth. Valporic acid and sodium butyrate can be added to enhance production but will arrest cell growth.
Q. How can cell aggregation be removed in BalanCD HEK 293?
A. If severe cell aggregation is observed, supplement with Anti-Clumping Supplement. Anti-Clumping Supplement is designed for use at a 1:200-1:1000 dilution (1-5 mL/L) range. Typically used at 2 mL/L. Supplement additional amount if necessary.
Q. What is the Anti-Clumping Supplement? My cells are starting to clump at high cell concentrations and I consider using this supplement, but I am not sure if it will interfere with virus infection and production.
A. Anti-Clumping Supplement may be added to culture if cells start to aggregate. If Anti-Clumping Supplement is added, it must be eliminated from the culture media prior to transfection. If cells aggregate post transfection, Anti-Clumping Supplement can be added at least one day post transfection. Up to 10 mL/L of Anti-Clumping Supplement can be added if there are larger aggregates.
Q. What is the best way to measure pH once the powder has been hydrated in WFI? Can phenol red be added to measure the pH?
A. We use a pH meter to measure the pH of our media. Phenol red can be added to the medium except when an assay involving fluorescence is performed since phenol red will contribute to the background.
Q. What is the stability data of the BalanCD HEK 293 medium at room temperature (20°C) and 37°C?
A. We currently do not have stability of this product at 20°C or 37°C in powder format. We recommend 2-8°C storage.
Q. Does adding GlutaMAX-I improve the media quality as opposed to L-Glutamine at the same 4 mM concentration? Can GlutaMAX-I and L-Glutamine be used interchangeably?
A. We typically recommend the addition of L-Glutamine. GlutaMAX is the dipeptide form of Glutamine which allows for longer stability in culture. The L-Glutamine form is more readily available to the cells early in culture when it is most critical. However, customers use GlutaMAX interchangeably without any negative effects.
Q. What is the concentration of Pluronic or equivalent in BalanCD HEK 293 medium?
A. BalanCD HEK 293 contains 0.1% poloxamer; however, an additional 0.05% to 0.1% can be supplemented if necessary.
Q. Does BalanCD HEK 293 medium contain phenol red?
A. No, BalanCD HEK 293 does not contain phenol red.
Q. What is FUJIFILM Irvine Scientific’s policy for disclosing formulation information?
A. Different levels of details can be shared depending on the request, and FUJIFILM Irvine Scientific will work with our customers to address their formulation inquiries on a case by case basis. Please contact us with your formulation requests.