293 Protein-free Medium-Protein-Free Media

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293 Protein-free Medium


Catalog #
Additional Information

Storage Conditions: 2 to 8°C, in the dark.


293-PFM is a protein-free medium developed for the long-term growth of Human Embryonic Kidney 293 (HEK 293) and related cells. The cells, in a suspension culture, can be subcultured into 293-PFM from serum-supplemented media with adaptation.

Quality Control

293-PFM is performance tested in a growth and maintenance assay using 293 cells in a dynamic cell culture system. Additional standard evaluations are pH, sterility, osmolality and endotoxin.

Physical Conditions

Standard physical conditions for 293 cells grown in 293-PFM are 37

293-PFM is a complete, protein-free cell culture medium optimized for growth and recombinant protein or adenovirus production of 293 cells (including 293-F, 293-H) in suspension culture. It is a chemically-defined, and contains no components of animal origin. It is supplied as a complete powdered medium in a variety of sizes.

It is critical that cell viability be at least 85% and cells be in the mid logarithmic phase of growth prior to adaptation. The procedure is as follows:

  1. Subculture the cell suspension grown in conventional serum supplemented media into a 50:50 ratio (v/v) of protein-free media (PFM) and serum supplemented media at approximately 1x105 cells/mL. Incubate culture at 37°C in a humidified atmosphere of 5% CO2. Allow cell density to reach in excess of 4-8x105 cells/mL.
  2. Subculture the above cell suspension by adding fresh 293-PFM to obtain a cell density of approximately 4x105cells/mL with at least 85% viability.
  3. Continue to subculture the cell suspension in 293-PFM (at an inoculum of 4x105cells/mL) until the serum concentration is decreased to 0.1% with at least 85% viability, each time allowing the cell density at 4 - 8x105 cells/mL.
  4. Pass the cells in PFM and after 3-4 days post planting, when the cell density is 4-8 x105 cells/mL.
  5. After several passages, the cell yield should be 1-3x106 cells/mL after 3-4 days in culture. At this point, the cells are considered to be adapted to PFM.
Cultures may be grown in spinner flasks with impeller speed set at 75-95 rpm or inshake flasks on an orbital shaker platform rotating at 120-135 rpm.


* Note: Adaption of cells grown in different serum-free media or protein-free media (other than NeoBiolab™) may be affected by selection of subpopulations(s) to specific components.



There are two options for freezing cells in serum-free medium. Method A is the

preferred method of NeoBiolab™.

  1. Harvest cells in the mid logarithmic phase of growth.
  2. Freeze cells at 1 x 107 cells/mL in a mixture of 90% 293-PFM(50% of conditioned PFM + 40% of fresh PFM) + 10% DMSO.
  3. Use of a controlled rate freezer is recommended to cryopreserve cells in a controlled and reproducible manner. If controlled rate cryopreservation equipment is not available, 293 cells in the described cryogenicstorage medium may be preserved using the following protocol:
                  a.  1 hour at 4°C
                  b.  2 - 4 hours at -20°C
                  c.  overnight at - 70°C
                  d.  store in liquid nitrogen


Thawing Cells:

  1. Remove vial from Liquid Nitrogen and immediately transfer to 37°C water bath.
  2. While holding the tip of the vial, gently agitate the vial, being careful not to allow water to into the vial.
  3. When completely thawed, transfer cells to 15mL tube.
  4. Slowly add 10mL warm 293-PFM and spin at 1000g for 5min
  5. Decant media and resuspend pellet in 293-PFM.
  6. Culture cells in flask on an orbital shaker platform rotating at 120-135 rpm.

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