Additional Information
Storage Conditions: 2 to 8°C, in the dark.
Application
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
Background
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.
Protocol
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:
- 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.
- Subculture the above cell suspension by adding fresh 293-PFM to obtain a cell density of approximately 4x105cells/mL with at least 85% viability.
- 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.
- Pass the cells in PFM and after 3-4 days post planting, when the cell density is 4-8 x105 cells/mL.
- 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.
Cryopreservation
There are two options for freezing cells in serum-free medium. Method A is the
preferred method of NeoBiolab™.
- Harvest cells in the mid logarithmic phase of growth.
- 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.
- 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:
- Remove vial from Liquid Nitrogen and immediately transfer to 37°C water bath.
- While holding the tip of the vial, gently agitate the vial, being careful not to allow water to into the vial.
- When completely thawed, transfer cells to 15mL tube.
- Slowly add 10mL warm 293-PFM and spin at 1000g for 5min
- Decant media and resuspend pellet in 293-PFM.
- Culture cells in flask on an orbital shaker platform rotating at 120-135 rpm.
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