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Protein-Free CHO Medium, Liquid
With 4500 mg/L D-glucose
With Pluronic® F68
With 4 mM L-glutamine
With sodium bicarbonate
For the suspension culture of CHO cell
Catalog Number PF 486A
Storage Temperature 2~8°C
Protein-Free CHO Media were developed to support the cell growth of Chinese Hamster Ovary cells in suspension cultures. Protein-Free CHO Medium differs from normal (serum supplemented) media in its amino acid, vitamin, and mineral composition, and includes peptides, fatty acids, and growth factors as substitutes to serum. When transplanting CHO cells that had been formerly cultured in a serum supplemented medium, direct adaptation or sequential adaptation should be administered for the appropriate amount of time. When trypsin is used in sub-cultivation of cells in an adherent culture, trypsin inhibitor is supplemented in place for serum for neutralizing effects. A centrifuge process followed by cell cleansing eliminates trypsin from the formulation. The absence of trypsin allows culture media to be transferred into a new container and continue to sustain cell growth with added Protein-Free Medium. If needed, the medium is to be centrifuged at 800 rpm for 5 minutes prior to transfer.
PF 486A contains 4500 mg/L D-glucose, 4 mM L-glutamine, sodium bicarbonate, and HEPES. Pluronic® F-68 is supplemented to prevent damage to CHO cells in a suspension culture. Hypoxanthine and thymidine should be supplemented separately when required for cell growth. PF 486A vary in their compositions and consequently yield different effects on different cell lines. The optimal product for a certain cell type can only be confirmed through direct application. PF 486A is produced free of materials of animal origin, thus making them appropriate for the research and production of medical quality recombinant proteins. In selecting the appropriate culture environment of Protein-Free Medium, (1) the origin of the cell host (the kind of cell), (2) the type of culture container to be used (tissue flask, multiplayer flasks, roller bottle, spinner flasks, or bioreactors), and (3) the characteristics of the CHO cell line is important factors to be considered.
The Protein-Free medium should be stored at 2~8°C in the dark. Deterioration of the liquid medium may be recognized by (1) precipitate or particulate matter throughout the solution, (2) cloudy appearance, (3) color change, and/or (4) pH change. The nature of supplements added may affect storage conditions and shelf life of the medium. Product label bears expiration date.
Instructions for Use
(1) PF 486A is generally supplemented with 2~8 mM of L-glutamine (LS 002-01), (2) nutritional requirements can be altered according to the gene expression system, (3) the concentration of D-glucose (LS 001-01 or LS 001-02) can be adjusted up to 6.8 g/L, (4) polyamine supplement (LS 018-01), fatty acid supplement (LS 017-01), sodium butyrate (LS 033-01) is recommended for encouraging the expression of recombinant proteins, (5) when cultivating in a CO2 incubator, adjustment of pH should be made by altering NaHCO3 concentration, (6) when preparing a suspension culture in a spinner flask, allow 1/4 of the tray to be open to ensure aeration.
Biological Performance Characteristics
The growth-promoting capacities of the Protein-Free CHO media are tested in a liquid medium using CHO cell. Growth rates are examined through three subculture. Cells are counted and growth is plotted as a logarithmic function of time in culture, and seeding efficiency, doubling time, and final cell densities are determined. During the testing period cultures are examined microscopically for a typical morphology and evidence of cytotoxicity.
Protein-Free CHO media are only used In Vitro, and are sensitive to light and heat. Adhering to the following suggestions can increase the product shelf life. (1) Limit light exposure, (2) avoid keeping at room temperature (around 37°C), (3) store in a darkroom at 2~8°C, (4) use in small amounts at a time.
Adaptation of CHO cells to Protein-Free media
A sequential adaptation protocol may be necessary if direct adaptation does not work.
- Direct Adaptation (1)
- If CHO cell confluence reaches more than 80% in serum supplemented medium, remove culture medium.
- Cell, remove using Trypsin or Trypsin-EDTA for 2~3 minutes at 37°C and cell neutralizes using serum supplemented medium. And then remove supernatant by centrifugation cells at 800 rpm for 5 minutes.
- Cell suspended using Protein-Free medium (contained L-glutamine). And remove supernatant by centrifugation cells at 800 rpm for 5 minutes.
- Resuspend cells to a viable cell density of 2~5X105 cells/mL in Protein-Free medium. Incubate the cells in spinner flask at 40 rpm.
- Direct Adaptation (2)
- If CHO cell confluence reaches more than 90% serum supplemented medium, convert culture medium into Protein-Free medium.
- Gather and culture suspension cell in T-flask of one while subculture the cells using Protein-Free medium.
- Confirmed growth cell is subculture. Resuspend cells to a viable cell density of 2~5X105 cells/mL in Protein-Free medium. Incubate the cells in spinner flask at 40 rpm.
- Sequential Adaptation
- If CHO cell confluence reaches more than 80% serum supplemented medium, remove cell using Trypsin or Trypsin-EDTA. Incubate cells at viable cell density of 3~4X105 cells/mL in a 75:25 (v/v) mixture of serum supplemented:Protein-free medium.
- Repeat subculture 2~4 times in same conditions and monitor the culture until the density reaches 90 % viable cells/mL compare with existing culture. Then subculture at viable cell density of 3~4X105 cells/mL into a 50:50 (v/v) mixture of serum supplemented:Protein-Free medium.
- Repeat process 2, do subculture into a 25:75 (v/v) mixture of serum supplemented:Protein-Free medium.
- Repeat process 2, do subculture into a Protein-Free medium only.
- If cell growth is confirmed, cell freeze using freezing medium (contained Protein-Free medium) and examines cell activity after thawing.