Can you customize my serum-free media?
All the formulations we offer may be tailored to meet your specific requirements. Whether your efforts are focused on optimizing yields or improving productivity, we can work with you to address your needs.
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When adapting cells from serum-containing to serum-free media, is the direct or sequential adaptation method better?
The sequential adaptation method is recommended. When cells are transferred to serum-free medium from a serum-rich environment, the cells are deprived of components in the serum-rich environment. Sequential adaptation allows the cells to gradually adapt to their new serum-free environment.

Suggested Sequential Adaptation Protocol:

1. Start with cultures (suspension or adherent) that are at maximum cell density and high viability (>80%.) If cells are attachment-dependent, trypsinization procedures should be used.
2. Split cells 1:2 using the desired final serum-free medium as the diluent.
3. Incubate cells until the maximum cell density is achieved.
4. Split cells again. The ratio may vary depending on whether the cells are growing attached or in suspension culture. For attached cells try 1:2 or 1:3 split based on confluency. For suspensions, try 1:3 to 1:5 or approximately 3-4 X 10⁵ cells per mL.
5. Continue incubation until maximum cell density is achieved.
6. Monitor the growth and viability of the cells relative to cells cultured in the original or control medium. Cells which have been successfully adapted to serum-free media should have a viability greater than 85% with a doubling time equivalent to the control medium (serum-containing or other serum-free medium) from which the culture was adapted.
7. Low viability or poor doubling times indicate that the culture may not yet be adapted. Three to five successive splits using 1:2-1:3 may be necessary. It is not uncommon for some cells to continue to require 0.1-0.5% serum.
8. Time for adaptation to serum-free conditions will vary with the growth characteristics of the particular cell type. If at anytime during the weaning process the culture viability drops below 80% or the time to double increases significantly, weaning may have occurred too quickly. The cells may require several passages at the previous dilution before renewal of the sequential weaning process resumes.

Should I be concerned with mycoplasma in my cell culture?
Mycoplasma contamination should always be a concern. Mycoplasma contamination can change the morphology of the cells and interfere with bioassays, cell growth, viability, and production.
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How can I test for mycoplasma?
Irvine Scientific offers MYCOTRIM® TC. The MYCOTRIM® TC system allows routine screening of cell cultures, as well as media and reagents which can potentially contaminate cell culture. Use of this test kit is efficient, while minimizing labor and expense. MYCOTRIM® TC requires only 1 mL of supernatant to inoculate the flask, which is then incubated for a maximum of 10 days.

The triphasic system allows reinoculation within the same closed flask; this provides a high degree of sensitivity while protecting the environment and the sample from contamination. It is also provided with a color changing indicator as well as Dienes Stain to allow definitive identification of mycoplasma.
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How do we prevent mycoplasma contamination in cell culture?
Good aseptic techniques should be used in all laboratory applications. The best method for prevention is to test all materials that come in contact with the cells. This should be done on a regular basis to ensure that mycoplasma is not introduced into the cell culture and to identify any potential sources of contamination.
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Why would I heat-inactivate my serum?
Heat inactivation is a process used to inactivate protein complement components which are present in the serum. The proteins from the complement cascade have the potential to impact or interfere with certain cellular processes.
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What effects could freezing and thawing have on my sera?
We generally discourage multiple freezing and thawing of sera. Sera contain proteins, lipids, and other components which may be differentially separated or precipitated out of solution depending upon the rate of thawing or freezing. This may adversely affect the performance of the sera. The best storage is in a freezer at -20° C or lower.
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What are the minimum requirements for a custom media formulation?
We generally require a 100L minimum order to manufacture a custom formulation. Our Pilot Operations can make smaller quantities to meet special investigational needs for our customers.
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What is the timeline on a custom formulation?
Customs are usually delivered to the customer between 4 to 6 weeks after we receive the final signed documents with a purchase order.
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Why was L-Glutamine eliminated from many of your formulas?
L-Glutamine is easily hydrolyzed in liquid. By-products of hydrolysis can be toxic to the cell. Additionally, the necessary concentration of L-Glutamine may be compromised after a period of time. Since L-Glutamine is an essential amino acid, it is best for the end-users to supplement media with the recommended concentration of fresh L-Glutamine.
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