- What are the differences between serum-free, protein-free, and chemically-defined media?
- Why should I use serum-free media?
- Why should I consider mammalian-free media?
- What does Irvine Scientific have to offer in their serum-free, animal-component-free, or chemically-defined lines of media?
- Can you customize my serum-free media?
- When adapting cells from serum-containing to serum-free media, is the direct or sequential adaptation method better?
- Should I be concerned with mycoplasma in my cell culture?
- How can I test for mycoplasma?
- How do we prevent mycoplasma contamination in cell culture?
- Why would I heat-inactivate my serum?
- What effects could freezing and thawing have on my sera?
- What are the minimum requirements for a custom media formulation?
- What is the timeline on a custom formulation?
- Why was L-Glutamine eliminated from many of your formulas?
Serum-free media do not contain or require the addition of serum. Animal component-free are media in which none of the components are animal derived. Protein-free media do not contain any proteins. Chemically-defined media contain components which are all known (or defined chemically) and they are usually, but not always, protein-free.
Why should I use serum-free media?
Serum-free media offers the customer better lot-to-lot consistency since it contains fewer undefined components, such as serum. The end user also need not take time to qualify lots of FBS for activity. Serum-free media are lower in protein content than medium supplemented with serum, which can simplify the purification process and increase the yield of the end product.
Why should I consider mammalian-free media?
Mammalian-free media has more defined components compared to “serum-free” media, thereby improving lot-to-lot consistency. It also eliminates the time that a researcher needs to test new lots of FBS, which can take two to four weeks. An additional advantage to using mammalian-free media, such as IS CHO-V, IS 293-V, or IS MAB-V, is the reduced threat of contamination from adventitious agents present in many animal-derived proteins. This regulatory advantage, coupled with high protein expression, makes these products an excellent alternative to using media with serum.
What does Irvine Scientific have to offer in their serum-free, animal-component-free, or chemically-defined lines of media?
Irvine Scientific offers many serum-free media, including products for three main types of cells currently used by Biotech or Pharmaceutical companies: 293 cells, CHO cells, and Hybridoma cells. Within each cell line, we offer a variety of media:
293 cells: we offer IS 293™ serum-free medium and IS 293-V™ animal-component-free. Both media are regulatory friendly and formulated for optimal cell growth and viral production.
CHO cells: For Chinese Hamster Ovary cells we offer four products to meet your cell culture needs: IS CHO™, IS CHO-V™, IS CHO-V-GS™ and IS CHO-CD. IS CHO™ is a serum-free medium, IS CHO-V™ is known as mammalian-component-free and is specifically formulated for use with the dhfr-selection system. IS CHO V-GS™ is a mammalian-component-free medium which has been optimized for the glutamine synthetase selection system. IS CHO-CD is a chemically-defined medium free of L-glutamine, Hypoxanthine and Thymidine to allow for use in a variety of applications.
Hybridoma cells: We offer four formulations to meet your needs.
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.
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:
- 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.
- Split cells 1:2 using the desired final serum-free medium as the diluent.
- Incubate cells until the maximum cell density is achieved.
- 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.
- Continue incubation until maximum cell density is achieved.
- 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.
- 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.
- 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.
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.
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.
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.
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.
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.
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.
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.