Historically hybridoma cells have been grown in base media plus fetal bovine serum (FBS), but there are problems associated with FBS supplementation, including FBS lot-to-lot variability and inconsistency. FBS and animal products in general are plagued by inconsistent product lots, mainly due to the fact that the source material (cows themselves) can be so different based on where they live, what they eat, etc. Some lots of FBS may contain little contamination, others significantly more and some lots may be higher in various nutrient or vitamin components. All of these inconsistencies have a major impact on cells, which makes manufacturing difficult because yield isn’t consistent. Customers can get around this by screening multiple lots and buying in large quantities, but these tests are time consuming and purchasing large lots ties up resources that could be spent elsewhere.
In addition, FBS contains contaminating antibodies (IgG, IgM, etc.) that reduce the overall purity of the produced monoclonal antibody. In some cases, low IgG serum can be used to address the issue, but it is quite costly and still isn’t completely IgG free. Another resource drain associated with FBS is the sourcing and documentation surrounding purchasing FBS and ensuring that it is free of mad cow disease. These issues have driven many researchers to switch to serum-free culture for monoclonal antibodies production in hybridoma cells.
Ok, serum-free is best, but how do we get there?
When using hybridoma cells to produce antibodies there is little time for media optimization or process development for each antibody produced. So when considering a transfer to serum-free culture, the solution must be “one size fits all”, easy to implement and provide clear benefits. It also needs to be cost-effective.
The most obvious solution would be to use a commercial serum-free hybridoma media. This method provides an all in one solution and eliminates the work of optimizing media internally. However, commercial media is more expensive and can result in a reduction in antibody yield. It can also be challenging to transition cell lines to this media-requiring long adaptation periods. Furthermore, a single commercial medium may not be able to support the growth of diverse hybridoma lines as well as a serum-supplemented medium. So many have chosen to formulate their own serum-free media by either starting with a commercial serum-free media or an optimized base media such as DMEM/F12 and then adding key supplements.
To ease the transition from serum and still maintain good antibody yield, serum-free media should be supplemented with products that help to replace some of the critical ingredients in serum. Innovative animal-free products such as recombinant albumin and recombinant transferrin offer the ability to replace some of the key factors in serum involved in cell growth and viability. Since they are recombinant, they are also completely IgG free. Companies including Sigma, Fisher Scientific, InVitria, Sheffield Bioscience and Mediatech sell recombinant albumin and recombinant transferrin that can be added to serum-free media to provide an alternative to using serum in hybridoma culture. InVitria offers a complete IgG-free solution supplement that is specifically designed for this purpose called Zap-Hybridoma.
Other products that can be very helpful in transitioning hybridoma cells to serum-free culture are insulin, transferrin, selenium and ethanolamine combination products, often called ITS or ITSE based on the ingredients (please see table below for product options). In our previous blog “Combining Insulin and Transferrin in Cell Culture Supplements,” we discuss the use of these supplements to remove or reduce serum from culture.
Glucose is also a necessary ingredient in successful serum-free hybridoma culture to provide energy for cells. Glucose should be added to initial culture media and since it will also be consumed throughout the run, adding an additional glucose feed during the run should be considered. Another energy boosting product is Glutamine. Glutamine should be added at the beginning of the hybridoma cell culture run.
Additional amino acid supplementation including may also improve cell densities, consider experimenting with the addition of a combination of these or utilize a nutrient/vitamin combination product formulated for hybridoma, such as Thermo Fisher Hyclone’s Cell Boost 3.
The advantage of animal-free supplements is that they eliminate the problems associated with serum, like inconsistency, IgG contamination and sourcing challenges, without the reduction in antibody yield that is common with serum-free media. They also reduce the transition time from serum containing to animal free culture conditions.
Does anyone have a serum-free hybridoma culture protocol to post here for the community? Hybridoma Animal-free Media Supplement List: This table includes animal-free and animal derived products for comparison
|Type||Product||Cost Per Liter of Media||Animal-free, Animal Derived|
|Recombinant Albumin||InVitria Cellastim||$1-6||Animal-free|
|Sigma recombinant albumin||Animal-free|
|Fisher Scientific recombinant albumin||Animal-free|
|Sheffiled Bioscience rAlbumin ACF||Animal-free|
|Mediatech cellgro rhAlbumin||Animal-free|
|Recombinant Transferrin||InVitria Optiferrin||$0.25-$1||Animal-free|
|Sigma recombinant transferrin||Animal-free|
|Fisher Scientific recombinant transferrin||Animal-free|
|Recombinant Hybridoma Supplements||InVitria Zap-Hybridoma||$12-15||Animal-free|
|Insulin-Transferrin Supplments||InVitria ITSE Animal-free||$10-12||Animal Derived|
|Corning/Mediatech ITS||$15-20||Animal Derived|
|Life Tech ITS-A||$20-25||Animal Derived|
|Life Tech ITS-G||$20-25||Animal Derived|
|Life Tech ITS-X||$20-$25||Animal Derived|
|Gemini BIO ITS||$20-25||Animal Derived|
|Sigma ITS||$12-15||Animal Derived|
|ThermoFisher Hyclone ITS||$12-15||Animal Derived|
|BD ITS +||$12-15||Animal Derived|
|Sciencell ITS||$15-20||Animal Derived|
|Serum Replacement||Millipore Hybricyte||Pricing not available||Animal Derived|
|MP Biomedicals TCH||$150-200||Animal-free|
|Nutrient/Vitamin Products||ThermoFisher Cell Boost 3||$5-7||Animal-free|