Catherine Shaffer holds a master's degree in biological chemistry and has worked as a research scientist. She is also an award-winning science fiction author and part-time reporter for local public radio.
Mammalian cell culture is a vital tool and often the initial phase in the research workflow for a cell biologist. Whether scientists perform the culturing themselves or rely on a shared resource lab, the fundamentals of cell culturing have remained largely the same for decades. Cells are grown in a synthetic medium that provides the cells with their nutritional requirements. At a minimum, those requirements are components like amino acids, vitamins, salts and an energy source like glucose. However, to support growth, cells need an animal serum or its equivalent. Serum has more than 1,000 different components, including proteins, lipids, carbohydrates, growth factors, enzymes and other constituents that are still undefined. Serum carries out functions like regulating cell permeability and acting as a carrier for nutrients and other molecules entering the cell. Although effective serum-free media are available, media containing animal serum are used for a majority of cell-culture applications in the life sciences. Here we discuss key factors to consider when selecting a serum for your research, to ensure you achieve accurate and consistent results.
Components of serum
Animal serum is a standard ingredient in cell-culture media, but serum itself is anything but standard or consistent.
Serum varies considerably from lot to lot, and a “homogeneous” population of cells can be extremely sensitive to those variations. Differences between lots of serum used from one experiment to the next can influence research results. Components such as hormones, uric acid, urea, bilirubin, creatine and cholesterol are all highly variable, with consequences for cells such as changes in the rate of growth or unwanted differentiation.
Selection starts with samples
When choosing a serum for a cell-culture project, the most important part of the process is testing multiple lots and selecting a single lot to be used throughout the project, including by any collaborators at other institutions whose results may be pooled or compared.
Alison Killilea heads the Cell Culture Facility at the University of California, Berkeley. She says that each year the facility requests samples from four different suppliers. “We test these serums on the growth of three to four different cell lines, making sure we choose a primary cell line, a transformed cell line and 293T cells, a line commonly used for transfection experiments.” Different lots of fetal bovine serum (FBS) are tested by growing three different cell lines for three weeks to determine which serum gives the most consistent number of cells when all are plated at the same density.
In addition to identifying the serum that the specific cells being tested are most responsive to, it’s important to purchase enough serum from the same lot for the entire project. For large research groups, that can be a challenge. Last year, Killilea’s lab experienced an issue of not being able to purchase a large enough lot of the serum that tested as the top performer for its work. Ultimately the lab had to go with a different product to continue its research.
Some manufacturers offer lot-matching services, so that new lots with similar performance and properties can be purchased to replace previous batches.
Quality is highly variable
Many factors in the manufacturing process contribute to the quality of a serum product. David Fiorentini, vice president for scientific affairs for Israel-based Biological Industries, says his company carries out extensive quality-control testing and differentiates its sera by country of origin. “All FBS filtered in the primary facility in Israel is certified by the European Directorate for the Quality of Medicines (EDQM). All sera are processed in a cGMP compliant facility,” says Fiorentini.
Biological Industries monitors the quality of serum in a number of ways. The company purchases raw materials from approved suppliers only and adheres to written specifications for those materials. The sera are processed in clean rooms. Each production lot is fully traceable and QC tested for physical, chemical and biological performance as well as sterility and adventitious-agent testing.
At MTI GlobalStem, an important part of the QC process is testing batches of serum on a wide variety of different cell types that are used by researchers. “For our stem cell customers, that’s really the whole point of having a serum that can feed the cells and keep them growing in culture without differentiating,” says Donna Trollinger, director of marketing for MTI GlobalStem.
Researchers should also pay attention to a serum’s country of origin, and make sure the serum is verified to be free of mycoplasma and viruses. Country of origin is important because each country has its own regulatory agencies that approve other countries of origin for sera. For example, some countries of origin are approved for the European Union, but are not approved by the U.S. Department of Agriculture.
Processing
Serum can be processed through heat inactivation, gamma irradiation, dialysis and other methods to optimize its properties for specific applications. Heating a serum stock is a common procedure used to inactivate complement proteins that would interfere in assays. However, heat processing may also inactivate other, undefined components of the system, so it’s a good idea to avoid heat-inactivated serum unless it is required for your specific application.
“Heat inactivation started back in the 1970s, before modern filtration systems made it obsolete. But some people still use those old protocols and unknowingly affect cell growth rates, because heat inactivation can destroy growth factors,” says Killilea.
Gamma irradiation is another common treatment used on serum. The main purpose of gamma irradiation is to inactivate viruses. Some FBS is dialyzed to remove low molecular weight components, or processed by chromatography to remove IgG.
Economics
The cost of serum is fairly volatile, because it’s essentially a by-product of the beef industry. As with the price of beef, environmental influences such as drought affect the market price of FBS. “Since 2011, the price has gone up 400 to 500%,” says Jason Walsh, director of strategic growth for Corning Life Sciences.
Some customers may find a less-expensive product meets their needs adequately. “In some cases, newborn-calf serum or calf serum is possible instead of FBS. Much cheaper!” says Fiorentini.
Putting it all together
Researchers have a challenging task when choosing serum for a cell-culture application. There are numerous factors to take into consideration plus labor-intensive testing of samples from various manufacturers and lots. In addition, they must consider special processing needs for their application, the country of origin and the cost. Chris Scanlon, global marketing development manager for research serum at Thermo Fisher Scientific says that researchers have a tendency to be misguided when choosing serum for the research, “They put too much emphasis on origin as a quality marker when there are many other elements that better define serum quality (i.e. growth, cloning, plating). He also recommended taking advantage of their FBS Specialist team that work directly with their customers to find the right serum to meet their research and budget needs.”
MilliporeSigma also urges customers to work with the company for their serum needs. “What MilliporeSigma offers is a sampling and reservation program,” says Barbara Mullenslader, global product manager for MilliporeSigma. “We provide customers with a sample of a particular batch to test. We will set aside that batch during testing. That’s how we eliminate the researcher’s risk of getting a batch that doesn’t perform well.”
In the end, selecting an appropriate serum to use in your cell-culture experiments requires hands-on testing of various samples to see how the cells respond. It also requires some scanning of the literature to understand optimal conditions for growing the cell types (particularly if a cell line is new to you), and to determine whether the cells require a particular serum type or variation. Lastly, planning for anticipated usage can be critical, especially if large-scale or long-term experiments are to be run.