Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Serum is a key component for growing and maintaining cells in culture. Containing a mixture of proteins, hormones, minerals and other growth factors, serum is a nutrient boost for cultured cells. It is added to media as a growth supplement, and specialized forms can be used for different experimental conditions.
Determining the serum conditions that provide optimal cell growth can be a challenging task. “Selection of sera products is dependent upon the growth requirements of the cell type it’s being used for, with sensitive cell lines often requiring the highest quality serum,” says Brian Douglass, global head of cell-culture product management and marketing in life sciences at GE Healthcare. Many suppliers offer serum, in various formulations and with different purity levels. Fetal bovine serum (FBS), or fetal calf serum (FCS), is the most widely used form, but there are features and options to consider. Here we discuss some key considerations when selecting sera for culturing cells.
Endotoxin and other nasty contaminants
Sera are complex, containing a diverse array of factors of both known and unknown origin. “Purity and performance are paramount in sera and media,” says Jhony Orbulescu, global fine chemicals technical sales director at MP Biomedicals. “Sera must be sterile and free of any possible pathogens.” Contaminants not only affect cell health but also impact experimental results. “Many factors influence cell growth, but the main driver is the level of endotoxin in the serum,” says Douglass. Endotoxin is derived from lipopolysaccharides that make up the outer membrane of most gram-negative bacteria. “Endotoxin is naturally present in all sera products but can be reduced with efficient collection practices and effective filtration and processing of blood,” Douglass explains.
At Valley Biomedical, a small company supplying sera and other cell-culture products, the No. 1 product is human serum from type AB blood. “AB blood-type serum lacks antibodies for both A and B blood-type antigens,” says Valley Biomedical’s director of sales and marketing, Brian Gnegy. “It is often used in tissue engineering, transplantation and cell-therapy applications.” To render this serum sample suitable for these applications, he says, it is “0.1-µm sterile filtered into 100-ml bottles and tested for sterility, mycoplasma and endotoxin.”
Sleuthing for sera
Different types of cells require different types of serum, so the first step is to make sure the cells are growing and responding consistently in the media. Most suppliers can help researchers find the best serum for their application. Gnegy says Valley Biomedical “offers free samples of most of our cell-culture products and allows the customer to test the lot to see if it will work for their project.” After a type and a specific lot of serum have been identified (often determined by how well the cells propagate), suppliers can assist researchers with securing enough material to complete their experiments. “Customers will frequently test and reserve the lots of serum that support the most robust cell growth while minimally impacting assay performance,” Douglass says. “End users of sera are typically risk-averse and … prefer to use what has historically worked rather than potentially jeopardize results by using sera from another vendor.” Orbulescu adds, “We offer the opportunity to our customers to try different sera, and if they are happy with a specific formulation or lot, we can reserve that lot for a specific customer.”
Because of its complex nature, serum is predisposed to lot-to-lot variability. “How the serum is collected and processed as well as the donor animal’s diet and country of origin will impact the quality of the serum and therefore affect cell performance,” says Douglass. MP Biomedicals minimizes lot-to-lot variation with “strict controls on the suppliers of the raw materials,” says Orbulescu.
Proper documentation
Reputable suppliers provide documentation detailing the serum’s lot number (which refers to its particular processing batch), processing details, storage information and a certification list of test results. At GE Healthcare, Douglass says that “each lot is tested for a range of biochemical parameters, some of which are required by regulatory authorities for compliance, such as 9 CFR testing for virus,” but other parameters may be important for individual experiments.
After separating serum from other blood components, a batch or lot of serum undergoes controlled testing to confirm that it’s suitable for research use. For example, the serum is filtered—usually three times through 0.1-µm filters—to remove bacteria and fungi. To check contaminant levels, manufacturers may culture a sample of the filtered serum. Documented test results should show the absence of growth. Test results also should confirm the absence of toxins that are too small to be filtered out, such as endotoxin. (In some cases, the low-level presence of toxins or other substances may be acceptable to researchers.) Serum also is tested for viruses; these cannot be removed by filtration, so it’s important that the serum is documented as virus-free.
According to the International Serum Industry Association (ISIA) website, the rounds of processing and testing that serum lots undergo can take several months, so it’s important for suppliers to store serum properly, frozen, when it’s not in use. ISIA offers a Certificate of Traceability for serum where suppliers can document an unbroken chain from the serum’s origin to the end user). The organization also offers a Certificate of Origin, which certifies the location where the blood was collected from the animals. Suppliers that comply with ISIA’s quality guidelines can display an ISIA Quality Seal on their products. (Please note that not all suppliers have applied for certification, and lack of certification does not imply lesser quality sera.)
Sera stratification
Certain cell types and test conditions require specialized sera. For example, GE Healthcare’s HyClone Super Low IgG FBS is designed for experiments requiring low levels of immunoglobulins, and Characterized FBS and Defined FBS are suitable for working with sensitive cells, says Douglass. Other companies also sell sera that are stratified to address different growth requirements and prices. For example, Gibco sera from Thermo Fisher Scientific are offered in five varieties, which vary in endotoxin concentration. More robust cell lines that can tolerate low levels of endotoxin without affecting experimental results could translate into cost savings.
Douglass explains that “engineered serum is a viable alternative to the costly serum that is often in limited supply and obtained at high prices to end users.” GE Healthcare’s engineered serum products include Cosmic Calf and FetalClone products, which work well for researchers looking for lower-priced supplements that work as well as natural serum.
Important though serum is for many cell-culture labs, there is growing interest in serum-free culturing. “The lot-to-lot variability of serum, combined with the potential of introducing adventitious virus contaminants, means that we are now also seeing serum-free defined media growing in popularity,” says Douglass. “Even existing serum customers are interested in migrating to low-serum or serum-free media formulations to gain even more control over tightly monitored processes.” But serum-free conditions may not provide all the factors cells need to be happy, which means serum will continue to be a vital ingredient in cell-culture media for some time to come.
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