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Gamma Irradiation of Fetal Bovine and Other Animal Serum

When animal sera such as fetal bovine serum (FBS) are used to supplement cell culture media, there may be introduced a low but finite risk of infecting the culture with an adventitious agent such as a virus or mycoplasma. Serum lots are pools of serum collected from many different animals, and there is always a chance that one or more of the donors might have been infected. Testing for viruses and mycoplasma may not detect a very low-level contaminant. There are, fortunately, ways of mitigating this risk so that the advantages associated with use of animal serum may be realized while reducing the potential risk. In this article, we discuss the use of gamma irradiation as an effective approach for inactivating potential adventitious contaminants of fetal bovine serum (FBS) and other animal sera.

Gamma irradiation is a physical inactivation approach that is commonly used for disinfection of plastic cultureware and for achieving pathogen reduction in tissue culture reagents such as porcine trypsin and fetal bovine serum. Gamma radiation consists of highly energetic photons of electromagnetic energy, typically derived from the radioactive decay of 60cobalt. Plastic culture dishes and pipettes may be effectively sterilized through use of high gamma radiation doses. For animal-derived reagents such as serum, there is a trade-off between efficacy for pathogen reduction and the possibility of adversely impacting the performance of the reagents. In order to understand this, we need to consider the ways that gamma irradiation kills microbes.

Gamma radiation interacts with microbes through two different mechanisms. One mechanism involves the direct action of the photons with nucleic acids of the microbes, causing strand breakage and other damage.                         

The second (indirect) mechanism is a consequence of the interaction of the photons with water to yield oxygen radicals, which subsequently cause damage to microbial macromolecules and lipids. While each of these mechanisms causes microbial inactivation, the latter effect also has great potential to damage essential components of the reagent itself, rendering it unfit for use. 

Fortunately, the irradiation of fetal bovine and other serum is done in such a way as to favor greatly the direct mechanism, limiting the harm done to the serum by oxygen radicals. By irradiating serum at very cold temperatures in the sealed containers used to package the serum, oxygen radical formation and diffusion can be limited (but not entirely eliminated). It is for this reason that the dose used for treatment of serum is typically limited to 25-40 kGy (1 kGy = 100,000 rad). Higher radiation doses would be more effective for pathogen reduction, but likely at the cost of decreased serum performance.

How effective is the typical gamma radiation dose in achieving pathogen reduction? This may be answered by spiking virus and mycoplasma into serum and then assessing the log10 reduction of the infectious agent caused by the irradiation process. Relatively small doses of gamma radiation are required to render mycoplasma non-infectious. At the typical irradiation levels, essentially complete (6-10 log10) inactivation of mycoplasma is to be expected. Viruses are more resistant to this treatment and the levels of inactivation are, to some extent, dependent on the virus family. Most of the typical viral contaminants of bovine serum, for instance, are readily inactivated at 25-40 kGy. This includes bovine viral diarrhea virus, Cache Valley virus, reoviruses, parainfluenza type 3, and foot and mouth disease virus. Some of the small, non-enveloped viruses are relatively resistant, and families such as the circoviruses, parvoviruses, and polyomaviruses may survive gamma irradiation at the usual doses. The table below (data from Nims, Gauvin, and Plavsic1) demonstrates the effectiveness of a typical gamma radiation dose (35 kGy) for viral inactivation. To appreciate these values, it may be recalled that 2 log10 inactivation represents 99% killing, and 4 log10 represents 99.99% killing, and so on.  

In summary, these data indicate that gamma irradiation is a very useful approach for mitigating the risk of introducing viruses and mycoplasmas into cells through use of animal serum as a component of culture medium. Users are encouraged to investigate the use of gamma irradiated serum for their specific cell culture applications. If it is found that irradiated serum works as well as non-irradiated serum, it makes sense to take advantage of this treatment option. Rocky Mountain Biologicals contracts with an experienced service provider using calibrated equipment and validated processes to perform our gamma irradiation. The added cost of irradiating the serum is a small price to pay for avoiding the rare but potentially debilitating introduction of a microbial contaminant into your cell stocks.

1Nims RW, Gauvin G, Plavsic M. Gamma irradiation of animal sera for inactivation of viruses and mollicutes – a review. Biologicals 39:370-377, 2011