Heat-inactivation of fetal bovine serum and other animal sera historically has been used to inactivate heat-labile complement that might be present in the serum. Most typically, heat inactivation consists simply of raising the temperature of serum to 56 ˚C in a water bath and strictly maintaining that condition for 30 minutes. In the early days of cell culture, such treatment was routinely performed, without much regard for whether the presence of intact complement proteins might actually represent an undesirable characteristic of the serum. Heat treatment was also advocated in the past as a risk mitigation approach for achieving pathogen (especially mycoplasma and virus) reduction in serum. Serum users are now encouraged to ask the question: is heat inactivation of serum required for my specific cell culture application? What are the downsides of performing heat-inactivation? These are the questions that we will attempt to address in this article.   

There are a variety of heat-labile and heat-resistant complement (C) proteins in serum which play various roles in immunological functions. When these proteins are introduced into cell culture media through use of animal (especially non-fetal) sera, there may be undesired impacts on immunological endpoints (detection of neutralizing antibodies, etc.) or on the proliferation of viruses. In other instances, the presence of complement may have no impact on the performance of the serum.

In the case of fetal bovine serum, it turns out that the levels of complement proteins are relatively lowii. For applications not involving immunological endpoints, the presence of such low levels of complement may have negligible impact on serum performance. It should not be assumed that heat-inactivation of fetal bovine serum is needed or even appropriate. The best way to answer this question is to empirically determine in your own laboratory whether, for a given cell culture application, non-heat-inactivated serum or heat-inactivated serum provides the optimal results. This investigation, conducted perhaps with a few different batches of treated and untreated sera, will allow you to determine whether heat-inactivation is needed for your specific application. 

If it appears that heat inactivation of the serum does provide better results, then it may pay to purchase serum that has been so-treated. Heat inactivation must be performed with care, as excessive temperature or time at temperature will likely degrade the serum. The potential for causing denaturing of essential nutrients during this treatment is always present, so a strict protocol must be followed if consistent serum quality is to be maintained.

It has been showniii that mycoplasma and certain viruses (including such known contaminants of bovine serum as bovine viral diarrhea virus, parainfluenza type 3, infectious bovine rhinotracheitis, reovirus type 3, and adenovirus) are rendered essentially non-infectious at the temperatures/treatment times used for heat-inactivation. There are, however, other ways of inactivating such potential contaminants that are less potentially damaging to the serum (see our article on gamma irradiation of serum for risk mitigation, for instance). Heat-inactivation should not be employed for this purpose alone.  

The unavoidable trade-off with heat inactivation

In summary, heat-inactivation of serum is no longer considered to be a requirement for most cell culture applications. If it must be done, due to the particular research endpoints being followed, it is recommended that heat-inactivated serum offered by serum providers be utilized, rather than purchasing non-heat inactivated serum and then leaving this important treatment step to the inexperienced.

idel Zoppo GJ. In stroke, complement will get you nowhere. Nature Med 5: 995-996, 1999

iiLinscott WD Triglia RP. The bovine complement system. Adv Exp Med Biol 137: 413-430, 1981.

iiiDanner DJ, Smith J, Plavsic M. Inactivation of viruses and mycoplasmas in fetal bovine serum using 56  ͦC heat. BioPharm 12: 50-52, 1999.

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