Fetal bovine serum (FBS) is the liquid fraction of clotted blood from fetal calves, depleted of cells, fibrin and clotting factors, but containing a large number of nutritional and macromolecular factors essential for cell growth. Bovine serum albumin is the major component of FBS. Growth factors in FBS are essential for the maintenance and growth of cultured cells [1, 2]. FBS also contains a variety of small molecules like amino acids, sugars, lipids, and hormones.

FBS is used in a wide range of applications. One of the primary uses of FBS is in eukaryotic cell culture, with concentractions up to 20% [3] or even higher, where it provides many essential nutrients and growth factors that facilitate cell survival and proliferation. However, it is important to note that FBS in human cell cultures may introduce research artifacts; human cells cultured with human sera behave differently from those cultured with FBS [4]. FBS is also used in the research, manufacture, and control of human and veterinary vaccines and of biotech drugs, and used to stop trypsin digestion [5] or to serve as a protectant in cryopreservation [6]. Cell culture media without any serum have been in use for many years. Fetal bovine serum might not be the best supplement for cell culture. For example, bovine serum albumin with insulin-transferrin-sodium selenite and/or epidermal growth factor in culture medium improves bovine embryo quality and trophoblast invasion as compared to fetal bovine serum [7].

Fetal serum contains more growth factors and has lower gamma globulin (i.e., antibodies) content and non-fetal serum. These are important because the growth factors facilitate cell survival and proliferation while antibodies could bind to the cells in culture. In addition, fetal serum contains lower levels of complement proteins (complements) than those from adults or newborns. These complements have the undesirable effects of lysing cells in culture and interfering with immunoassays.

Serum and plasma are both derived from whole blood by using centrifugation to remove components including red blood cells. The difference between plasma and serum is that coagulation proteins are present in plasma but have been removed from serum. Plasma is generally prepared by adding anti-coagulants to the blood before centrifugation, but the clotting proteins are not removed. Serum is prepared by allowing the blood to coagulate before centrifugation or by extensive/progressive centrifugation. Thus fibrinogen and proteins associated with clotting are not present in serum.

In humans, the following 22 proteins comprise 99% of the total protein content of serum and plasma: albumin, total IgG, transferrin, fibrinogen, total IgA, alpha-2-macroglobulin, total IgM, alpha-1-antitrypsin, C3 complement, haptoglobulin, alpha-1-acid glycoprotein, apolipoprotein-B, apolipoprotein-A1, lipoprotein (a), factor H, ceruloplasmin, C4 complement, complement factor B, pre-albumin, C9 complement, C1q complement, and C8 complement [8, 9]. The remaining 1% includes hundreds of proteins. The most abundant serum protein, albumin, at 50 mg/ml, comprises about half of the total protein mass [10].

The International Serum Industry Association (ISIA; www.serumindustry.org), a trade organization of serum providers, established guidelines for quality control standardization. These guidelines require the following tests be performed using specific methodologies, and the test results must be available as the Certificate of Analysis (Table 1).

Endotoxins, also called lipopolysaccharide (LPS) or lipooligosaccharide (LOS), comes from the outer membranes of Gram-negative bacteria, and contributes to the clinical manifestations of a variety of pathogenic Gram-negative bacteria. In vivo, endotoxins induce fever and the inflammatory response while in cell culture they introduce variation in cellular responses.

Endotoxin levels are measured by the limulus amebocyte lysate (LAL) test or the newer synthetic tests, called recombinant factor assay [11]. Amebocytes are analogous to white blood cells in vertebrates and LAL is prepared from the blood of Limulus polyphemus (American horseshoe crab; Figure 1). LAL is very sensitive to endotoxins and coagulates in the presence of even a minute amount of endotoxin. The conservation status of the American horseshoe crab has been assessed [12] and discussed in popular culture [13]. Many of the horseshoe crabs populations are at risk and thus care should be taken to protect them from loss [14].

Figure 1. Horseshoe crabs.

We discuss some of commonly used speciality sera below. Serum preparations dedicated to specific research topics, such as lipoprotein deficient serum from Kalen Biomedical for cholesterol research [15], or for specialized gene expression systems, such as tetracycline-screened fetal bovine serum [16, 17], are not discussed.

Regular FBS contains a large number of extracellular vesicles, some of which are exosomes [18, 19]. When performing exosome research with cultured cells it is critical to use FBS without exosomes. However, it is important to be aware that exosome-depleted FBS may affect and support cell growth differently than regular FBS [20, 21]. Exosome-depleted FBS can be either purchased directly from several commercial suppliers, or prepared through either ultracentrifugation or ultrafiltration, though the effects of the depletion processes and/or the commercial sources may not be consistent in terms of cell growth and physiology [22].

Other components of FBS can be depleted, depending on the experimental requirements. For example, Galmozzi A et al depleted heme from FBS with ascorbic acid [23].

A common treatment of FBS is heat-inactivation, where FBS is heated at 56°C for 30 minutes in a water bath with occasional shaking. The purpose is to inactivate whatever components of the complement system are present in the FBS [24], and other potential unknown inhibitors of cell growth. Previously heat-inactivation was also intended to remove mycoplasma contamination, which is no longer an issue since all serum products are now filtered through pore sizes small enough to remove mycoplasma. It must be noted that heat-inactivation may have undesired effects as well, such as reducing the ability of cultured cells to attach to surfaces [25].

Several suppliers have urged users not to heat-inactivate FBS for most cell culture needs. In each case it is advisable to evaluate the need for heat inactivation in a particular application, since different cells have different responses towards heat-inactivation of FBS [26-29].

Furthermore, like all other protocols with regard to FBS, the heat inactivation process must be done with care, since a temperature too high or prolonged heating will inactivate growth factors, and generate precipitates.

A Pataskar et al cultured MD55A3 melanoma cells derived from metastatic melanoma tumour resections in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum from Sigma [30]. LM Silva et al purified and maintained neutrophils in media with heat-inactivated fetal bovine serum [31]. D Schneider et al maintained Burkitt’s lymphoma cell line Raji, and leukemia lines K562, REH and NALM-6 in media supplemented with 10% heat-inactivated fetal bovine serum from Hyclone [32]. HY Chew et al maintained A431, HCC1569, MCF7, MDA-MB-231, MDA-MB-468, SCC25, SKBR3, T47D, RENCA and CT26.WT cell lines in media with 10% (v/v) heat-inactivated FBS [33]. Freeman SA et al used heat-inactivated FBS throughout in their study on macrophages [34].

Activated carbon can bind to lipophilic molecules and thus has been used to remove hormones such as androgen, estradiol, progesterone, cortisol, testosterone, triiodothyronine (T3), and thyroxine (T4) from FBS. These serum lipid hormones tend to interfere with immunoassay systems and insulin assay methods. L Zhao et al measured tumor cell growth in culture media supplemented with 10% charcoal-stripped serum [35].

Dialysis can remove all molecules with molecular weight less than 10,000 MW from FBS. This includes both polar and non-polar molecules. Hormones, cytokines, glucose, amino acids, and many others are removed. Dialysis also removes antibiotics and other exogenous molecules in FBS.

Dialyzed FBS from Thermo Fisher Scientific was used in the culture of glucose-starved cells [36], pancreatic ductal adenocarcinoma for SILAC mass spec study [37] and other SILAC studies [38], and in the culture of HeLa cells with Met analogue l-AHA pulse-chase for the click-chemistry detection of long-lived proteins [39], among others [40].

Gamma irradiation can be part of the process to sterilize FBS. FBS is usually filtered through 0.1 um filters multiple times to eliminate microorganisms. Gamma irradiation can inactivate the viruses commonly present in bovine species. However, some viral species, such as parvovirus, are resistant to gamma-irradiation.

Though FBS has a quite low IgG content, even this level may be too high for some certain applications. The IgG level can be substantially reduced further by capture chromatography. FBS with low IgG is used for antibody production, recombinant protein synthesis and other applications.

Stem cell culture has stringent requirements in terms of growth factors. Some of the growth factors present in FBS promote stem cell differentiation. Several companies offer embryonic stem cell qualified FBS intended to maintain undifferentiated stem cell. However, it is important to test specific lots of FBS for their applicability in maintaining the pluripotency/totipotency of different types of stem cells. Thermo Fisher provides embryonic stem cell-qualified FBS, as, for example, used in E14TG2a mouse ES cell culture by Yasuda S et al [41].

Major cattle countries are the main providers of FBS. The countries include the US, Australia, New Zealand, Canada, several in South and Central Americas.

The production and collection of serum products are regulated by government agencies. FBS is labeled as USDA-Grade or European-Grade. USDA-Grade FBS can be imported into any country free of bovine spongiform encephalopathy and foot and mouth diseases, while European-Grade FBS can be sold in most European and Asian countries.

FBS is best stored frozen, between -5 to -20°C, and can be thawed at a temperature between 2 to 8°C. It is often useful to aliquot and freeze the serum in smaller portions, often 50 ml tubes, to avoid many freeze and thaw cycles. Occasionally, some aliquots of FBS remain in a liquid at freezing temperatures. This is due to the lack of a nucleation center (particulate matter) for the crystallization (freezing) to start. If you simply flick the tubes with your finger, they usually solidify almost instantly.

It is common to have some precipitate after thawing. The precipitates, likely due to denaturation of some of the serum proteins, can be cleared by brief centrifugation, and this generally does not affect the quality of the serum.

Many suppliers provide FBS with different grades, country of origin, and treatment. FBS, itself, is a complex mixture, and variability between different grades, suppliers, and lots is to be expected. Thus, it is essential to establish a process for selecting and evaluating FBS. It is a good idea to examine the literature to determine how a particular selection of FBS was previously used in similar research, or for the same cell culture and to pay close attention for potential lot-to-lot variation in the FBS. To assist with this, Labome has conducted a survey of FBS usage from scientific publications, discussed below, to help our visitors select the most suitable FBS.

Labome surveys literature for the materials used. Here the publications which cited fetal bovine serum are summarized. The publications are a random subset of over 10,000 publications.

The major suppliers of FBS are Life Technologies (now part of Thermo Fisher Scientific), Thermo Fisher Scientific Hyclone (now part of GE LifeScience), and MilliporeSigma (Table 2).

Life Technologies is a global biotechnology company headquartered in Carlsbad, California. It’s formed in 2008 with the merger of Invitrogen Corporation and Applied Biosystems Inc. Many of their products are under the brand name Gibco. They provide cell culture products, such as cell culture medium, FBS, and other relevant reagents. The company merged with Thermo Fisher Scientific in 2014.

Figure 2. Permission from Life Technologies Gibco, the copyright owner.

FBS from Life Technologies has been used to study nucleolus as a phase-separated protein quality control entity in HEK293T and HeLa cells [46]. Pandolfini L et al cultured HEK293T and A549 cells in DMEM supplemented with 10% Gibco FBS (10270-106) and Caco-2 cells in Eagle’s Minimum Essential Medium supplemented with 20% Gibco FBS (10270-106) [45]. HEK293T cells are quite often maintained with 10% fetal bovine serum from Thermo Fisher [44].

GE Healthcare Hyclone FBS is widely used. De Cecco M et al used it at 15% to maintain the senescent fibroblasts LF1, IMR-90 and WI-38 in Ham's F-10 media [68]. Nicetto D et al used Hyclone FBS (SH30071) to terminate trypsin digestion and maintain embryonic cells [5]. Laflamme C et al maintained HEK-293 cells in DMEM high-glucose from GE Healthcare (SH30081.01) containing 10% bovine calf serum from GE Healthcare (SH30072.03) [48].

Figure 3. Permission from Thermo Fisher Scientific, the copyright owner.

MilliporeSigma FBS products have been used to culture HAP1 wild-type and knockout cells [49], terminal Schwann cells [69], HEK293T cells (ATCC CRL-3216) and COS-7 cells (87021302) [70].

Frohner IE et al maintained Neuro-2a (N2a) and Cos-7 cells from American Type Culture Collection in DMEM containing 10% FBS from Bovogen, France [49]. Laflamme C et al cultured U2OS cells in DMEM high-glucose containing 10% tetracyclin-free fetal bovine serum from Wisent (081150) [48]. Dong JX et al maintained primary hippocampal cultures on Matrigel from Corning with 5% fetal bovine serum from Atlanta Biologicals (S11550) [52]. Lee A et al cultured murine C2C12 myoblasts with DMEM supplemented with 10% fetal bovine serum from VWR (89510-186) [67]. Zhao N et al cultured U2OS cells from ATCC (HTB-96) in DMEM medium with 10% (v/v) fetal bovine serum from Altas Biologicals [71]. Liu X et al cultivated murine 4T1 cells in DMEMsupplemented with 10% fetal bovine serum from Biochrom [63].