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FCS-free – cell culture media without fetal calf serum

Summary

Fetal calf serum (FCS) is used as a nutrient solution for cells. However, obtaining the serum involves great animal suffering, as it is collected from the blood of unborn calves by puncturing their hearts with a needle. All blood is aspirated until the calf dies. But there are cell culture media without fetal calf serum. They offer not only ethical but also scientific advantages over FCS because their use increases experimental reproducibility and eliminates the risk of contamination with animal viruses and bacteria or of adverse reactions with human cells.

Background

Researchers are increasingly working with cell or tissue cultures in order to avoid animal experiments. Such cultures are used in toxicity tests of chemicals or in drug development, for example. In order for the cells to stay alive and keep growing, a so-called nutrient culture medium is used, which is a liquid containing various nutrients. Up until now, blood serum from unborn calves has been used as the “gold standard” for this purpose. It supplies the cells with hormones, growth factors, proteins, amino acids, minerals, and trace elements, among others. However, its exact composition is not known to this day and can vary greatly depending on the origin (1). Serum from unborn calves (fetal calf serum = FCS; or fetal bovine serum = FBS) contains fewer substances that inhibit cell growth and is therefore preferred to that from adult animals (2).

The cruel collection of calf serum

Calf serum is a lucrative by-product of meat production. Immediately after a pregnant cow is slaughtered, the fetus is excised from the uterus. A thick needle is then pushed between the ribs of the still-living calf going through the skin and muscles, directly into the beating heart. The blood is aspirated until the animal is drained of blood and dies. This procedure is performed while the calf is still alive because a larger amount of blood, which won’t clot, can be drawn from the beating heart. This is done without anesthetic, although scientific studies suggest that calf fetuses are already capable of suffering, at least in the last third of pregnancy (3). According to some reports, pregnant cows can be intentionally sent to slaughter for more than 20 different reasons (4). In many countries it is common for cows and bulls to sometimes graze together (3). If a whole herd is brought to the slaughterhouse, it always contains some pregnant animals.

According to a study at the University of Leipzig, up to 15% of cows slaughtered in Germany every year are sent to slaughter pregnant due to management errors or unnoticed insemination (5). In May 2017, a legal ban on the slaughter of cattle in the last trimester of pregnancy was passed in Germany (6). Previously, only the transport of such cows was prohibited, but this has still happened illegally on many occasions. The unborn calves suffocate painfully in the uterus during slaughter. The animal welfare community has therefore been calling for a uniform EU slaughter ban for pregnant animals for years, as well as legal provisions regulating sanctions in the event of violations. For example, the slaughter of pregnant sheep and goats is still permitted. In addition, the ban on slaughter must urgently be extended to the entire pregnancy period. However, there is no data about FCS collection in Germany. The manufacturers obtain the serum from abroad.

The lucrative business with calf serum

The demand for nutrient media for cell and tissue cultures, and therefore also for calf serum, is high and constantly increasing, since in vitro research (in vitro = "research in a test tube") is becoming more and more important. The size of the global FCS market is estimated at $1.07 billion in 2023 and is projected to reach $1.4 billion in 2027 (7). According to an estimate from the years 2002-2007, the worldwide annual use was around 600,000 to 800,000 liters of FCS, for which 1-2 million calves were killed (3,8). There are no more recent figures, but it can be assumed that consumption is much higher now. About half a liter of blood is obtained from each calf. The origin of the calf serum and the current demand determine the price (9). Serum from New Zealand or Australia is particularly expensive as the island cows' blood is free from diseases such as foot and mouth disease.

Why is FCS used and why its use must be stopped

Fetal calf serum is used both as a nutrient medium and in the cryopreservation (cold treatment) of cells (10). It is also suitable as a nutrient medium because it offers a wide range of macromolecules, transport proteins for lipoid substances and trace elements, binding proteins and attachment factors, low-molecular nutrients, as well as hormones and growth factors.

  • A possible contamination of the serum with pathogens, depending on the origin, can lead to a slower cell culture growth or even cell death (11,12).
  • There is a lack of standardization due to fluctuations in the composition. Furthermore, contamination by microorganisms such as bacteria,viruses or prions is possible, making FCS difficult to reproduce and not ideal as a nutrient medium (13).
  • In the worst case, patients' health can be endangered if the calf's blood has been contaminated with pathogens, which come into contact with components used during drug production. The use of FBS in cell and tissue culture for human clinical use is discouraged in the US and EU due to the potential for contamination with non-human pathogens, the risk of an adverse immune response, and product reproducibility issues (14,15).
  • Scientific results can also be skewed by the heterogeneous composition of the nutrient solution (11,16). The varying composition is due to the different origins of the mother cattle and the use of different cattle breeds and pastures. Furthermore, the animals come from different countries and are confronted with different environmental conditions.
  • But the most important reason why FCS should not be used is that it involves considerable suffering and millions of calf fetuses. Based on scientific studies, it can be assumed that fetuses can feel pain (3). Even the EU Animal Testing Directive includes fetuses from mammals in the third trimester as they are "at increased risk of experiencing pain, distress and fear". (17). In order not to support this immense animal suffering, animal-free culture media should be used. Furthermore, FCS is a by-product of the beef industry. Its use can therefore not meet ethical standards.

FCS-free culture media

A large number of FCS-free culture media have been developed over the past two decades. In the FCS-free database alone, available to researchers and the public free of charge, around 900 such culture media have been described to date, of which more than 400 are free from any animal components (18).

Animal-free culture media are ethical because they do not cause animal suffering. In addition, serum-free culture media have a precisely defined composition. In this way, it is possible to create controlled and reproducible cultivation conditions and there is no risk of pathogen transmission. In general, there are five types of culture media:

  • media with animal serum (e.g. fetal calf serum)
  • human blood media
  • serum-free media
  • chemically-defined media
  • media with vegetable substances

In addition to the FCS-free database, other platforms such as NC3Rs and PETA Science Consortium International also offer information about various FCS-free culture media (19,20). In addition, the Danish Society for the Protection of Laboratory Animals provides a list of companies that offer serum-free culture media (21). Some frequently used types of such media or media components are listed below.

Human platelet lysate (hPL) from unused blood donations

One possibility is to use human platelet lysate (hPL) instead of FCS as a cell culture media supplement. hPL is manufactured from human platelet extracts obtained from a “buffy coat”, an inexpensive and uncomplicated byproduct in blood donation centers (22). Because hPL is derived from human serum, it is better suited as a nutrient medium for human cells. It is also rich in specific growth factors. Because donated human platelets have a shelf life of only four days, approximately 50-60% of all platelet donation units expire and cannot be used for patients (23). Expired donations are usually thrown away. Processed into hPL, they could save lives - those of unborn calves. Large parts of the global demand for FCS-free culture media could be covered by hPL. It contains growth factors that are superior to those of fetal calf serum. hPL offers better growth conditions than FCS for many human cell types, such as mesenchymal stem cells (24,25). Because human donor units are pooled during hPL production, its variation in composition is extremely low, which enables good reproducibility of the nutrient solutions and uniform growth of cell cultures (22). In addition, the blood of the donors has been tested for pathogens in advance, so there is no risk of pathogen transmission. The disadvantage, however, is that the cells first have to adapt to the hPL-containing medium and heparin, which is mainly obtained from the intestinal mucosa of pigs, has to be added to prevent coagulation (26). However, synthetic heparin or special heparin-free culture media can also be used (27,28).

Human serum

Another FCS alternative are human sera such as the human AB serum (HABS). AB serum is an antibody-free, AB-blood group human serum. HABS supports the proliferation of human osteoblasts, chondrocytes, bone marrow cells, endothelial cells, and cancer cells, particularly gliomas and melanomas (29). Human sera consist exclusively of human material. They are therefore more suitable for cultivating human cells than FCS, since both the serum and the cells belong to the same species. The same physiological and natural conditions that the cells experience in vivo (in the body) are created in vitro (in the test tube). Human serum is particularly suitable for cell culture in the field of cell and immunotherapy or tissue engineering, where components of animal origin are not desired (30,31). Like hPL, human sera are produced from expired blood donations and are available in large quantities (32).

Serum-free media

Serum-free media are culture media that do not use human or animal serum (like FCS). Instead, they contain a combination of proteins, hormones, and other media supplements like insulin, transferrin, and albumin (33). Serum-free media are very suitable for the cultivation of different cell types, e.g. fibroblasts, epithelial cells, and endothelial cells (34). Animal and human sera represent a rich mixture of compounds, the exact composition of which is unknown and may vary. In contrast, all components of serum-free media are known, which increases the reproducibility of cell culture experiments. A potential drawback is that some additives in serum-free media, such as albumin, may be of animal origin (35). This not only poses animal welfare problems, but also a risk of contaminating the cells with animal viruses. Therefore, many serum-free media today are also free of any animal materials.

Chemically defined media

In contrast to "standard" serum-free culture media, which can contain undefined compounds such as hormones, growth factors, and various proteins, chemically defined media require the identification of all components and their concentrations (36). Therefore, a chemically defined medium must be completely free of animal or human serum. Culture media that do not contain components from another species (i.e., no animal materials when human cells are being grown) are also called xeno-free media. Such media are not only ethical, since no animals were killed for them, but also eliminate the risk of contamination with foreign pathogens and the transmission of human diseases (37). Some chemically defined media are also protein-free and/or peptide-free and contain only low molecular weight components (38). Chemically defined media provide excellent scientific conditions for studying human cells, as they are free from unknown factors that can interact with the cells and alter the results. Therefore, they offer a high experimental reproducibility. A limitation here may be that the development of such media can be expensive and time consuming (37). However, chemically defined media suitable for a wide range of human cells are already commercially available (36). One problem is that most biotechnological companies do not disclose the exact composition of their chemically defined media, leaving scientists unaware of which nutrients are affecting their cells and experiments. However, there are freely available recipes for various chemically defined growth media, as well as different approaches to creating and optimizing such media formulations (39).

Plant and synthetic FCS substitutes

Some plant-derived and synthetic substances can be used instead of FCS as media supplements. A common type of supplements are the hydrolysates, which are the products of the enzymatic digestion of soy, pea, rice, rapeseed, or other plant proteins (40). Hydrolysates often contain a mixture of peptides, amino acids, minerals, carbohydrates, lipids, and proteins. The relatively low cost of plant hydrolysates makes them attractive as serum replacement components for large-scale protein production. However, the composition of hydrolysates is not fully characterized, so a deeper understanding of their components and how they affect cell growth is key to their success as potential serum substitutes (40).

Many companies offer various synthetic substances as FCS substitutes. These are usually growth factors or lipoproteins that support cell growth and proliferation (41,42). Synthetic supplements are of better quality than FCS and their manufacture is much more consistent and reproducible.

There are a few ways to replace FCS with other animal substances such as extracts from meat, milk, insects or spiders. However, these materials are also associated with animal exploitation and animal cruelty and should therefore be avoided.

Difficulties in switching to FCS-free culture media

As discussed above, the use of FCS raises multiple ethical and scientific concerns. In addition, an important advantage of using human cell cultures is the lack of animal testing.FCS reduces this advantage by introducing animal components and animal suffering. There are complex reasons why FCS is still widely used despite these disadvantages. One problem is that the cells in the cell cultures first have to adapt to the new culture medium (10). During this adaptation phase, which varies depending on the cell type, growth losses occur, which can lead to additional expenditure for the laboratories and institutes. However, these growth difficulties would usually last no more than a few weeks. In addition, many laboratories are insufficiently informed about the use of alternative culture media to fetal calf serum and tend to revert to well-known FCS-containing media, because FCS has been the gold standard since the 1960s. According to a global survey on the use of animal-derived materials in science, only 43% of scientists have considered replacing animal serums such as FCS in their experiments (43). A lack of knowledge about the availability of animal-free alternatives and a preference to stick to established protocols were the main reasons why survey participants continued to use FCS and other animal-based reagents. Other problems included the higher cost of non-animal reagents and insufficient education about how to handle such substances.

Conclusion

Millions of calves are cruelly killed every year for the production of FCS. FCS is a standard component in cell culture media and can cause many scientific problems, such as contamination with animal viruses and bacteria or unknown reactions of the cells, in addition to the ethical ones. There are a variety of different FCS-free culture media and culture media supplements. Some contain human materials like hPL or human serum, while others are completely free of human and animal components. Unlike FCS, these culture media are ethical and have many scientific advantages. There is a need to improve the awareness on the availability of these non-animal reagents and to provide researchers with adequate funding and training for their use. In this way, it will be possible to eliminate the use of FCS from cell culture.

09 March 2023
Julia Schulz (Vet) & Dr. Dilyana Filipova

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