The Unethical use of Fetal Bovine Serum

“Lack of reproducibility of scientific experiments is a pervasive issue that affects cell culture work due to the plethora of uncontrollable components used in these systems.”

“FBS and other serums have the potential to greatly impact the quality and reproducibility of cell-culture work. The role of FBS should be elevated, given the significant impact its purity—or lack thereof—has on vital experiments.”

https://www.genengnews.com/insights/shifting-quality-of-fbs-may-impact-your-experiments/

Fetal bovine serum is the most commonly used sera in cell cultures. It is made from the blood taken from the unborn fetus found inside a pregnant cow at the time of slaughter. A cardiac puncture is performed to drain the blood from the heart of the fetus which is typically alive during the process. This is done without any form of anaesthetic. The blood, or serum, is added to cell cultures in order to provide the necessary “nutrients” for the cell to grow. As shown below, FBS was used in the original “SARS-COV-2” papers:

“Inoculated Vero cells were cultured at 37°C, 5% CO2 in 1× Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% fetal bovine serum and penicillin-streptomycin.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045880/

“For swabs, 1.5 ml DMEM containing 2% FBS was added to each tube.”

https://www.nature.com/articles/s41586-020-2012-7

While FBS is used in nearly every cell culture, there are many moral and scientific issues regarding its use. A few articles help to highlight these problems:

Fetal Bovine Serum

“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].”

“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.”

“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].”

https://www.labome.com/method/Fetal-Bovine-Serum.html

From this article, we see FBS:

• is commonly used in very high concentrations
• can introduce research artifacts
• affects human cells differently than those which use human sera
• is not considered the best supplement for cell culture even though it is widely used
• contains a large number of extracellular vesicles and exosomes
• exosome-depleted FBS may affect and support cell growth differently than regular FBS

Reproducibility: Respect your cells!

“Most academic labs culture cells by using fetal bovine serum (FBS), a liquid extracted from clotted cow blood and collected from abattoirs when pregnant cows are slaughtered. What ends up in the serum depends on factors such as diet, geographical location, time of year, whether the animals receive hormones or antibiotics and the gestational age of fetal calves. Substantial amounts of FBS are added as a supplement to the culture media in which cells grow; 5–15% of the volume of growth media is typical. FBS composition can affect how thick an engineered tissue becomes, cause spontaneous artefacts that mimic cell activity and even influence how surface receptors respond to a given compound. “FBS is like a big dark cloud over our heads, not knowing what’s real and what’s not,” says Khodabukus, now a postdoctoral researcher at Duke University in Durham, North Carolina.”

“Serum is arguably the most common supplement in cell-culture media, and also the least consistent. Human serum harbours thousands of distinct proteins originating from a wide range of cells and tissues, as well as thousands of small-molecule metabolites, all in varying concentrations. FBS probably has similar complexity, with plentiful factors to support a fast-growing fetus, too.

FBS is not only variable, it also differs from the fluid that cells are exposed to in their natural environment. Most cells are in contact not with blood directly but with the interstitial fluid that bathes organs, says Adam Elhofy, chief science officer at Essential Pharmaceuticals in Ewing, New Jersey, a company developing a serum replacement for multiple cell types. Hormones, growth factors and other signalling molecules are abundant in serum, but tightly regulated in organs, he says (see ‘Bovine serum’s wide range’).”

“Everyone agrees it would be a great thing if we can move away from FBS and to something more defined,” says Jon Lorsch, head of the US National Institute of General Medical Sciences in Bethesda, Maryland. “The question is how feasible it is, and we don’t know the answer to that question.”

“Even if the option is available, many researchers are unwilling to take the time, or the risk, to wean their cells off serum, says Paul Price, a culture-media consultant in Mount Pleasant, South Carolina, who has designed serum-free formulations. “Every year since 1980, people have been saying that serum is dead,” he says. “Serum is still very popular because people like the idea that they can grow cells and not have fabulous technique.” Culture is tough on cells: researchers pipette them from dish to dish, freeze and thaw them, add digestive enzymes to detach them from substrates and more. Serum is a balm for such abuses, says Price.”

https://www.nature.com/articles/537433a

From this article, it is clear that FBS:

• is variable depending on many factors that go into its production
• can affect tissue thickness
• causes artefacts that mimic cellular activity
• can influence how surface receptors respond to a given compound
• differs from the fluid cells are exposed to in their natural environment
• is used for its ease of use without requiring fabulous technique

Would 20 nm Filtered Fetal Bovine Serum-Supplemented Media Support Growth of CHO and HEK-293 Cells?

“However, the continued use of serum in cell culture features many drawbacks too. In particular, the composition of serum is poorly defined, and it is prone to significant batch-to-batch variation.(1,3,4) Further, serum may harbor a wide array of contaminants, such as bacteria, mycoplasma, viruses, endotoxins, and prions.”

“The most commonly used type of serum in cell culture is fetal bovine serum (FBS) due to its strong growth-promoting capacity and relatively low immunoglobulin levels.(9) It has recently been reported that nearly 80% of the late clinical stage cell therapies based on mesenchymal stem cells use FBS.”

“The use of 50 nm large-size virus retentive triple-layer hydrophilic PVDF (polyvinylidene difluoride)-based DV50 filters to filter 10% FBS in Dulbecco’s modified eagle’s medium (DMEM), has been reported, featuring robust clearance of large-size viruses but poor clearance of small-size 25 nm viral particles.”

“Currently, FBS is not processed through 20 nm virus retentive filtration, and, overall, there is currently poor understanding of how the cell culture would be affected by the nanofiltered FBS as its proteomic composition will inevitably change during processing.”

https://pubs.acs.org/doi/10.1021/acsabm.0c01372

From this article, we see FBS:

• has a poorly defined composition
• is prone to significant batch-to-batch variations
• may harbor a wide array of contaminants
• filtration can not remove small “viral” particles
• composition will inevitably change due to nanofiltration and the effect on cell cultures is not understood

The above 3 articles alone highlight many reasons why FBS should not be used in cell culture or at the very least cause concern about the validity of any culture using it. Once you read the description for how it is made, you will realize there are ethical issues as well. Keep in mind that in most instances, the fetus is alive during this process:

The use of fetal bovine serum: ethical or scientific problem?

“The general procedure of a cardiac puncture is the following (see diagram). At the time of slaughter, the cow is found to be pregnant during evisceration (removal of the internal organs in the thorax and abdomen during processing of the slaughtered cow). The reproductive tract is removed from the carcass, and is dropped down a special stainless steel chute leading to the calf processing area, a room that is separated from the rest of the abattoir floor. The calf is removed quickly from the uterus and the umbilical cord is tied off, the fetus is cleaned from amniotic fluid, and is disinfected.”

“A cardiac puncture is performed by inserting a needle between the ribs directly into the heart of the unanaesthesised fetus and blood is extracted under vacuum into a sterile blood collection bag via a tube. In the absence of a vacuum pump, fetal blood may be obtained by means of gravity or massage. In this case the blood collection bag is placed at a level below the fetus. Once the blood has been obtained, it is allowed to clot at low temperature, after which the clotted substance is separated from the serum by refrigerated centrifugation. The fetus is processed for animal feed and extraction of specific substances like fats and proteins, among other things. A much less common technique is umbilical cord puncture.”

“Since the fetus is expected to be alive during blood collection, its possible suffering is considered. The described procedure may cause pain in the fetus, thus raising ethical questions. First, literature on the resistance of fetuses to lack of oxygen is discussed. The bovine fetus experiences anoxia, acute lack of oxygen, since oxygen-rich blood supply to the placenta ceases upon death of the maternal animal. Lack of oxygen may interfere with neural processes such as transmission of stimuli, and eventually leads to death.”

“Since FBS is undefined, its application in culture media may alter the outcome of scientific experiments involving cell cultures and make it difficult to compare similar experiments performed with different batches of serum (6, 76). Hence, FBS may interfere with the advancement of biological science (92, 93). FBS should be replaced or its use reduced in cell culture both on scientific and moral grounds. As long as fetuses may be expected to experience discomfort from the methods employed in FBS harvest it is a morally problematic product. Even if a fetus was stunned prior to blood harvest the scientific problems discussed above remain. Hence it is worthwhile to consider methods of reducing or replacing FBS in cell culture.”

“Batch-to-batch variations (68-70) make it necessary to pre-test every batch before purchase. The presence of many different growth and growth inhibition factors may lead to overgrowth of e.g. fibroblasts in mixed cultures. From a scientific point of view it may be questioned what the effects are of the absolute molecular composition of FBS relative to the serum of the species, gender and developmental stage the cultured cells are derived from (70). Proper cell growth does not necessarily coincide with proper cellular function (3). Fetal bovine serum can interfere with genotypic and phenotypic cell stability (71, 72), and can influence experimental outcome (5, 6, 71, 73-77). Serum can suppress cell spreading, attachment (78) and embryonal tissue differentiation (76). Finally, serum can be contaminated with viruses, bacteria, mycoplasmas, yeast, fungi, immunoglobulins, endotoxins, and possibly prions (11, 67, 72, 79-85). These undesired substances can affect scientific experiments and bulk production of proteins. Many substances present in FBS have not yet been identified (67, 86, 87) and of many substances, which have been identified, the function on the cultured cells is unclear (79).”

“Fetal bovine serum is both a scientifically and a morally problematic product. Its application in cell culture experiments represents a scientific problem as FBS is undefined and may interfere with the outcome of experiments. Harvesting bovine fetal blood by cardiac or umbilical cord puncture without the refinement of pain relief, such as stunning with a captive bolt, can be considered immoral. This has consequences for cell culture applications, which currently rely on FBS as a medium component. The thought that cell culture techniques requiring FBS are a replacement to the use of animals is a misconception. If refinement of fetal blood harvesting methods can be endorsed, several scientific and technical problems inherent to the biological origin of sera remain. It should therefore be questioned why animals are used at all for such product. On moral and scientific grounds the most promising alternative to FBS is the use of defined media.”

https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.forskautandjurforsok.se/docs/Forskarrummet/Serum/the-use-of-fetal-bovine-serum-ethical-or-sceintific-problem.pdf&ved=2ahUKEwjd27PG0PbuAhWScc0KHXmJCFEQFjAKegQIChAC&usg=AOvVaw3C4fQwIdWNztcYFh2zIXuv

This last article really highlights the moral issues regarding the collection and use of FBS. It also highlights many of the scientific problems such as FBS:

• is undefined and may alter the outcome of cell cultures
• interferes with genotypic and phenotypic stability
• supresses cell spreading, attachment, and differentiation
• can be contaminated
• has many unidentified substances and the ones that are known have an unknown effect on cell cultures

To put this all together and summarize briefly, FBS has significant batch-to-batch variability, unknown composition and characterization, numerous contaminants equaling a lack of purity, concerns over the inability to reproduce results, and many known and unknown effects on the cells including interfering with genotypic/phenotypic stability. These are but a few of the various problems related to fetal bovine serum and that list does not include the morally reprehensible mutilation of the unborn fetuses. Needless to say, just like the use of antibiotics which have profound impacts on cell cultures, the same can be said of FBS, potentially even more so. It is clear that any cell culture using FBS in the “isolation” of a “virus” is of a highly questionable quality.