Primer: FBS in cell-based meat R&D
One of the most important elements of a cell culture system is the media, which provides nutrients and other compounds essential for cell survival, proliferation and differentiation. For cell-based meat, one challenge for creating a scalable and cost-effective media is the replacement of fetal bovine serum (FBS) [1], commonly used in research labs. In this article, we will show why cell culture media is so important, and why FBS has achieved such prominence. We will then discuss the limitations of FBS and explore the animal-free media formulations currently available. We conclude that FBS elimination, while a necessary part of cell-based meat R&D, is likely a less pressing bottleneck to large scale production than other challenges, like reducing media costs. This is contrary to the way the challenge is often described in other sources.
The Components of Cell Culture Media
For cells to thrive in vitro, culture conditions must mimic those in an animal tissue. The culture medium is arguably the most important part of the cell culture system, as it provides a number of factors that are essential for the cell’s construction, internal processes, and energy production (see Figure 1). In native tissue, a cell’s natural environment varies between cell types and species. Therefore, cell culture medium should ideally be tailored to the cell type and the animal species being studied. However, defining a precise list of medium components optimized for a particular type of cell is costly and time consuming.
Nonetheless, there are some types of compounds that we know are required for most types of cell culture systems:
Inorganic salts like calcium (Ca2+), magnesium (Mg2+), potassium (K+) and sodium (Na+) are crucial for a number of cellular processes. For example, the active transport of complex compounds through cellular membranes is dependent on the continuous exchange or inorganic salts through the cellular membranes. The first culture medium, developed in 1882 by Sidney Ringer, was a balanced salt solution, containing nothing more than a mix of small ions at defined concentrations. With such a simple solution, Ringer could keep frog cardiac muscle cells alive for several hours. A number of other balanced salt solutions have been developed by other researchers like Locke’s solution, Tyrode’s solution, the Krebs–Ringer bicarbonate solution, Gey’s solution, Earle’s solution, and Hanks’ solution. Many of these are still used today as basal media.
Amino acids like leucine and glutamine are the building blocks of proteins. There are two types: non-essential, which can be created by cells, and essential, which must be consumed (or added to media in the case of cell culture). Of the 20 amino acids, 13 are essential for humans: Arginine, Cysteine, Cystine, Glutamine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Tyrosine and Valine. Non-essential amino acids may also be added to the media, since they may cause cells to behave differently.
Carbohydrates, i.e. sugars, are the main source of energy for cells. Most of culture media includes carbohydrates like glucose and galactose, or less often fructose and maltose.
Lipids, also known as fats, are important components for cell membrane integrity, and as a secondary source of energy. Cholesterol is a particularly important lipid, as it facilitates cellular signaling and intracellular transport.
Transition metals are important for the structural integrity and function of a large number of proteins and enzymes. For example, iron is essential for the structure of hemoglobin’s dioxygen carrier site. Selenium is also present in selenoproteins, which have antioxidant properties. Finally, Copper and Zinc are present in metalloproteins, which have varied functions ranging from the regulation of genetic expression to enzymatic degradation of the extracellular matrix.
Vitamins are involved in a wide variety of cellular processes. There are 13 types of vitamins: A, C, D, E and K, as well as 8 vitamins belonging to the B complex. Some notable functions are vitamin C’s regulation of iron absorption, and vitamin K’s importance for blood coagulation.
Polyamines such as spermidine, spermine and putrescine are low-weight amines present in all cell types. They promote cell growth by regulating protein and DNA synthesis.
Carrier proteins facilitate the transportation of other compounds through the cellular membrane. Albumin is a particularly important carrier protein, as it has been shown to bind to a wide variety of compounds, and has toxin-neutralizing, antioxidant, and shear-stress-reducing effects. Two other important carrier proteins are transferrin and lactoferrin, which carry iron to the cell membrane.
Growth factors and hormones regulate cell proliferation, differentiation, and homeostasis (the maintenance of ongoing physiological functions). There are many known hormones and growth factors, each with a specific role for a particular cell type. For example, insulin and glucagon are important regulators of sugar metabolism in muscle and fat cells. Nerve growth factor (NGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF) have all been found to increase cellular proliferation in cell culture.
Cell attachment and spreading proteins provide structure for cells to grow on. In native tissue, cells are attached to a complex mesh of molecules called the extracellular matrix (ECM). The ECM provides physical support for cells, and transmits signals that regulate cell behavior and development. In vitro, it is common for the glass of petri dish supporting the cell culture to be coated with extracellular matrix proteins, such as laminin or fibronectin. The culture medium can also be supplemented with these proteins to promote cell adhesion.
The Utility and Importance of FBS
FBS was used as an ingredient for cell culture media for the first time by Theodore Puck and colleagues in 1958. They showed that a medium made up of 15% FBS by volume improved cell survival and genome stability for a variety of human and animal cells. Following Puck’s work, a broad list of the major ingredients of FBS have been progressively identified (Table 1). Unsurprisingly, many of the components necessary for cell survival and proliferation are often naturally present in FBS. Because FBS is extracted from the blood of cow fetuses, it allows media to mimic the environment in which bovine embryonic and fetal cells would develop naturally.
It turns out that cellular requirements are similar enough between species (including humans, other mammals, most animals used for lab testing, and even insects) and cell types that FBS can serve as an almost universal promoter of cell growth and survival. This greatly reduces the need for researchers to develop culture media tailored to the specific cell type they are studying. Instead, researchers can start with an FBS-based media and focus solely on the more interesting scientific problems. This convenience has led to widespread adoption in biomedical research.
Table 1: Components known to often be present in FBS. Highlighted compounds were mentioned as important ingredients of culture medium in the previous section. Modified from: Brunner et al., 2010.
Despite its convenience, FBS’ production and usage poses some scientific, economic, and ethical problems. These problems make FBS problematic for any industrial-scale process [2].
Scientific limitations. Despite the convenience of FBS for biomedical research mentioned above, FBS is not a perfect R&D tool. FBS composition varies between batches, depending on the geographical area where it was produced, and the diet, breed, and age of the animal. It's impossible to know what exactly will be contained in a particular batch of FBS, negatively affecting reproducibility of experiments. For example, a lab might be unable to replicate the results of another group even though they are studying the same cellular process in the same cell line because the FBS they use has a different amount of a particular component. Additionally, FBS can be contaminated by viruses, fungi, bacteria or prions if the cow is infected or if pathogens are introduced during dissection and collection.
An unsustainable economic model and supply chain. The current price of cell culture-grade FBS varies between $1,000 and $1,200 per liter, making it cost-prohibitive to use at scale. Although it is not yet clear how many liters of cultured media are needed to produce one pound of meat, it is obvious that such an expensive production input would prevent cell-based meat from being cost-competitive with conventional meat. Currently, the wholesale price of beef in the US is around only $3.44 per pound. Additionally, since FBS is a by-product of beef production, it is impossible to lower prices by scaling up production. FBS price is volatile and on the rise because of factors on which buyers have little to no control, such as changes in weather conditions that can reduce yield, feed prices, and cattle imports and exports. In addition, further work on livestock breeding and nutrition will likely increase meat yield per animal, reducing the number of animals slaughtered for the same amount of beef, and thus the supply of FBS. Finally, the FBS market is known for its opacity and loose regulation, which has led to mislabeling and product adulteration.
Ethical concerns. FBS, as its name indicates, is collected from fetuses of pregnant cows. If pregnancy is detected after slaughter, the whole uterus is dissected from the carcass and then moved to a separate aseptic environment. There, the fetus is removed from the uterus and blood is extracted by directly puncturing the heart with a syringe. Blood is then let to clot at low temperature, and FBS is collected by refrigerated centrifugation. This naturally raises animal welfare concerns. To ensure good quality of the product, blood must be collected while the fetus’ heart is still beating, meaning they are still alive. Additionally, mother cows often live most of their lives in crowded, unsanitary conditions, and are themselves slaughtered for meat. This is problematic given that one goal of cell-based meat is to move away from the need to slaughter or hurt animals.
Therefore, FBS use is a clear non-starter for cell-based meat companies. It may be convenient for startups to use FBS to get their initial R&D off the ground since allows them to work with cells without a highly optimized media. However, they will have to move away from FBS as they scale up.
Alternatives to FBS
Long before the beginnings of cell-based meat, other industries successfully developed FBS-free, chemically defined culture media. For example, in the production of monoclonal antibodies, Chinese Hamster Ovary (CHO) cells are often cultured in serum-free media. Serum-free media was important for this industry because of the problems listed above, but also because unknown FBS components can lower the purity of harvested recombinant proteins. Some examples of commercially available serum-free media for CHO cells are GC3 and WCM5, which can be bought for $250/L for research purposes, and likely much cheaper for industrial production.
FBS-free media is also needed in stem cell culture for applications like cell-therapies. An additional issue with FBS in this industry is that some batches can contain unwanted differentiation factors, causing the stem cells to differentiate prematurely. This makes it difficult to maintain long-term stability of the culture. In anticipation of eventual large-scale production, researchers have developed FBS-free chemically-defined media optimized for stem cell growth, such as TeSR and Essential 8 (see Appendix). However, these media are currently designed primarily for academic laboratories and are produced in relatively small quantities. This makes them cost prohibitive for large scale culture. Retail price for Essential 8 and mTeSR1, a TeSR variant, are between $400 and $600 per liter, making them more expensive than some FBS-based culture media.
Cell-based meat has a similar need to develop serum-free, chemically defined media optimized for the growth of whatever cell lines the industry ends up using. Costs aside, given that similar industries have succeeded in developing such media, we can expect the same to happen for cell-based meat. On a theoretical level, we understand all of the components that make FBS so effective, and we can supply each of these components individually. Therefore, development of an FBS-free media is unlikely to be a major bottleneck for the industry, at least compared to others issues like lowering media costs [3].
Appendix
Table 2: Some examples of animal-free media used for cell culture in academia and/or industrial settings. Each are created by supplementing a commercially available basal medium with a number of other components. Modified from: Yao and Asayama, 2016.
Tommaso Lucchesi is an innovation consultant based in Paris, France. You can get in touch at tommasolucchesi07@gmail.com.
Footnotes
[1] We will focus on fetal bovine serum here since it is the most common animal serum. However, other serums like horse serum are often used as well.
[2] Serum is sometimes used in industrial vaccine production. However, FBS’ problems are less acute for vaccine production, since IP allows vaccines to be sold at extremely high margins. Therefore, lowering production costs is not as important.
[3] Some strategies for lowering media costs include:
A recent report by Liz Specht of the Good Food Institute highlights that 96% of the cost of Essential 8 medium cost is due to two growth factors: TFG-β and FGF-2. One of the most promising strategies would therefore be to find cheap replacements for these growth factors, or to lower their costs by scaling up their production.
Increasing cell densities to lower the amount of media required per pound of meat.