Due to its high concentration of factors that promote embryonic growth, foetal bovine serum (FBS) is the most often utilised growth supplement for cell culture media. It provides a variety of known and unknown substances that, when used at the right concentrations, have been demonstrated to meet unique metabolic requirements for cell culture. Growth factors, proteins, trace elements, vitamins, and hormones are all included in the complex mixture of biomolecules that makes up FBS. Both in vivo and culture, are crucial for the growth and upkeep of cells.
Fetal bovine serum is used for a variety of purposes. The field of bio pharmaceuticals and vaccines is the most significant. It is utilised in the discovery, production, and control of numerous cutting-edge pharmaceuticals as well as human and veterinary vaccines.
Numerous studies also make use of foetal bovine serum. Bovine serum is frequently utilised in the production of bio-pharmaceuticals, including vaccinations, using a method known as "Cell culture."
FBS is obtained from the foetuses of pregnant cows that have been slaughtered following international veterinary inspection standards and OIE (World Animal Health Organization) regulations.
In essence, FBS is a natural product. Every FBS batch has distinctive qualities that set it apart from other batches.
However, FBS is becoming less and less accessible. Prices for meat and calves are rising as beef consumption rises in some nations and cattle breeding methods continue to advance. Fewer pregnant cows are being slaughtered as a result of these combined reasons. For many years, the collection of FBS in new countries offset the decline in supply; however, there are no longer any new producer nations accessible. FBS is currently in short supply and will continue to decline.
Cell growth is unaffected by the source of the serum. Biowest studied cell growth in FBS from seven different nations across three continents and found that all cell lines tested had the same average performance regardless of the nation of origin. For one particular cell line, one batch of FBS might be effective, but not for another. Each cell line has a unique "serum quality." Because of this, analysing FBS is frequently done while working with delicate cell lines. Biowest conducts the industry's most thorough analyses of biochemical variables and tests on cell lines, publishing the results on Certificates of Analysis.
Legalities
Country where the serum comes from is bound to receive USDA approval. This indicates that serum is made from blood obtained in nations whose ruminant serum exports to the United States have been authorised by the United States Department of Agriculture (USDA). Australia, Canada, Chile, Costa Rica, El Salvador, Guatemala, Honduras, Mexico, New Zealand, Nicaragua, and Panama are among the nations that are permitted to export foetal bovine serum to the United States.
Vaccines and FBS
With the most recent advancements in science, such as stem cells, cell therapy, novel vaccines, and proteomics, the demand for FBS is increasing.
Because Australia is thought to be a "safer" origin for BSE and cow infections, Australian FBS prices are higher. The first nations outside of the USA where FBS was created were Canada and Australia. North American BSE instances in the 1980s developed into a compelling argument in favour of Australian FBS. It was said that Australia was undoubtedly the safest origin because of its "isolation."
FBS imports are less controlled in the EU than they are in the USA. The US FBS sector rejected a USDA request to adopt the same import regulations for FBS as the EU in 1994. The International Serum Industry Association (ISIA) encourages adherence to the OIE standards and supports attempts to unify import regulations. FBS is still more than twice as expensive in the USA as it is in Europe.
One or more active compounds are the main components in every vaccine. Other than this, water is the primary component of vaccinations. The majority of injectable vaccines comprise 0.5 millilitres, or a few drops, of liquid. The total weight of the remaining ingredients is zero grammes or a few milligrammes at most.
Contrary to food products, vaccine ingredient lists may include items utilised in manufacturing even if they are not present in the final product. There are very trace amounts of additional components (usually a few milligrams). None of the materials needed to make the vaccinations or cultivate the active components may make it into the finished product. If they do, there are simply traces of them.
Ingredients in vaccines can seem strange. However, it's critical to keep in mind that a lot of the ingredients in vaccinations are produced naturally by the body. For instance, many vaccinations contain salts based on sodium and potassium, which are vital for life (see the section on "Acidity regulators"). Although formaldehyde is typically thought of as a chemical produced by humans, it is also naturally present in the circulation in minute amounts.
Vaccine verdicts
There is no evidence to suggest that any of the chemicals in vaccines, which are all present in extremely minute amounts, are harmful. The rare individuals who may be extremely allergic to a vaccine ingredient, even if it is present in minuscule amounts, are the exception to this rule (for example, egg proteins or antibiotics used in vaccine manufacture). Although some of the components in vaccines may be dangerous, the majority of them are present in vaccines in concentrations that are entirely acceptable for human bodies. Even common salt (sodium chloride), which is necessary for the body to operate normally, is dangerous in big doses.
Active components are the components of the vaccination (also known as "antigens") generated from viruses or bacteria. So that it can produce antibodies to attack the disease, they put the immune system to work. Only a few micrograms (millionths of a gramme) of active chemicals are present in each vaccine. One paracetamol pill includes 500 milligrammes of the medication to give you a sense of how little these amounts are. The amount of the active component in this is thousands of times greater than what is present in the majority of vaccines. A single teaspoon of the active ingredient might be used to make hundreds of thousands of unique vaccinations.
The complement system was once inactivated for immunoassays by heating serum to 56°C for 30 minutes. Other unidentified inhibitors of cell growth in culture have also been reported to be rendered inactive by heat activation. However, the procedure is pricy and labour-intensive. As too high of a temperature or for too long of a period may damage some growth factors, the procedure must be strictly followed.
Due to laboratory history or the convenience of keeping only one type of serum, heat inactivation of serum is occasionally utilised.
To inactivate the complement protein present in newborn calf serum, Coriell previously employed only heat-inactivated serum for their cell cultures. Since switching to foetal bovine serum, we have discovered that most cell lines do not require heat-inactivated fetal bovine serum. The majority of lymphoblast and fibroblast lines do not appear to be affected by whether the serum is heat-inactivated or not, yet occasionally we do identify a differentiated cell line that grows better in heat-inactivated fetal bovine serum. The original cell line's serum can be found in the online Coriell catalogues (either heat-inactivated or "not inactivated"). The culture conditions are modified to mirror current practice when we create an expansion slot of the cell line ("not inactivated") for most cell lines.
Bottomline
So to conclude, you can utilise heat-inactivated fetal bovine serum without any issues if your lab typically keeps it on hand. In actuality, the heat-inactivated serum is used in a large number of labs that use Coriell cell lines. And FBS has proven to be the most effective supplement.
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Wondering what even is fetal bovine serum? You have just landed on the right page then! Fetal bovine serum (FBS) is made from blood taken from a cow foetus during the slaughtering using a closed collecting system. The most popular serum supplement for eukaryotic cell in vitro culture is foetal bovine serum. This is because it contains fewer antibodies and more growth factors, making it more adaptable for use in a variety of cell culture applications.
The Fetal bovine serum contains a significant amount of the globular protein known as bovine serum albumin (BSA). Fetal bovine serum's wide range of proteins keeps cultivated cells in a condition where they can live, grow, and divide.
FBS is not a completely specified media component because it is a biological product, and as a result, its composition changes across batches. Serum-free and chemically defined media (CDM) have been created as a result of this to reduce the likelihood of the transfer of accidental agents. Because many cell lines still require serum to thrive and many serum-free media formulations can only support the growth of specific types of cells, the efficacy of serum-free media is constrained.
The meat industry produces FBS as a by-product. FBS is made from blood drawn in industrial slaughterhouses from cattle that also supply meat intended for human consumption, as is the case with the great majority of animal serum used in cell culture
FBS manufacturing
Blood is drawn from the bovine foetus after it has been taken from the dead cow as the initial step in manufacturing FBS. The blood is drawn aseptically into a blood bag or sterile container, where it is left to coagulate. The standard procedure for collection is a cardiac puncture, which involves inserting a needle into the heart. As a result, there is less chance that the surroundings or the foetus itself will contaminate the serum. The residual blood cells and fibrin clot are subsequently separated from the clear, straw-coloured serum using centrifugation. Before the additional processing is required to make the serum acceptable for cell growth, it is frozen.
Filtration is used in the second step of processing, often in the form of a chain of filters with three sterile 0.1-micron membrane filters serving as the final filter. A thorough Certificate of Analysis is provided together with the aseptically processed FBS, which has undergone rigorous quality control testing. The certificate provides comprehensive test findings as well as details on the serum's country of origin. Commercial suppliers issue different certificates of analysis, but they all typically contain the same information: filtration statement, country of blood collection, country of manufacture, cell growth performance testing, and microbial sterility testing, as well as screening for mycoplasma and virus, endotoxin, haemoglobin, IgG gamma-glut amyl transferase and total protein assays. FBS may also be examined for collection country.
Reservations
concerns have been raised. The methods for collecting foetal bovine serum have been the subject of dispute. The comprehensive regulation and procedures used to guarantee that serum is obtained ethically are described in-depth in the literature that has been published by the International Serum Industry Association (ISIA). Although the act of killing the dam and the time that passes during the slaughter process before harvesting will cause the foetus to become unconscious or die before serum harvesting, it has been hypothesised that exposing live unborn calves to oxygen could cause them to become conscious before being killed, sparking an ongoing ethical discussion about the practice.
While the Industry Association acknowledges that some organisations have reservations, they contend that since all serum collections are conducted in registered slaughterhouses that are under the authority of the competent authority in the country of collection, it is highly unlikely that a problem would arise.
Fetal bovine serum (FBS) is most usually employed as the leading growth supplement in insect cell culture. It can create excessive foaming, interact with transfection chemicals, and is expensive, but it increases cell development and offers shear force protection crucial in shake and stirrer cultures.
Due to the great sensitivity of cells cultured in vitro, the foetal bovine serum is commercially accessible from numerous suppliers. Customers typically test certain batches to ensure that they are suitable for their particular cell type. It is customary to adopt the cells to the new batch of material when switching from one batch to another, for instance by combining 50% of the old serum with 50% of the new serum and giving the cells time to adjust to the new material.
To maintain the stability of components like growth factors, a serum is frozen during storage. Some precipitation may be noticed when serum is thawed. This is a typical occurrence, and it has no negative effects on the serum's quality. By transferring the serum to sterile tubes and centrifuging for 5 minutes at 400 g, the precipitate may be eliminated. It is advised that the serum be defrosted in a refrigerator at 2-8 C to reduce the quantity of precipitation.
Compared to non-Fetal serum, the foetal serum has a higher concentration of growth hormones and a lower concentration of gamma globulins, or antibodies. These are crucial because growth factors promote cell survival and growth, whereas antibodies might adhere to cultured cells. Additionally, the prenatal serum has lower concentrations of complement proteins (complements) than adult or neonatal serum. These complements lyse cells in culture and interfere with immunoassays, which are both negative outcomes.
serum and plasma. Coagulation proteins are present in plasma but not in serum, which is how they vary from one another. Anticoagulants are typically added to the blood before centrifugation to make plasma, but the clotting proteins are left in place. Before extensive/progressive centrifugation or by allowing the blood to clot, the serum is prepared. As a result, a serum is devoid of fibrinogen and proteins related to clotting.
They are complement of 22 proteins that make up 99 per cent of the total protein content of serum and plasma in humans. Numerous proteins make up the remaining 1 per cent. At 50 mg/ml, albumin, the most prevalent serum protein, makes up around half of the total protein mass.
Donor Bovine Serum
Bovine donor serum is taken from healthy, living animals that are at least a year old. The donor cattle are meticulously chosen and kept in confined herds, guaranteeing total control over their upkeep, food, and health. All cattle must test zero-negative for Bovine Viral Diarrhoea (BVD) and Infectious Bovine Rhinotracheitis (IBR) before being admitted to the separated herd (BVD). The animals are not medicated or given sub-therapeutic doses of antibiotics; instead, they are employed solely in the production of donor bovine products such bovine serum. To ensure long-term stability and to maintain growth performance consistency throughout its shelf-life, serum products need to be stored and handled properly. Additionally, it is commonly preferred to heat inactivate serum in order to deactivate complement there.
New-born Bovine Calf Serum
Under strict collection and processing guidelines, new-born bovine calf serum is produced from examined, healthy bovine calves that are under 14 days old. In situations when low antibody concentrations are not crucial, it is employed for the routine maintenance of specific cell lines. Fetal Bovine Serum (FBS) can be replaced with New-born Calf Serum (NBCS), which is derived from 3- to 10-day-old calves (FBS). The complement system, antibodies, and other active enzymes are rendered inactive during the heat inactivation process.
Bovine Serum
Protein obtained from cows called bovine serum albumin. In laboratory research, it is frequently used as a reference for protein concentration.
The BSA precursor protein is 607 amino acids (AAs) long in its whole form. The precursor protein is separated from an N-terminal 18-residue signal peptide during secretion, leading to the first protein product's 589 amino acid residues. The mature BSA protein, which has 583 amino acids, is produced by cleaving an additional six amino acids.
In the past, serum was heated to 56°C for 30 minutes to deactivate the complement system for immunoassays. However, the procedure is pricy and labour-intensive. As too high of a temperature or for too long of a period of time may damage some growth factors, the procedure must be strictly followed.
Since switching to foetal bovine serum, we have discovered that most cell lines do not require heat inactivated fetal bovine serum
Bottomline
So to conclude, considering the above factors the major drawback of FBS is the possibility of hazardous pathogens such as prions, viruses, Mycoplasma, or other unexplained zoonosis contaminating FBS/media and triggering severe immune reactions following MSC therapy is one of the main worries.
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The blood component called serum is what remains of the cells after coagulation and elimination. In addition to serum proteins, it also contains hormones, amino acids, and growth factors. The serum is one of the most crucial supplements for promoting cell development and proliferation in vitro cell culture because of its comprehensive mixture. Fetal bovine serum is very interesting (FBS).
It is particularly high in growth factors and low in antibodies, which could affect the effectiveness of cell culture.
Activated charcoal is used to completely remove free, low molecular weight hormones and proteins from charcoal-stripped plasma and serum. This method does not guarantee the removal of hormones linked to proteins.
Studies on hormone-responsive malignancies typically use a charcoal-stripped fetal bovine serum (CS-FBS) to establish hormone-free cell culture conditions. Although the underlying mechanisms are still unknown, CS-FBS may have an impact on the development of cancer cells. When examining androgen responsiveness and androgen metabolism in cultured prostate cancer (CaP) cells, charcoal-stripped fetal bovine serum (CS-FBS) is frequently utilised.
Without eliminating nutrients, charcoal-stripped FBS eliminates nonpolar molecules such as viruses, certain growth factors, hormones, and cytokines (salts, glucose, amino acids). Highly effective for studies on hormones, cancer, cell signalling, medication response, etc.
The process of filtering fetal bovine serum (FBS) through an activated carbon-absorbent filter reduces the quantities of steroid hormones such as estradiol, progesterone, cortisol, testosterone, T3, and T4 without losing other serum components in an unintended way. FBS Charcoal Stripped helps research processes affected by steroid hormones (oestrogen stimulation, obesity process), as well as other research areas that profit from low hormone levels, like particular kinds of viral infections.
Widely utilised in eukaryotic cell culture, fetal calf or bovine serum is typically added to growth media as a supplement. A good cell culture depends on the nutritional and growth-promoting components found in fetal bovine serum. Additionally, the fetal bovine serum has a variety of tiny molecules, including hormones, lipids, carbohydrates, and amino acids.
The blood component obtained following coagulation and the removal of cellular components is called fetal bovine serum (FBS). FBS is one of the most crucial supplements for promoting cell growth and proliferation in vitro cell culture because of its distinctive and thorough mix.
A 12,000 to 14,000 Dalton cutoff membrane is used to dialyze pre-selected fetal bovine serum, also known as fetal calf serum (FCS), versus physiological saline. Throughout the operation, the glucose concentration is carefully monitored to keep the process under control. Serum peptide precipitation and inactivation are not prevented by exhaustive dialysis.
Storage and Handling
FBS - Dialyzed 12-14 kD is provided in sterile, gamma-irradiated PETG or PETE bottles. We advise keeping the serum refrigerated between -5 and -20 degrees Celsius. The serum should not undergo repeated freeze-thaw cycles since this could cause the product's quality to decline. We advise using one of our smaller container sizes or separating the serum into smaller aliquots suitable for single-use if occasional consumption of the product is anticipated. When handling the serum, always use aseptic procedures, and a liquot into sterile containers.
Precaution
Safe laboratory practices and personal protective equipment should be used when handling biohazardous materials like human cells.
Wearing protective gear is advised.
Limitations
Not to be used in diagnostic procedures; only for laboratory research use.
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For both routine cell culture and particular research applications, Fetal Bovine Serum - Premium, Heat Inactivated is a low-endotoxin, low-hemoglobin FBS that offers exceptional value. FBS of the highest quality is frequently utilised and exhibits great performance with a variety of cell types.
Heat inactivation aims to eliminate complement activity in serum without damaging the product's growth-promoting properties. Most cell cultures do not require the removal of complement activity from the serum, but cultures that are sensitive to the activity could need it.
Numerous immunological components, including serum complement, which under certain circumstances may inhibit or kill cells, are present in adult serum. The goal of heating serum is to deactivate serum complement.
FBS is kept at a temperature of 56° for 30 minutes as part of the heat inactivation (HI) process.
The efficacy of the serum will be negatively impacted by heating it for more than 30 minutes or to a temperature greater than 56°C, and this will almost certainly result in more cryo-precipitate forming.
The goal is to neutralise any complement system components found in FBS as well as other potential unidentified cell growth inhibitors. Before, mycoplasma contamination was also meant to be removed by heat-inactivation, but this is no longer a concern because all serum products are now filtered through pores tiny enough to do away with mycoplasma. It should be emphasised that heat-inactivation could also have unintended consequences, such as lowering cultivated cells' capacity to adhere to surfaces.
For the majority of cell culture needs, several vendors have advised customers against heat-inactivating FBS. Since various cells react differently to the heat-inactivation of FBS, it is always advisable to assess if heat inactivation is necessary for a given application
FBS has several different applications. FBS has numerous crucial nutrients and growth factors that help cells survive and proliferate in eukaryotic cell culture, where it is used at concentrations of up to 20 per cent or even more. Notably, FBS in human cell cultures has the potential to introduce research artefacts; human cells cultured with human sera exhibit distinct behaviours from those cultured with FBS. It is utilised in the development, production, and control of human and veterinary vaccinations as well as biotech pharmaceuticals. Serum-free cell culture mediums have been in use for a long time.
After blood is extracted from a cow foetus, it coagulates, leaving behind FBS. It is the serum supplement that is most frequently used for in vitro cell culture (for eukaryotic cells). It also has uses in stem cell research and is crucial for the study of both human and animal vaccinations.
Steps of Heating the Serum
· Charcoal therapy
These serum lipid hormones frequently obstruct insulin detection techniques and immunoassay platforms. Tumour cell growth in culture conditions supplemented with 10% charcoal-stripped serum was measured by L Zhao et al.
· Dialysis
All compounds in FBS with a molecular weight of less than 10,000 MW can be eliminated through dialysis. Both polar and non-polar molecules fall within this category. The removal of hormones, cytokines, carbohydrates, amino acids, and numerous other substances. Antibiotics and other foreign compounds in FBS are also eliminated by dialysis.
· Gamma-irradiation
Sterilization of FBS may involve gamma irradiation. To remove bacteria, FBS is typically filtered through 0.1 um filters several times. The viruses frequently seen in bovine species can be rendered inactive by gamma radiation. However, some viral species, like the parvovirus, are gamma-irradiation resistant.
· Poor IGG
Even while the IGG content of FBS is fairly low, it can still be too high for some applications. By using capture chromatography, the IgG level can be further, significantly lowered. For the creation of recombinant proteins, the generation of antibodies, and other uses, FBS with low IgG is employed.
There are strict growth factor requirements for stem cell cultivation. Some of the growth factors in FBS encourage the development of stem cells. Numerous businesses provide FBS suited for maintaining undifferentiated embryonic stem cells. It's crucial to evaluate particular FBS lots to see if they can sustain the totipotency or pluripotency of various stem cell types.
Bottom Line
So to conclude, the immune analysis of SARS-CoV2 antibodies is hampered by the 30-minute heat inactivation of serum at 56°C. If the sample was inactivated by heating, the likelihood of false-negative results should be taken into account for highly suspected cases. Additionally, the heat inactivation procedure must be carried out carefully, just like all other protocols using FBS, because overheating or extended heating will inactivate growth factors and produce precipitates
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