Serum is the centrifuged fluid component extracted from either clotted or defibrinated whole blood. Bovine serum comes from blood taken from domestic cattle.
Whilst the procedure of making serum may seem to be straightforward, the processing of serum takes place under very tightly controlled conditions. The process has been carefully developed and uses sophisticated facilities and equipment, and is accompanied by extensive testing particularly stringent when processed bovine serum is intended for use in the production of medicinal products.
Bovine serum is a by-product of the meat industry. Bovine blood may be taken at the time of slaughter, from adult cattle, calves, very young calves or (when cows that are slaughtered are subsequently found to be pregnant) from bovine fetuses. It is also obtained from what are called "donor" animals which give blood more than once.
Blood is available from bovine fetuses only because a proportion of female animals that are slaughtered for meat for human consumption are found (often unexpectedly) to be pregnant.
Bovine serum is categorized according to the age of the animal from which the blood was collected as follows:
Fetal Bovine Serum (FBS) comes from the blood drawn from a bovine fetus via a closed system of collection at the slaughterhouse. Fetal Bovine serum is the most widely used serum supplement for the in vitro cell growth factors because of its high content of embryonic growth promoting factors.
There is a wide range of applications for fetal bovine serum. The most important is in the field of biopharmaceuticals and vaccines. It is used in the research, manufacture and control of human and veterinary vaccines and of drugs, many of which are at the cutting edge of drug development.
Fetal bovine serum is also used extensively in research. A technique known as "Cell culture" is widely applied in the manufacture of both vaccines and bio-pharmaceuticals in which bovine serum is broadly used.
Maintaining cells in vitro in a healthy condition and over time is a complex task. They will only survive, grow and multiply if they are well fed and provided with an appropriate and protective environment. Complex mixtures of substances ("media") are used to bathe the cells in order to both feed and protect them. Different cells have different requirements. In many instances the presence of serum in the mixture is essential if the cells are to grow adequately and normally. Bovine serum is much the most widely used, because high quality bovine serum is available in sufficient volume and has been found to support cell growth very well indeed.
FBS is collected from the fetuses of pregnant cows, slaughtered in compliance with the OIE (World Animal Health Organization) guidelines and internationally accepted standards of veterinary inspection.
With more than 25 years of experience with serum as its main activity, Biowest is dedicated to quality and service; to vertical integration, and providing a wide range of origins.
Biowest takes full responsibility of ensuring a vertically integrated supply chain to the highest standard of quality, traceability and safety. In 2004, Biowest was acquired by the Viking/Serascandia group to become the first vertically integrated serum company. In the same year, Biowest denounced cases of misrepresentation to the authorities and informed the affected victims; highlighting the importance of serum industry traceability guidelines and oversight, and promoting the creation of the International Serum Industry Organization.
The choice of the FBS supplier is based on Quality, Traceability and Safety along the whole supply chain, endorsed by comprehensive documentation; backed up by audits of the supply chain back to the serum source; and covering several years.
The OIE (World Organization for Animal Health) is an intergovernmental organization created in 1924.
In 2011, the OIE totaled 178 Member Countries and Territories.
Its missions are:
The serum's source is from a USDA Approved country. This means that serum is produced from blood collected in countries that have been approved by the United States Department of Agriculture (USDA) to export ruminant serum products to the United States. Eligible countries that export fetal bovine serum into the U.S. include: Australia, Canada, Chile, Costa Rica, El Salvador, Guatemala, Honduras, Mexico, New Zealand, Nicaragua, and Panama.
Some cattle viruses can cross the placenta of the cow and infect the calf fetus, thus contaminating FBS and potentially making it unsuitable for use in cell culture. Some of these cattle viruses have a limited distribution and only exist in certain parts of the world. These are viruses of concern when importing FBS from infected countries - See Table of Diseases of Importation Concern for FBS. Other cattle viruses exist in all countries, and regardless of country of origin, the FBS must be tested or treated to assure freedom from these and other viruses (See USDA 9 CFR 113.53 and EMEA-CPMP-BWP-1793-02). The treatment of choice for most FBS is gamma irradiation at 25-45 kGy to guarantee freedom from viruses.
FMD is relevant for regulatory import purposes but not for cell growth. The USA and a few other countries only allow FBS imports from countries free from FMD, WITHOUT vaccination while most other countries also accept FMD-free WITH vaccination. Both types of FMD-free countries are free of FMD outbreaks and circulating FMD viruses, as verified by the OIE and declared equally safe.
The serum's origin has no influence on cell growth. Biowest has compared cell growth in FBS from seven different countries on three continents, and confirmed that regardless of the country of origin, all cell lines tested had the same average performance. One batch of FBS may work well for one specific cell line, but not for another. "Serum quality" is specific for each cell line. That is why testing of FBS is widely used when dealing with sensitive cell lines. Biowest performs the most extensive analysis of biochemical parameters and testing on cell lines in the industry, making available the results on the Certificates of Analysis.
Australian FBS prices are higher because Australia is perceived to be a "safer" origin for BSE and cattle viruses. Canada and Australia were the first countries outside the USA where FBS was produced. In the 1980s, BSE cases that occurred in North America became a strong argument in favor of Australian FBS. The point was made that being "isolated", Australia was necessarily the most secure origin.
However, BSE has never been found in young animals, let alone in fetuses, and BSE transmitted by animal feed has been eradicated. The remaining BSE cases are the spontaneous BSE events, which occur in all cattle populations (Stanley B. Prusiner, Nobel Prize 1997). All countries find spontaneous BSE cases if their sanitary surveillance systems are sensitive enough. Additionally, the OIE veterinary authorities have declared all blood and blood product, regardless of country of origin, to be safe in terms of BSE. So, the BSE arguments in favor of Australia were clearly ill founded from the beginning, and have become obsolete.
Nevertheless, Australian FBS is still sold as "safer", at much higher prices than other origins. Paradoxically, Australia is actually among the countries with the most types of viruses of import concern, including Blue Tongue, Akabane, Aino, and Bovine Ephemeral Fever (see FAQ #13). Like in the tale "The Emperor's new clothes", by Danish writer Hans Christian Andersen, those who believe that paying more will assure them a better serum, are being misled.
A huge price difference exists between countries classified as "FMD-Free-without-vaccination" for which demand exceeds supply, and countries listed as "FMD-Free-with-vaccination" where supply historically has exceeded demand. The price difference has been several hundred per cent, whereby serum users in the US have paid billions of USD more than serum users in the EU. Proposals from USDA to harmonize US and EU import rules for FBS relating to FMD (following OIE guidelines), have repeatedly been turned down by the US serum industry.
Wrong beliefs about risks associated to BSE have lead some FBS providers to promote the Australian origin as "safer" without any scientific basis, making this origin several times more expensive than any other. The Australia "safer" status is being promoted, despite the virus status of Australia (see FAQ #13); and despite the higher risk of misrepresentation associated with such huge price differences.
The price differences persist due to longtime marketing efforts promoting certain origins, and regulations limiting trade, even among countries that according to the OIE's classification have identical veterinary risk status (regarding viruses of import concern for FBS). ISIA has among its objectives the education of serum users, and EU and USA are working to harmonize trade rules, all of which will contribute to harmonizing price levels for most FBS origins.
FBS Imports into the EU are less restricted than in the USA. A proposal made by USDA in 1994 to apply the same FBS import policies as the EU did not get support from the US FBS industry. The International Serum Industry Association (ISIA) supports efforts to harmonize import rules, suggesting compliance with the OIE recommendations. In the meantime, FBS in the USA remains more than twice as expensive as in Europe.
The availability of FBS is diminishing. Meat and calf prices are increasing as consumers in some countries increase the consumption of beef; and as cattle breeding techniques are permanently improving. These combined factors lead to a reduced frequency of pregnant cows at slaughter. The collection of FBS in new countries has, for many years, compensated for the decrease in supply; but now there are no more new producer countries available. The present world supply of FBS is short and will continue decreasing.
The demand for FBS is growing along with the latest research developments, including stem cells, cell therapy, new vaccines and proteomics.
For most cell culture applications, heat inactivation of serum is not recommended. Heat inactivation degrades complement proteins that may interfere with immunological assays. Heating serum for prolonged periods of time can reduce or destroy growth factors, as well as increase the formation of deposits which are commonly mistaken for microbial contamination.
. The stirring is crucial for good serum quality. If the serum is not correctly mixed, a precipitant of salts, proteins and lipids will appear, and allow crystalline and flocculation deposits to form. These deposits are not toxic for cell culture but affect the serums appearance and consistency.
. Heating the serum for long periods can destroy the growth promoters, which can also enable deposits to form.
FBS is sometimes gamma irradiated to eliminate all potential virus risks.
At standard dosage of gamma irradiation, no loss of performance is observed.
The sampling for sterility tests of all Biowest products conforms to the European Pharmacopeia requirements.
Biowest tests the serum for the viruses (BVD, IBR, PI3) by using a cell culture method, so that live viruses, if present, are detected in the serum.
A simple PCR test, not combined with the cell culture test, is used to detect DNA and RNA viruses that are alive and dead.
Bovine spongiform encephalopathy (BSE), more commonly known as "mad cow" disease, is a fatal neurodegenerative disease (encephalopathy) in cattle.
According to the OIE (World Animal Health Organization), there is no risk of BSE being transmitted in several products, including: milk, hides and skins, deboned meat, blood and blood by-products, such as fetal bovine serum (OIE Terrestrial Code 11.4 BSE). Most OIE member countries have adopted this position, including the European Union and the United States (USDA).
For some time, BSE was argument used in favor of certain origins. After OIE has determined that BSE is not transmitted by blood products, these arguments have become obsolete.
FBS is essentially a natural product. All FBS batches are unique, with specific characteristics, different from all other batches.
The serum should be stored frozen and protected from light. The recommended storage temperature is -10°C to -40°C. At temperatures below -40°C, the bottles may become brittle increasing the risk of breakage.
Serum may be stored between +2 to +8°C for up to 8 weeks without diminishing its performance (*).
Internal studies in Biowest have shown that during 26 weeks of storage (between +2 and +8°C) the serum's capacity to promote cell growth was not significantly altered.
(*) Art to Science, Vol.19, No.2, "Serum Stability at Refrigerated Temperatures (2-8°C).
Turbidity and flocculent material may be present after thawing or after prolonged freezing and/or refrigeration. Our experience indicates these modifications do not affect the biological performance of the serum.
Our collection procedures and rapid freezing may leave some fibrinogen in the serum. Fibrinogen is the soluble precursor of the clot-forming protein, fibrin. The fibrin may appear after thawing or heat inactivation. Fibrin does not alter the capacity of the serum to promote cell growth. It is recommended to use the serum without treatment (filtration or centrifugation).
Serum that is incubated at 37°C for extended periods will become cloudy and deposits may appear. They are composed of calcium and phosphorous. To the best of our knowledge, this does not alter the performance of the serum in cell cultures.
Remove the serum from frozen storage and let it thaw overnight at room temperature. The following day, slightly agitate the serum to homogenize it without making it foam.
The agitation is the key to avoid the formation of crystalline or flocculent deposits.
If a deposit does appear, it is not toxic to cell cultures. It only affects the appearance and consistency of the serum.
Except where notified on product labels, Biowest sera and liquid media are sterile filtered. Sterility is not guaranteed after opening. The use of aseptic manipulation techniques is recommended.
In addition to normal testing, Biowest also tests FBS for stem cell work suitability. However, it is recommended that batches used for stem cells be tested on specific cell lines for the specific conditions required. Dr Yamanaka in his Nobel Prize winning work related to stem cell research, selected Biowest FBS after testing our batches.
Some preparations of trypsin contain phenol red. Since the product is shipped with dry ice, there could be a significant CO2 build up in packaging. This CO2 may enter the solution and lower the pH slightly, giving an orange (around pH 6.5) vs. pinkish (around 7.3) color. The solution, if orange (acidic) can still be used as sodium hydroxide may be added to adjust the pH.
Traceability is important because it provides an unbroken chain of identity of the product from the country and slaughterhouse of origin, to the processing, packaging and labeling facilities ready for sale and final distribution.
Biowest completely controls the supply chain from harvesting the raw material to filling sterile bottles.
In April 2012, Bureau Veritas certifies compliance with the ISIA guidelines.
Our traceability with SAP management software: SAP Business One guarantees complete product traceability, from raw material until the end product. Playing a key role to complete supply network traceability - enabling Biowest to determine batch genealogy and global traceability.
The slaughtering of pregnant animals is, in most cases, an unplanned event. The frequency depends on farming techniques. In most cases, the condition of gestation is unknown; especially where animals are produced naturally; in the case of cattle moving freely around, grass fed, heifers and steers together. No cows are killed with the main purpose of harvesting the fetal blood. The blood is collected as a by-product from cattle slaughter operations for the bovine meat. The pregnancy condition is unknown when an animal enters the slaughtering line. The percentage of pregnant cows going to slaughter is very low and decreasing year by year as cattle farming techniques constantly improve.
The collection of the blood is done without causing pain to the fetus. The fetus's life ends through oxygen depletion, generally agreed to be a gentle method of life termination. Cell culture technology can reduce the use of live animals in science. Research is also being done to produce meat by cell culture, which could ultimately lead to the end of breeding animals for food.
Given that the death of some pregnant animals is unavoidable, the fetus can be used to serve science, and generate jobs. This contribution benefits research and development and cannot be underestimated.
Since the 1990s, from time to time, cases of illegal practices have been rumored. Only two cases are known to have been reported to authorities. In 2004, Biowest realized we had purchased and resold misrepresented origins; and in 2013, GE realized it had been selling misrepresented and adulterated FBS products, after acquiring the company PAA with activities in Austria, Canada and Australia. In both cases affected victims were informed, and product was recalled. Other cases are known to be under investigation. These cases illustrate the importance of having complete control of the supply chain.
FBS can be adulterated by the addition of water, growth factors and/or serum of other species. Unscrupulous companies in order to enhance performance and/or lower the cost of the product can carry out one or more of these or similar activities. These practices are extremely serious as they affect critical results of life science research, and the purity of final products obtained in biopharmaceuticals, diagnostics, or vaccines. When such cases are detected, victims having used adulterated product must be informed to make it possible to evaluate the damage and take corrective actions.