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A recent misconduct with fetal bovine serum demands to call again for serum-free cell and tissue culture applications

In 2011, GE Healthcare, a unit of General Electric Co., acquired PAA Laboratories, Linz, Austria. Earlier this year, GE Healthcare published an “Urgent Field Safety Notice”, stating that lots of FBS produced at PAA facilities from March 2008 to March 2013 are subject to label non-conformances. These products may contain added adult Bovine Serum Albumin (BSA) of United States origin, water, and/or cell growth promoting additives. For FBS product shipped into countries other than the United States, current product labeling states that the origin of the product is either Australia or EU approved serum sources. In addition to, or instead of product of this origin, the product may contain adult BSA of United States origin and/or may contain FBS from sources including United Sates, Canada, Argentina, Brazil, and/or Mexico.

   This fraudulant action prompted us to inform and to alert the cell culture community!

   Fetal bovine serum (FBS) is a universal growth supplement of cell and tissue culture media. FBS is a natural cocktail of most of the factors required for cell attachment, growth, and proliferation, effective for most types of human and animal (including insect) cells. Although in use for more than 50 years, FBS has never been fully characterized. Recent proteomic and metabolomic studies revelead approx. 1.800 proteins and more than 4.000 metabolites present in serum. However, the use of serum in cell culture also bears a number of disadvantages. These disadvantages can either be seen from: (a) a scientific, cell biological point of view, since serum in general is an ill-defined mixture of components in culture media, with qualitative and quantitative, geographical and seasonal batch-to-batch variations, (b) from biosafety aspects, since FBS may contain adverse factors, like endotoxin, mycoplasma, viral contaminants or prion proteins, (c) from ethical perspectives in terms of animal protection arguments regarding the harvest and collection of FBS from bovine fetuses, and (d) in terms of recent concerns about the global supply vs. demand of FBS. As a consequence, a number of strategies were developed to reduce or replace the requirement for FBS in cell culture media.

   FBS is a by-product of the beef packing industry. Thus, the supply is dictated by many factors, including beef consumption, dairy product consumption, feed prices, environmental factors such as drought, cattle import and export, governmental farm policies, and the outbreak of diseases (foot and mouth disease, BSE) [1-3]. The availability of FBS has changed dramatically over the past few years [4]. Therefore, all efforts and attempts should be undertaken to overcome the expected shortfall in FBS supply. In the case of FBS, supply vs. demand models do not follow typical economic principles. Normally, supply can be adjusted to meet the demand. However, in case of FBS, supply and demand operate independently of each other. In addition, there is a severe geographical mismatch between the supply of and the demand for FBS. Demand is highest in U.S. and Europe, while the major sources of FBS are far away – in Brasil, Argentina, South Africa, Australia, New Zealand, and Central America, since in those countries huge meat cattle herds – bulls and cows – roam freely together, and as a result, many cows are pregnant at the time of slaughter [5,6]. The same holds for the geographical distances between raw serum producers and FBS processors. The latter are also mainly located in the U.S. and in Europe. It is estimated that approximately 500.000 litres of FBS are sold per year, which means that more than 1,000.000 bovine fetuses have to be harvested. And the numbers are still increasing.

   The FBS market is only loosely regulated [7-9]. That creates the opportunity for abuse. And abuses have occurred and happen until now. In 1994 it was reported [9], that approx. 30.000 litres of “New Zealand” serum was sold worldwide. However, only 15.000 litres of high-quality FBS were annually collected in New Zealand. Thus, we still do not know whether FBS in general might be blended with newborn or adult bovine serum to meet the quality and/or the increasing demands. And in the past, no attempts have been undertaken to trace the origin of the collected sera by approved methods, e.g. analysis of stable isotopes in the sera, which would give the unequivocal evidence about the geographical origin of the raw serum.

   Now, a most recent case of fraud with FBS came up. As pointed out above, lots of FBS were blended with bovine serum albumin, water and growth promoting additives. FDA, the U.S. Food and Drug Administration reports, that 143 batches of FBS produced within the last 5 years with a total of approx. 280.000 litres are affected [10].

   Obviously, in the last 20 years nothing has changed [7-9]. This latest incident of false FBS supply and quality on the world market might be just the tip of the iceberg. Most importantly, the actual case might also have a substantial impact on thousands of cell and tissue culture experiments, which can hardly be ignored!

   Furthermore, this recent incident should be taken as an opportunity to question the future use of FBS in cell culture media and/or to increase quality and safety of those sera, which are still in use, e.g. because of directives in the vaccine production. We therefore appeal to cell and tissue culturists to reduce or completely avoid FBS in their cultures, e.g. by alternatives to FBS, like serum-free cell and tissue culture [2,5,11,12], or the replacement of FBS by the use of serum substitutes [13]. Especially, cultures that will be initiated in the future, like induced pluripotent stem cells (iPS) (http://www.stembancc.org), should be grown from the very beginning under serum-free culture conditions. In 2003 and 2009, cell culture experts from all over Europe gathered at two Workshops discussing options for, and methodologies of serum-free cell culture. Two comprehensive Workshop Reports [14,15] were published, in which clear recommendations for a replacement of FBS, and the design of serum-free media, respectively, are provided. Following those recommendations will result in scientifically better results, safer products, contribution to ethical research without harming animals, and better availability of cell and tissue culture media and components with transparent and traceable composition.

* An earlier version of this Letter is published in Cytotechnology 65(5), October 2013.

References

  1 Dormont, D. (1999) Transmissible spongiform encephalopathy agents and animal sera. Dev. Biol. Stand. 99, 25-34

  2 Even, M.S. et al. (2006) Serum-free hybridoma culture: ethical, scientific and safety considerations. Trends Biotechnol. 24, 105-108

  3 Wessmann, S.J. and Levings, R.L. (1999) Benefits and risks due to animal serum used in cell culture production. Dev. Biol. Stand. 99, 3-8

  4 Fujimoto, B. (2002) Fetal bovine serum – supply vs. demand? Art to Science 21, 1-4

  5 Brunner, D. et al. (2010) Serum-free cell culture: The serum-free media interactive online database. ALTEX 27, 53-62

  6 Jochems, C.E. et al. (2002). The use of fetal bovine serum: Ethical and scientific problem? ATLA 30, 219-227

  7 Hodgson, J. (1991) Checking the sources: the serum supply secret. Nature Biotechnol. 9, 1320-1324

  8 Hodgson, J. (1993) Fetal bovine serum revisited. Nature Biotechnol. 11, 49-53

  9 Hodgson, J. (1995) To treat or not to treat: that is the question for serum. Nature Biotechnol. 13, 333-343

10 FDA, (2013) U.S. Food and Drug Administration; http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfRes/res.cfm?ID=117863

11 Gstraunthaler, G. (2003) Alternatives to the use of fetal bovine serum: serum-free cell culture. ALTEX 20, 275-281

12 Gstraunthaler, G. und Lindl, T. (2013) Zell- und Gewebekultur. 7^th Ed.,  Spektrum Springer Verlag, Heidelberg

13 Rauch, C. et al. (2011) Alternatives to the Use of Fetal Bovine Serum: Human Platelet Lysates as a Serum Substitute in Cell Culture Media. ALTEX 28, 305-316

14 van der Valk, J. et al. (2004) The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture. Toxicol. In Vitro 18, 1-12

15 van der Valk, J. et al. (2010) Optimization of chemically defined cell culture media – Replacing fetal bovine serum in mammalian in vitro methods. Toxicol. In Vitro 24, 1053-1063

Gerhard Gstraunthaler¹,Toni Lindl², and Jan van der Valk³

¹Division of Physiology, Innsbruck Medical University, Innsbruck, Austria, ²Institut für Angewandte Zellkultur, Munich, Germany, ³3Rs – Center Utrecht Life Sciences, Utrecht University, Utrecht, The Netherlands