FBS and Other Animal Components Used in Cell Culture

To maximise the quality and reproducibility of cell culture research, the components within cell culture media should be defined. These components include serum, hydrolysates, growth factors, hormones, proteins, peptides, lipids, adhesion factors, and amino acids.

On this page:
Fetal Bovine Serum
Transitioning to Serum-Free Media
Other Cell Culture Components
Companies that Sell Serum-Free Media or Cell Culture Supplements

Foetal Bovine Serum
Foetal bovine serum (FBS) is composed of an undefined mixture of macromolecules, including hormones, transport proteins, growth factors, lipids, minerals, elements, and detoxifying factors. As a supplement for cell culture media, FBS is intended to maintain cell viability and facilitate cell metabolism, growth, proliferation, and spreading in culture. It’s obtained from the blood of foetal calves. When pregnant cows are slaughtered, a large-gauge needle then is used to draw the blood out of the beating heart of the foetus.^1,2 It is estimated that 600,000 litres of FBS – obtained from up to 1.8 million bovine foetuses – are produced worldwide each year.³

In addition to ethical issues, FBS use is accompanied by batch-to-batch variability in biomololcue composition, potentially unexpected or undesirable outcomes, risk of contamination, and potential shortages. (See “FBS: Scientific and Availability Limitations” below.) Chemically defined, serum-free media or human platelet lysates can replace FBS in cell culture media. For optimal definition and reproducibility, a chemically defined, animal-free medium that avoids all animal-derived supplements should be used.

Types of media

From Chary A, Groff K, Stucki AO, et al. Maximizing the relevance and reproducibility of A549 cell culture using FBS-free media. ALTEX. 2022;83:105423.

Click here to download a printable version of a factsheet on alternatives to FBS in cell culture applications.

Click here to view a 2019 webinar on replacing foetal bovine serum in cell culture media while maintaining robust cell growth and cellular functions. Presentations are by Dr Jan van der Valk (3Rs-Centre Utrecht Life Sciences, Utrecht University), Dr Carol Treasure (XCellR8), and Dr Sandra Coecke (European Commission Joint Research Centre, EURL ECVAM).

FBS: Scientific and Availability Limitations
At workshops held between 2003 and 2016, the following limitations of serum and ways to replace FBS in in vitro assays were discussed.^4,5,6

• Variability: The complex mixture in FBS causes significant batch-to-batch variation. Each batch has an undefined composition of biomolecules (in which, according to proteomic and metabolomic studies, an estimated 1,800 proteins and more than 4,000 metabolites are present).⁷ The qualitative and quantitative differences in proteins between lots may be due to geographical and seasonal variation and may also explain discrepancies among results from in vitro studies that use FBS as the cell culture supplement.^6,8
• Unexpected and undesired outcomes: Cell culture media can affect cell morphology and functionality and can favour certain cell pheno- and genotypes. The presence of FBS in media may induce phenotypic changes that can potentially complicate the analysis of data obtained from cultured cells and reduce human relevance. Transitioning cells to defined media reduces experimental variability and, depending on the media and cell type, could facilitate the growth of cells that more closely resemble an in vivo phenotype. For example, it has been shown that serum can suppress TGF-β1, thus preventing chondrogenesis in fibroblast-like type-B synoviocytes.⁹
• Risk of contamination: The use of FBS or other animal-derived substances is especially problematic in the manufacture of biologics for human therapies due to the risk of contamination by animal proteins or pathogens.⁷ In this context, FBS poses a biosafety risk, as it is possible for exogenous agents (i.e. endotoxins, mycoplasma, or viral particles) to contaminate cultured cells or for bovine proteins to contaminate biologics.^1,8,10
• Potential shortage: Due to the extensive use of FBS in vaccine development, research laboratories, and drug production, there is a potential for future shortage.^3,6

Transitioning to Serum-Free Media
When culturing cells, researchers should consider the following:

• For some cell types, serum-free media have already been developed, and their components are published in the literature or available commercially. (See below for a list of companies.)^11–16
• For cell lines in which serum-free media have not been developed commercially or published in the literature, researchers will need to obtain a basal medium, optimise the concentration of supplements, and transition the cells to the new medium. Guidance on this process is available in Rafnsdóttir 2023 and van der Valk et al. 2010.^6,17 Researchers can test FBS-free media in parallel with their currently used medium containing FBS to identify an equivalent chemically defined medium or a medium that induces cells to have more human-relevant characteristics. Afterward, the concentration of media supplements used should be published in order to facilitate their use in other laboratories.
• Although some cell lines can be transferred directly to a different medium, cells normally must be gradually transitioned to FBS-free conditions in order to reduce stress and provide time for the cells to adjust to the new environment.^6,18,19 Cells are often cultured in decreasing dilutions of the original medium containing FBS in order to transition to the new medium.^15,16,20
• When validating tests for regulatory use, animal component–free media should be incorporated into any new validation efforts or test guidelines (TG) using cells cultured in vitro. The Organisation for Economic Co-operation and Development has published TGs that use cells maintained in serum-free media, including several involving reconstructed human tissues (e.g. TGs 431, 439, 442D, and 492), and a project to transition cell lines used in TG 442E to animal component–free media is ongoing.^21–25 Numerous three-dimensional reconstructed human tissues, such as those from MatTek, Epithelix, and SkinEthic, are maintained in serum-free media. Furthermore, the EU Reference Laboratory for Alternatives to Animal Testing presubmission form for tests asks applicants to justify why animal-derived serum is used and explain why they are or are not considering possible replacements.²⁶

Other Cell Culture Components
In addition to FBS, there are other commonly used cell culture components derived from animals that should be replaced with chemically defined components or components of human origin:

• Bovine pituitary extract (BPE) is an undefined mixture of growth factors that enhances cellular proliferation and helps maintain cells’ phenotype. Depending on the application, it can be eliminated or replaced by supplementation with other proteins or coculturing cell types.²⁷
• S9 fraction induces metabolic activity in cultured cells. Historically derived from the livers of rats, it can now be obtained from human livers and cell cultures and is sold by companies such as Scinora and Ewomis.
• Dissociation reagents derived from animals, such as trypsin and Accutase, are often used to detach adherent cells from culture plasticware. Non-animal options include TrypLE, DIMX, TrypZean, STEMCELL Technologies Animal Component-Free Cell Dissociation Kit, and CellPrime rTrypsin.
• Extracellular matrix (ECM) products include Matrigel, which is made by inducing tumours in mice, has undefined compomponents and batch-to-batch variability. Numerous companies offer non-animal ECM, such as denovoMATRIX, PeptiMatrix, Obatala, MilliporeSigma, and UPM Biomedicals.

Companies That Sell Serum-Free Media or Cell Culture Supplements

For additional FBS-free media formulations and products, see the Fetal Calf Serum-Free Database. Disclaimer: A number of the companies below sell animal-derived products in addition to alternatives to FBS. PETA Science Consortium International e.V. does not endorse any of the organisations listed.

• REFERENCES

  ¹Brunner D, Frank J, Appl H, Schöffl H, Pfaller W, Gstraunthaler G. Serum-free cell culture: The serum-free media interactive online database. ALTEX. 2010;27(1):53-62. https://www.ncbi.nlm.nih.gov/pubmed/20390239.

  ²Rauch C, Feifel E, Amann EM, et al. Alternatives to the use of fetal bovine serum: Human platelet lysates as a serum substitute in cell culture media. ALTEX. 2011;28(4):305-316. doi:10.14573/altex.2011.4.305

  ³Brindley DA, Davie NL, Culme-Seymour EJ, Mason C, Smith DW, Rowley JA. Peak serum: Implications of serum supply for cell therapy manufacturing. Regen Med. 2012;7(1):7-13. doi:10.2217/rme.11.112

  ⁴van der Valk J, Mellor D, Brands R, et al. The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture. Toxicol Vitr. 2004;18(1):1-12. doi:10.1016/j.tiv.2003.08.009

  ⁵van der Valk J, Bieback K, Buta C, et al. Fetal Bovine Serum (FBS): Past – Present – Future. ALTEX. 2018;35(1):99-118. doi:10.14573/altex.1705101

  ⁶van der Valk J, Brunner D, De Smet K, et al. Optimization of chemically defined cell culture media – Replacing fetal bovine serum in mammalian in vitro methods. Toxicol Vitr. 2010;24(4):1053-1063. doi:10.1016/j.tiv.2010.03.016

  ⁷Gstraunthaler G, Lindl T, Van Der Valk J. A plea to reduce or replace fetal bovine serum in cell culture media. Cytotechnology. 2013;65(5):791-793. doi:10.1007/s10616-013-9633-8

  ⁸van der Valk J, Gstraunthaler G. Fetal Bovine Serum (FBS) — A pain in the dish? ATLA Altern to Lab Anim. 2017;45(6):329-332. doi:10.1177/026119291704500611

  ⁹Bilgen B, Orsini E, Aaron R, McK Ciombor D. FBS suppresses TGF-beta1-induced chondrogenesis in synoviocyte pellet cultures while dexamethasone and dynamic stimuli are beneficial. J Tissue Eng Regen Med. 2007;1(6):436-442.

  ¹⁰Gstraunthaler G. Alternatives to the use of fetal bovine serum: serum-free cell culture. ALTEX Altern zu Tierexperimenten. 2003;20(4):275-281. doi:10.14573/altex.2003.4.257

  ¹¹Chen G, Gulbranson DR, Hou Z, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8(5):424-429. doi:10.1038/nmeth.1593

  ¹²Burridge PW, Matsa E, Shukla P, et al. Chemically defined generation of human cardiomyocytes. Nat Methods. 2014;11(8):855-860. doi:10.1038/nmeth.2999

  ¹³Efe JA, Hilcove S, Kim J, et al. Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol. 2011;13(3):215-222. https://doi.org/10.1038/ncb2164.

  ¹⁴Lu J, Hou R, Booth CJ, Yang SH, Snyder M. Defined culture conditions of human embryonic stem cells. Proc Natl Acad Sci USA. 2006;103(15):5688-5693. https://doi.org/10.1073/pnas.0601383103.

  ¹⁵Weber T, Wiest J, Oredsson S, Bieback K. Case studies exemplifying the transition to animal component-free cell culture. Altern Lab Anim. 2022;50(5):330-338. doi:10.1177/02611929221117999

  ¹⁶Chary A. Culturing human lung adenocarcinoma cells in a serum-free environment. In: Movia D, Prina-Mello A, eds. Cancer Cell Culture: Methods and Protocols. Humana Press; 2023:165-172. doi:10.21820/23987073.2017.2.20

  ¹⁷Rafnsdóttir ÓB, Kiuru A, Tebäck M, et al. A new animal product free defined medium for 2D and 3D culturing of normal and cancer cells to study cell proliferation and migration as well as dose response to chemical treatment. Toxicol Reports. 2023;10(March):509-520. doi:10.1016/j.toxrep.2023.04.001

  ¹⁸Beltran Paschoal JF, Patiño SS, Bernardino T, et al. Adaptation to serum-free culture of HEK 293T and Huh7.0 cells. BMC Proc. 2014;8(S4):6561. doi:10.1186/1753-6561-8-s4-p259

  ¹⁹Marigliani B, Balottin LBL, Augusto E de FP. Adaptation of mammalian cells to chemically defined media. Curr Protoc Toxicol. 2019;82(1):1-11. doi:10.1002/cptx.88

  ²⁰Chary A, Groff K, Stucki AO, et al. Maximizing the relevance and reproducibility of A549 cell culture using FBS-free media. ALTEX. 2022;83:105423. doi:10.1016/j.tiv.2022.105423

  ²¹Organisation for Economic Co-operation and Development. Test No. 431: In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method. OECD Publishing; 2019. doi:10.1787/9789264264618-en

  ²²Organisation for Economic Co-operation and Development. Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. OECD Publishing; 2020. doi:10.1787/9789264242845-en

  ²³Organisation for Economic Co-operation and Development. Test No. 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method. OECD Publishing; 2018. doi:10.1787/9789264229822-en

  ²⁴Organisation for Economic Co-operation and Development. Test No. 492: Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage. OECD Publishing; 2015.

  ²⁵Organisation for Economic Co-operation and Development. Work Plan for the OECD Test Guidelines Programme. 2023. https://www.oecd.org/chemicalsafety/testing/work-plan-test-guidelines.pdf

  ²⁶European Commission. EURL ECVAM test method submission – test presubmission form. https://joint-research-centre.ec.europa.eu/eu-reference-laboratory-alternatives-animal-testing-eurl-ecvam/alternative-methods-toxicity-testing/validation-and-submission-process/eurl-ecvam-test-method-submission_en

  ²⁷Sharma M, Stucki AO, Verstraelen S, et al. Human cell-based in vitro systems to assess respiratory toxicity: A case study using silanes. Toxicol Sci. Published online July 27, 2023:1-41. doi:10.1093/toxsci/kfad074