Improving the human relevance of cell culture using animal-free culture media

Supplementing basic media with fetal bovine serum (FBS, also known as fetal calf serum or FCS) to support cell viability in vitro was developed in the 1950s [1] and remains a fundamental technique in cell culture. However, the composition of FBS is not fully characterised, and recurrent batch-to-batch variation leads to a lack of reproducibility and unintended experimental effects [2]. Culture media containing animal-derived supplements also has immunogenic potential due to the presence of xenogenic proteins [3] and cells grown in these conditions are generally not suitable for clinical applications such as cell therapies [4].

FBS is bioreactive and interferes with specific pathways or characteristics [5,6], causing unpredictability and increasing the risk of producing unreliable data from in vitro experiments [2,5]. Animal-free supplements and media that contain defined components have a lower risk of unintended experimental effects and batch-to-batch variation, increasing the reliability and reproducibility of data produced.

Other practical concerns associated with FBS use can be minimised with the adoption of highly regulated, defined media supplements. For example, the risk of contamination with mycoplasma, prions, bacteria and viruses [7,8], which can be a cause of concern for researcher safety and long-term culture effects, is minimised in animal-free, chemically defined media. Effects of the environment, such as extreme weather, outbreak of disease and farming policies can all cause downstream fluctuations in the global supply of FBS, whereas the supply chain for animal-free media is more stable.

The collection of serum can lead to considerable suffering for the animals in question. FBS is a by-product of the dairy industry. Blood is collected from unborn un-anaesthetised calves by cardiac puncture which has the potential to cause pain and discomfort in the animals, and which ultimately leads to their death [2,9]. Recent estimates state approximately 600,000 litres of FBS are produced annually worldwide, corresponding to two million bovine fetuses [2,10].

These scientific, ethical and practical drivers support the adoption of animal-free cell culture conditions that may ultimately lead to a more rigorous and reproducible in vitro research landscape.

Animal-free media supplements are commercially available and include:

• Growth factors – can be added in isolation as a cocktail and either human-derived or synthetic. Note that synthetic growth factors often have a higher level of standardisation.
• Human blood derivatives – serum albumin, serum, plasma and platelet lysates can be derived from human blood samples. Serum albumin can be synthesised from human-derived materials, and human platelet lysate can be isolated as a by-product of plasma preparations for example, from blood banks.
• Supplements derived from non-animal and non-human organisms – proteins, lipids and other components that are important for cell growth can be used to reduce or eliminate the need for animal-derived serums and can be extracted from plants, bacteria and yeast, for example plant-derived protein hydrolysates.  

Chemically defined media is completely free from serum (both animal and human-derived). All the chemical components and concentrations are known, eliminating batch-to-batch variation and improving quality control. However, some commercially available ‘serum-free’ media are not animal-free as they contain animal-derived supplements other than serum, such as keratinocyte serum-free media that may require the addition of bovine pituitary extract or immunological serum-free media that requires the addition of bovine serum albumin.  These products are not always optimal replacements for FBS-supplemented media as they may still incur batch variability.

• ​Supply – clear supply chains that are not influenced by unpredictable external factors. 
• Non-infectious – clear sources or synthetic constitutions reduces the risk of infection considerably, including reduced risk of mycoplasma contamination.
• Tailoring – defined media can be tailored to specific cell types to avoid differentiation or other phenotypic changes.
• Reproducibility – high level of consistency between batches.
• Physiological relevance – only human-derived or synthetic molecules (no xenogenicity).
• Non-immunogenic – defined media does not contain immunogenic molecules, an important consideration in cell therapy.

Researchers are already embedding animal-free culture components in their work (see Case Studies and Further Reading), particularly for applications in cell therapy. The benefits of adopting  animal-free cell culture and widespread implementation will lead to improved standardisation across the in vitro research landscape (see Good In Vitro Practice).

The perception that FBS is the most economical option for cell culture is not always correct, as rising demand has caused both supply issues and fluctuating costs [2]. As the demand increases for animal-free media, the costs will continue to decline, so it is important that users share their needs for these components with their suppliers.

Cells can require a ‘weaning’ period or protocol optimisation to adapt to serum-free or animal-free conditions, and this has been successfully demonstrated in cell types such as human retinal pigment epithelium cells [11] and human adipose stem cells [12]. Animal-free freezing mediums are also available for cryopreservation, reducing the need for serum for long-term cryopreservation (see Resources).

We are supporting the development of animal-free in vitro techniques through the launch of the 2020 CRACK IT Challenge “Animal-free in vitro: Replacement of animal-derived reagents in an established human cell-based in vitro assays associated with an OECD Test Guideline”.

Below are some examples of successful adoption of animal-free in vitro conditions from the literature.

• Adaptation of the KeratinoSens™ skin sensitisation test to animal-product-free cell culture. doi: 10.14573/altex.1701311
   
• A novel xeno-free culture system for human retinal pigment epithelium cells. doi: 10.18240/ijo.2019.04.06
   
• Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications. doi: 10.1007/s00441-012-1360-5

The FCS-free database, maintained by the 3Rs-Centre, Utrecht University, provides an overview of the range of commercially available FCS-free media for cell culture. 

Rafnsd´ottir et al. recently published the composition of a defined animal-free medium for use in the routine or long-term culture of normal or cancer cells containing only human proteins [13]. 

Companies offering human-derived or synthetic serum substitutes or non-animal media supplements:

• Helios Bioscience
• Kerry
• Lonza
• Merck
• Qkine

Companies offering chemically defined, xeno-free, serum-free media:

• Cell Applications
• Cytiva
• Lonza
• Merck
• TakaraBio

If you are a company or institution offering animal-free supplement technologies and would like to be added to the resource list, please get in touch.

• NC3Rs Blog: A change in (cell) culture: exploring alternatives to fetal calf serum
• Review: A consensus introduction to serum replacements and serum-free media for cellular therapies - ScienceDirect
• Review: Reproducibility: Respect your cells! | Nature
• Article: In Vitro Expansion of Human Mesenchymal Stem Cells: Choice of Serum Is a Determinant of Cell Proliferation, Differentiation, Gene Expression, and Transcriptome Stability - Shahdadfar - 2005 - STEM CELLS - Wiley Online Library
• Article: Effects of amotosalen treatment on human platelet lysate bioactivity: A proof-of-concept study - PubMed (nih.gov)
• Article: Epidermal growth factor, basic fibroblast growth factor and platelet-derived growth factor-bb can substitute for fetal bovine serum and compete with human platelet-rich plasma in the ex vivo expansion of mesenchymal stromal cells derived from adipose tissue - Cytotherapy (isct-cytotherapy.org)
• Article: Effects of different serum conditions on osteogenic differentiation of human adipose stem cells in vitro | Stem Cell Research & Therapy | Full Text (biomedcentral.com)
• Article: Autologous plasma versus fetal calf serum as a supplement for the culture of neutrophils | BMC Research Notes | Full Text (biomedcentral.com)
• Article: Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support (nih.gov)
• Review: Human-Derived Alternatives to Fetal Bovine Serum in Cell Culture - FullText - Transfusion Medicine and Hemotherapy 2013, Vol. 40, No. 6 - Karger Publishers
• Article: A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic, Induced Pluripotent and Adipose Stem Cells (plos.org)