Given that FBS originates from a fetal milieu, it contains various hormones and growth factors related to fetal development. These can affect and alter cells. For instance, Khodabukus and Baar (2014) engineered skeletal muscles using murine C2C12 myoblast cells and demonstrated that variations in serum origin can directly lead to changes in muscle phenotype. In another study on the healing of primary tendon tissue, van Vijven et al. (2021) showed that serum induces phenotypic drifts to such an extent that the validity of the study model can be fundamentally affected, especially in high serum concentrations. They cultured explanted mouse-tail tendon fascicles in serum-rich or serum-free medium and used transcriptome analysis to examine the altered physiology of the cells. Remarkably, a 1-week culture of tendon cells without serum showed little change in native gene expression. In contrast, FBS overrides the initial determination of primary cells concerning matrix integrity and cell morphology. Thus, this demonstrates that fetal serum-derived factors can render cell culture models unreliable. To avoid unpredictable effects of the numerous undefined serum factors in systems where precise control is required to achieve scientific aims, it is necessary to use chemically defined culture conditions from the beginning (Bieback et al., 2010). Further indication of this “rejuvenating” effect comes from a study by Kwon et al. (2016), demonstrating increased reprogramming efficacy at high serum concentrations, in which FBS modulates key molecular and cellular mechanisms. While this is a mechanism likely appreciated in this specific system, it is highly unwanted in other cell culture models, aimed to mimic physiological situations. FBS substitutes are less likely to induce unwanted reprogramming and differentiation processes (Zhao et al., 2010), even if they could initially increase the cost of media.

As early as 1958 it was pointed out that the undefined and variable nature of FBS can affect the quality and performance of experiments (Puck et al., 1958; van der Valk, 2022). Variability occurs even between lots from a single manufacturer (Zheng et al., 2006; Barosova et al., 2021). Therefore, cell culture scientists, whether wittingly or unwittingly, accept a degree of uncertainty in their experimental setup, which ultimately contributes to the ongoing reproducibility crisis (Baker, 2016a; 2016b; Barosova et al., 2021; Liu et al., 2023). Nevertheless, a global survey on the use of animal-derived materials and reagents confirms that FBS is still commonly used in scientific experimentation (Cassotta et al., 2022). It is rather surprising, considering that the use of animal-derived products for scientific purposes usually requires cost-intensive pre-testing to avoid the risk of serious disruption of experiments or whole production systems by batch-to-batch variations (Bhat et al., 2021). This is mitigated by intensive pre-testing of FBS lots, to single out the ones that raise the least issues. An alternative route is using more laborious manufacturing techniques to create “purer” FBS classes suggested by Berrong et al. (2023). Either way, both will result in higher costs for the end consumer.

The use of animal-derived components always bears the risk of introducing pathogens into cell cultures. In the case of FBS, viral antibodies were first reported in 1967 (Kniazeff et al., 1967), triggering thorough examinations that confirmed the presence of viruses (Boone et al., 1971; Molander et al., 1971; Kniazeff, 1973; Kniazeff et al., 1975). Unfortunately, viral contamination of commercially available FBS remains an ongoing issue even half a century later (Paim et al., 2021; Zhang et al., 2022). Of recent concern is the spread of a pathogenic avian virus among cattle in many FBS-producing countries, causing influenza in humans with high mortality (PAHO/WHO, 2025). Moreover, various contaminants, including viruses, prions, bacteria, fungi, endotoxins, and exogenous extracellular vesicles, can be present in FBS (Kirikae et al., 1997; Reuther et al., 2006; Urzì et al., 2022). In addition, Gregersen (2008) revealed that while Mycoplasma hyorhinis was unable to grow in a chemically defined, serum-free medium, low amounts of serum triggered rapid growth to high titers.

Furthermore, human cells cultured in FBS can incorporate substances that are xenogenic to humans (Martin et al., 2005). This raises concerns about the safety of stem cell transplantation into humans, as it can induce an immune response in the patient, which reduces the efficacy of subsequent cell therapeutic interventions (Heiskanen et al., 2007; Sundin et al., 2007; Martin K. E. et al., 2022). Therefore, the Food and Drug Administration (FDA) of the United States of America (USA) has defined requirements for clinical-grade FBS. These include health monitoring and approval of the geographic origin of the bred animals, along with imposing minimum requirements on (current) Good Manufacturing Practice standards (cGMP or GMP), traceable certificates of origin, and testing for contamination (Minonzio and Linetsky, 2014; DiNicolas, 2015). Comparable requirements are in place in the European Union (EU) and Canada (European Commission, 2011; European Medicines Agency, 2013; Viswanathan et al., 2017). Nevertheless, these highly elaborate processes lead to increasing financial, administrative, and logistical expenses, simply to try to standardize an intrinsically unstandardized product instead of redirecting these efforts to create a standardized replacement from the beginning. Even the European Commission recommends that “when manufacturers have a choice,” the use of materials from non-animal origin is preferred (or at least materials from other species than bovines) (European Commission, 2011). And since animal-free materials suitable for cell culture media exist, manufacturers do have that choice.

The production steps of FBS are challenging to retrace (Hodgson, 1991) and a lack of transparency in the manufacturing can facilitate fraud, as scandals of serum adulteration and mislabeled geographic origin have demonstrated (Gstraunthaler et al., 2013; Gstraunthaler et al.,2014; Besser et al., 2018). Such incidents not only result in questionable experimental outcomes, but they also undermine the trust in science and industry. Subsequently, the International Serum Industry Association (ISIA) has introduced FBS traceability auditing, which has been recommended for human serum collection as well (Jacobs et al., 2023). Nonetheless, information on global annual FBS production volumes is hard to find, only estimations are publicly available (see Table 1). The most detailed numbers were published by Hodgson in 1993, stating a global supply of more than 500,000 L of raw FBS (Hodgson, 1993). In 2007, Festen estimated a worldwide production of 600,000 L per year, with half of it originating from the USA, a fifth from Australia and New Zealand, and the remainder from Canada, Central/South America, and Europe (Festen, 2007). Later information provided by the ISIA states that the USA in fact provides around 26% of the global supply (ISIA, 2020). Considering the most recent estimation from 2016, which assumed production of 200,000 L for the USA in 2017 and 2018 (RMBIO, 2016), the annual global production is projected to be close to 800,000 L (van der Valk et al., 2018). This corresponds to approximately one to two million bovine fetuses that were slaughtered in the process since the amount of obtained serum depends on the size/age of the fetuses (Jochems et al., 2002).

However, it must be stated that there is no official information available, not even in a supposedly highly regulated market as the EU (European Parliament, 2023), even though an official EU guidance note states that “traceability to the slaughterhouse must be assured for each batch of serum or plasma. Slaughterhouses must have available lists of farms from which the animals originate. If serum is produced from living animals, records must be available for each serum batch which assures the traceability to the farms” (European Commission, 2011). Furthermore, the European Medicines Agency insists that “the traceability of serum from final container back to the abattoir of origin is of prime importance and a clear audit trail must be demonstrable including records of volumes at each stage” (European Medicines Agency, 2013). The lack of (or will to) transparency offers latent opportunities for fraud by leaving the true production scale of FBS unknown to the public.

Financial aspects join the ranks of disadvantages surrounding FBS. FBS prices are inherently volatile since they are interconnected with the price and supply of beef and dairy products (DiNicolas, 2015; Meat and Livestock Australia, 2016; RMBIO, 2016; Brann, 2022). Hence, if prices for these products rise (e.g., due to epizootics, natural disasters, or changing farm regulations) the price for FBS will inevitably rise as well. Trade sanctions, policies and tariffs are expected to further increase costs, especially since global tariff rates on beef were higher than those for many other agricultural-food products (Ridley et al., 2024) already before international trade tensions arose in April 2025 (Bekkers et al., 2025). In addition, beef production is expected to continuously decline over the next years in the USA, and even a relatively minor reduction by 5% could trigger a 30%–50% decrease in FBS production (Corning, 2025). This uncertainty by itself already hampers long-term cost calculations for customers and might slow down or even stop certain projects due to sudden price surges (Fife and Payne, 2025). Thus, like in every other business, rising prices prompt customers to consider reductions or replacements (Jayme, 2007; Brindley et al., 2012). In contrast, the prices of non-animal products are less susceptible to agricultural fluctuations, and easier process upscaling could further reduce costs. Therefore, it would be in the economic interest not only of customers to switch to less financially unpredictable products, but also of the serum-distributing industry to invest in animal-component-free media supplements, allowing them to diversify their business model and benefit earlier from the growing swing toward such products.

Even though the focus of this publication is mostly on the effects of FBS on biology and medicine, the welfare of the animals involved is part of the bigger picture. Fetal calf blood is the raw material for FBS and cannot be retrieved noninvasively. As there is no scientific consensus on whether bovine fetuses suffer during and after the slaughter of the dam, the animals should be given the benefit of the doubt (Maurer et al., 2016; Weber et al., 2021b; McCann and Treasure, 2022). Nonetheless, even if the fetuses would be dead during blood retrieval (Versteegen et al., 2021), animal welfare issues still prevail (Weber et al., 2021b), e.g., the lack of a mandatory certified killing method including mandatory anesthesia for the fetuses (Weber and Wagner, 2021). Furthermore, pregnant dams should not be transported to slaughterhouses at all, because this inflicts additional pain, suffering, and distress on both the dams and the fetuses, potentially also resulting in premature birth or even spontaneous abortion. As an immediate step, the Eurogroup for Animals demands a transport stop for pregnant animals that have exceeded 40% of their gestation period (Porta et al., 2024).

The aspect of animal-derived materials in in vitro methods aiming to replace animal experiments or cultivated meat aiming to replace traditional meat production is far from being of secondary importance, as the main ethical advantages of transitioning to new techniques could be compromised if the process remains dependent on the slaughtering and suffering of animals. Therefore, FBS should be phased out to avoid this ethical dilemma.

While FBS might have been a useful component in the past, there is hardly any reason for its continued use (Weber et al., 2022) as there are numerous replacement materials available in scientific literature, databases, and on the market.

Political, scientific, and economic strategies are continuously being implemented to promote the gradual phasing out of FBS to develop serum-free, defined culture media and to adapt cells to serum-free conditions (ECVAM News and Views, 2008; Knudsen and Ritskes-Hoitinga, 2021; NC3Rs, 2024). Thus, the scientific community should seek to raise awareness among cell culture researchers that FBS has been identified as a contributing factor to the reproducibility crisis and that animal-component-free replacements are available. Eventually, FBS should be limited to projects where rigorous scientific evidence demonstrates that no other viable substitute exists, rather than being used by default in routine cell cultures.

FBS provides an undefined environment for cells, and everyone should be aware that cells cultured in FBS-supplemented medium might undergo changes under its influence, resulting in an undefined cell type with unknown features and developmental potentials, which might not be physiologically representative anymore. However, every journey begins with the first step, and scientists around the globe have already gone beyond: Institutions like the “Fetal Calf Serum-free Database” make it easy to browse for existing serum replacements (van der Valk, 2021; 3Rs Centre Utrecht, 2025). This database is becoming the main place to find replacements for serum and even commercial suppliers see the giant market rising for a defined and reliable science. Meanwhile, the community of serum-free media users is growing every day, likely due to the raised awareness of its drawbacks and its role in the reproducibility crisis.

To demonstrate the feasibility of departing from FBS and establishing new and reliable gold standards for cell culture, this review presents FBS-free examples across the following fields:

• 2.1 Stem cells

In addition, strategies for adapting cells to new media are provided. To encourage researchers, financiers, and regulators to switch to FBS-free science, frequently heard statements and rebuttals on FBS use are listed in Table 2.

This review demonstrates that there is hardly any need for the continuous use of FBS anymore. FBS replacement can enhance efficiency and cost-effectiveness by improving reproducibility and thus ensuring the scientific relevance of data obtained from in vitro methods.

FBS can and will be fully replaced. It is not only a necessary contribution to standardize science, but an ethical necessity to ensure a transition to safer, more reproducible, and reliable research methodologies.

The stem cell field is a prototype example documenting how the use of serum, in general, can impair scientific progress. The plethora of factors influencing the embryonic milieu during the bovine fetus development have been shown to prevent controlled culture and differentiation of primitive stem cells, including hematopoietic stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) (Lebkowski et al., 1995; Shin et al., 2019). The undefined factors present in FBS and in other serum preparations can trigger spontaneous differentiation in the human system. Quite surprisingly, this issue has not been observed in murine ESCs (Koestenbauer et al., 2006). Thus, human ESCs and iPSCs should be cultured per se under chemically defined conditions, since this is the only option to ensure controlled differentiation and prevent spontaneous differentiation into unwanted lineages.

A multitude of different two-dimensional (2D) and/or three-dimensional (3D) cell culture systems are subsumed under the term complex in vitro models (CIVMs). They consist of micropatterned cells, 3D microtissues (spheroids and organoids), and microphysiological systems (MPS) like organ-on-a-chip (OoC) and 3D bioprinted systems (Ekert et al., 2020; Baran et al., 2022; Parvatam et al., 2025). CIVMs provide a closer and more accurate assessment of real-life physiological conditions and enable the study of higher complex systems. Thus, external factors that influence or threaten the integrity of these systems need to be replaced with defined substances. In fact, complete independence from animal-derived materials is an important prerequisite to consider CIVMs fully fit for purpose (Weener et al., 2024).

The cultivation of animal cells for human or animal consumption, whether from vertebrate or invertebrate origins, represents a promising solution with potential benefits to the trade-off situation of high demand for animal protein consumption and growing animal welfare concerns (Scollan et al., 2011; Cornish et al., 2016; Kristensen, 2018; Komarek et al., 2021). Since the world’s first “lab-grown hamburger” was produced and consumed at a press conference in London in 2013 (Post, 2014), there have been significant advancements in the field. For clarity reasons, the term “cultivated meat” will be used throughout this text, even though “in vitro meat,” “lab-grown meat,” “cultured meat,” and “clean meat” have been used in the literature (Sexton et al., 2023; Battle et al., 2024).

Production of cultivated meat is relatively simple in its principles, involving a few essential steps (Post, 2014). Briefly, cells are collected, ideally non-invasively, which is done by isolating them from living animals maintained in optimal species-appropriate conditions. Stem cells are the most valuable for generating cell sources that can be grown for scalable production (Jara et al., 2023). Moreover, immortalized cell lines can be developed via genetic modification (Guo et al., 2025) or selected through spontaneous mutations (Ramboer et al., 2014). Following a proliferation phase, the cells are differentiated and maturated primarily into muscle and fat cells, which represent the main components of traditional meat. The mature cells are harvested to eventually undergo a post-processing phase that involves scaffolding them to provide meat-like shapes. It should be noted that although most research and development focuses on culturing bovine cells, due to the significant greenhouse gas emissions by cattle compared to other farmed animals (Gerber et al., 2013), investigations involving cells from other species are also underway (Lee D. Y. et al., 2023; Nikkhah et al., 2023; Musgrove et al., 2024; Siddiqui et al., 2024).

This section explores current and potential advancements in optimizing growth media composition for cultivated meat production, with a particular focus on achieving complete independence from animal-derived materials, which otherwise undoubtedly hinder the low-cost scale-up and commercialization of cultivated meat (Kadim et al., 2015; Stephens et al., 2018; O’Neill et al., 2021). The successful production of cultivated meat necessitates media that are safe for consumers, food-grade, cost-effective, capable of supporting large-scale cell proliferation and differentiation, possess acceptable sensory qualities, and are obviously animal-component-free (O’Neill et al., 2021).

Rather than relying on a single solution, researchers can develop serum alternatives by adopting multiple strategies, leading to the formulation of complex and chemically defined media with promising results. In this regard, Yamanaka et al. (2023) developed a novel serum-free medium containing nutrients extracted from microalga Chlorella vulgaris and a conditioned medium containing cell-secreted growth factors to promote the proliferation of primary bovine myoblasts. Stout et al. (2022) have taken inspiration from the aforementioned chemically defined culture medium B8 developed by Kuo et al. (2020), given its simplicity, cost-effectiveness, and capacity to positively support cell growth, and used it as a starting point for developing effective serum-free media formulations to use in cultivated meat production. Indeed, they validated a simple, serum-free, and animal-component-free medium called Beefy-9 for culturing bovine satellite cells (BSCs). This B8-inspired medium is supplemented with recombinant human serum albumin (rHSA) expressed in rice (Stout et al., 2022). Recombinant bovine serum albumin (rBSA) may serve as a future alternative in the context of species-specific cell culture (Mogilever et al., 2025). In particular, Stout et al. (2022) established a protocol for passaging BSCs in Beefy-9 and demonstrated that it maintains cell myogenicity under serum-free conditions, resulting in short-term growth comparable to that observed with 20% FBS. Further optimizations of the performance/cost ratio in Beefy-9 were achieved by adjusting the concentrations of rHSA, resulting in an improved medium called Beefy-9+. Shortly after, Kolkmann et al. (2022) developed another serum-free and chemically defined medium that effectively supported bovine myoblast proliferation at 97% efficiency compared to 20% FBS-containing growth medium. Interestingly, the albumin concentration in the medium developed by the Kolkmann group is 5 mg/mL (Kolkmann et al., 2022), which is higher compared to 0.8 mg/mL and 3.2 mg/mL in Beefy-9 and Beefy-9+, respectively (Stout et al., 2022), suggesting that rHSA significantly enhances cell proliferation and growth (Kolkmann et al., 2022). To reduce costs, Stout’s group modified Beefy-9 by replacing albumin with rapeseed protein isolate, a bulk-protein solution derived from agricultural waste streams, obtaining a new, cost-effective medium, namely Beefy-R. This new medium improved BSC growth compared to Beefy-9 while maintaining cell phenotype and mitogenicity (Stout et al., 2023). Other serum-free variants of Beefy-9, fully defined in composition and capable of in-house preparation, have been described by Schenzle et al. (2025). They tested various combinations of growth factors, myokines, and hormones, which significantly increased the proliferation rate of BSCs. Especially, the use of methylcellulose and racemic alanine, two inexpensive, food-grade stabilizers, either alone or in combination, has been proposed to efficiently enhance the propagation of muscle cells cultured in both B8 and B9 media while keeping overall production costs low. In this regard, it has to be noted that the ongoing research in microbial cell factories, particularly the optimization of Escherichia coli strains for recombinant growth factor and serum protein production, as well as that of purification processes, offer new opportunities in the field, not only from a cost-effectiveness perspective but also in terms of scalability, as highlighted in a recently published review (Mainali et al., 2025). Further attempts to reduce costs came from the work published by Pasitka et al. (2024), who developed an optimized animal-component-free medium where they replaced albumin with methylcellulose and hydroxypropyl β-cyclodextrin, reducing medium costs by 38% (Pasitka et al., 2024). Notably, this medium supported high-density chicken cell cultures, achieving 28 million cells/mL and enabling multiple harvests over 20 days, performing comparably to albumin-containing serum-free media, thus offering a cost-effective and scalable option for cultivated meat production (Pasitka et al., 2024). Interestingly, in 2022, Mosa Meat, a leading food technology company in the Netherlands focused on cultivated beef, developed a chemically defined, serum-free medium that induced myogenic differentiation of 3D muscle organoids, mimicking serum starvation without resorting to transgene expression (Messmer et al., 2022). As the authors themselves highlighted, the hydrogels in the study were not entirely animal-component-free, but they expect that these findings can be reproduced in fully animal-free organoid cultures. In addition, Mosa Meat researchers developed a defined serum-free medium for cultivating beef fat in vitro (Mitić et al., 2023), which is a key ingredient to the flavor of traditional meat. They reported superior adipogenesis in bovine stromal vascular cells differentiated in this serum-free medium compared to those in the FBS-containing one, both in 2D and 3D cultures. These findings have collectively contributed to the Dutch company’s submission of its first application for EU market approval of cultivated beef fat in 2025 (Team IO, 2025). It is important to note that, as reported previously throughout this section, not all serum-free media are necessarily animal-component-free. This distinction is crucial since the inclusion of any animal-derived ingredients in cultivated meat products would most likely discourage consumers who do not follow animal-based diets (Jaiswal and Shrivastava, 2024). Therefore, researchers are encouraged to explore alternatives to animal sera from sources outside the animal kingdom to ensure that cultivated meat production is sustainable and genuinely free of animal suffering.

Ultimately, while this discussion has primarily focused on cultivated meat production and the associated issues with FBS and animal sera, and potential animal-free supplements, the landscape of cellular agriculture extends far beyond meat. This includes cell-based milk, egg proteins, and leather, each presenting unique challenges and opportunities that are both exclusive or shared with those of cultivated meat (Eibl et al., 2021; Fytsilis et al., 2024).

Despite the progress made, further technical challenges remain, including identifying the best cell source and achieving scalability (Stephens et al., 2018). Nevertheless, knowledge in the field of cultivated animal products is advancing rapidly. One approach that includes machine learning is described in Nikkhah et al. (2023). The authors use a unique approach of several techniques to optimize culture media formulation for growing cultivated meat using a cell line from zebrafish (Danio rerio). To this end, a multistep approach is used that includes radial base functions with genetic algorithms to determine the problem of the optimal base media for serum creation, while the overall design is modeled with the response surface method. The use of generative approaches in general for linear and nonlinear optimization has been proven to be effective in certain aspects of bioinformatics (Li et al., 2020), and thus also promises potential to be harnessed in this context. While the authors claim a good predictability, the standard pitfalls of machine learning approaches such as overfitting or bad sample bases need to be considered.

This demonstrates that not only did cellular agriculture profit immensely from the progress published by biomedical science (Heine et al., 2024), but it can vice versa inspire the development of animal-free media for other scientific fields. While competition and proprietary reasons might seem to speak against publishing results obtained by cellular agriculture companies on animal-component-free cell culture media, transparency on their ingredients will boost consumer trust in the nutritional value and safety of their products.

In addition to cell culturing, animals have long played a role in the generation of biotechnological products, including antibodies and vaccines. These animal-derived materials were crucial for various medical and scientific applications ranging from diagnostic tools to disease prevention. Nevertheless, past reliance on animals does not justify the continued use of animals as “means of production” in perpetuity. By examining antibodies and vaccines as two prominent examples, this chapter explores the ongoing transition to animal-free production methods and highlights the technological advances and their impact on sustainability and innovation.

Recombinant antibody production is the cornerstone of modern antibody production and enables the production of monoclonal antibodies without relying on animal-intensive hybridoma technology. In this approach, antibody-encoding genes are cloned into expression vectors, which are then introduced into eukaryotic host cells such as Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells.

The sequences of the antibody genes can be obtained animal-free using display methods (McCafferty et al., 1990; Barbas et al., 1991; Breitling et al., 1991; Boder and Wittrup, 1997; Ho et al., 2006; Akamatsu et al., 2007), by polymerase chain reaction (Larrick et al., 1989; Breitling and Dübel, 1997), or next-generation sequencing of hybridoma clones (Subas Satish et al., 2022; Mitchell et al., 2023). Recombinant production systems are highly scalable, compatible with regulatory requirements, and produce antibodies with consistent quality, rendering them indispensable for therapeutic, diagnostic, and research applications.

In the production of recombinant antibodies for the pharmaceutical industry, but also diagnostics and research, it is essential to avoid potentially harmful or unknown substances during the entire production cycle. Production should be defined, scalable, and batch consistent. These criteria can be achieved through animal-free, recombinant production without FBS or other animal-derived materials. Consequently, the EU Reference Laboratory for Alternatives to Animal Testing of the Joint Research Center stated in 2020 that “non-animal-derived antibodies are well-defined and better reagents that will improve the reproducibility and relevance of scientific procedures and lead to more efficient and effective use of research funds,” while recommending that “EU countries should no longer authorise the development and production of antibodies through animal immunisation, where robust, legitimate scientific justification is lacking” (Barroso et al., 2020). This gave impetus to ongoing research into the development of animal-free antibodies for diagnostics and research (Dübel, 2024; Groff et al., 2024).

CHO and HEK293 cell lines are commonly used to produce antibodies that match the human glycosylation pattern and post-translational modification. These cell lines are optimized for bioreactors and in most cases already adapted to FBS-free medium. If not, they can be gradually adapted to FBS-free medium via out-dilution of FBS (Weber et al., 2022). CHO cells are considered the gold standard for large-scale therapeutic antibody production due to their high adaptability to serum-free and suspension culture systems. They are robust and scalable for industrial production (Reinhart et al., 2019), and despite not being of human origin, produce antibodies with human-like glycosylation patterns. Furthermore, they are suitable for most FDA-approved therapeutic monoclonal antibodies with more than 34 biosimilars produced in CHO receiving FDA approval since 2015 (Gupta et al., 2023). Alternatively, HEK293 cells are ideal for rapid, transient expression of recombinant antibodies. Since they are a human-derived cell line, this indubitably ensures human-like glycosylation. HEK293 cells are often used in early-stage research or preclinical development (Backliwal et al., 2008; Jäger et al., 2013). Several commercially available media for protein expression in cells are free of animal components and do not require FBS as an additive (Reinhart et al., 2015). A selection of commercially available media suitable for CHO and HEK293 suspension cells can be found in Table 4. It has to be noted that Cervera et al. (2011) developed a supplement consisting of recombinant proteins and an animal-component-free lipid mix, which improves HEK 293 cell density grown in Freestyle™ 293.

Advances in cell culture technologies are rapidly expanding the capabilities of non-mammalian-based cell lines e.g., insect cells (Korn et al., 2020) and even plant cells (Donini and Marusic, 2019; Schillberg and Spiegel, 2022). Thus, it is essential to anticipate and explore innovative advancements in cell line development that could potentially revolutionize research methodologies. These advancements offer many advantages for the future, especially in research and diagnostic applications that are not dependent on human post-translational modification and glycosylation patterns.

Another prominent biotechnological application, besides recombinant antibody production, is the development of vaccines using immortalized cell lines, especially mammalian kidney-derived cells like HEK293, Madin-Darby canine kidney (MDCK), or Vero cells, but also insect-derived cells like Spodoptera frugiperda 9 (Sf9). Especially suspension cell cultures in combination with an animal-component-free medium are more appropriate for large-scale bioreactor-based production by creating a steadier and more controlled vaccine manufacturing process (Zhang et al., 2023). Fortunately, several animal-component-free media are available. Gélinas et al. (2019) utilized the transfection medium HyClone HyCell TransFx-H to express a promising Ebola virus vaccine in HEK293 cells. The 4Cell® MDCK CD medium is suitable for MDCK cells and also protein-free (Chaabane et al., 2021; Zinnecker et al., 2024). Chen A. et al. (2011) tested five commercially available animal-component-free media for the influenza vaccine production in Vero cells, with EX-CELL® Vero SFM achieving the highest cell concentration. Moreover, several media suitable for culturing insect cells are on the market as well (Chan and Reid, 2016; Cox, 2021). These and further examples are summarized in Table 5.

In addition to the commercially available media mentioned in Tables 4, 5, several disclosed formulations are also available: Schneider (1989) described the preparation of a basal medium for CHO cells as early as 1989, while Burteau et al. (2003) modified it with plant peptones to improve cultivation and productivity. More recently, Balabashin et al. (2020) described a CHO medium consisting of recombinant protein supplements and hydrolysates of non-animal origin.

For HEK293 (Oredsson et al., 2025), Vero (Rourou et al., 2009; Kuncorojakti et al., 2024; Oredsson et al., 2025), MDCK (Mochizuki, 2006), and Sf9 cells (Inlow et al., 1989; Boegel, 2019), animal-component-free media formulations can be found in the literature, which all contain proteins either from soy, yeast, human, or recombinant sources. In contrast, Wilkie et al. (1980) developed a chemically defined, protein-free medium for insect cell lines, with the detailed composition reprinted by Mitsuhashi (2018). Generally, protein-free media are recommended for cell culture designated for vaccine production to reduce extraneous antigens and avoid an unstable amino acid composition in the medium due to protein degradation (Wallis et al., 1969; Mitsuhashi, 2018). A white paper from Eppendorf consequently declares that the “holy grail” in vaccine production “being an economical, protein-free, serum-free medium that would provide strong growth support and have the property of scalability to large volumes, up to thousands of Liters, while coming in at an affordable price” (Sha, 2021). For completeness, the phrase “with a disclosed formulation” should be added to this list.

Despite the fact that FBS-supplemented medium has none of these traits, recent studies continue to use it for the cultivation of the cell lines mentioned above (Bermúdez-Abreut et al., 2025; Koide et al., 2025; Li et al., 2025; Yan et al., 2025; Yang et al., 2025). Even when cell culture collections display that a specific cell line like the conveniently named “Vero (AC-free)” is adapted to grow in animal-component-free medium (González Hernández and Fischer, 2007; UKHSA Culture Collections, 2025), one can still find publications using FBS-supplemented media instead (Riepler et al., 2020; Malekshahi et al., 2021). One cannot and should not blame the scientists for doing this, because supplementation with FBS has permeated deep into the general workflow of cell culturing. This shows the ongoing importance of disseminating information on replacing animal-derived materials, although replacements have been described before in the literature.

Toxicologists investigate the potentially harmful properties of substances, where it is essential to maintain strict control over experimental conditions. The introduction of unknown or potentially adverse components into research and test samples could significantly compromise the reliability and accuracy of findings. Therefore, it is imperative that cell culture media used in these studies are chemically defined and reproducible to facilitate good toxicological science (Rabbit et al., 2025). This enables toxicologists to isolate and study the specific effects of the research or test substances under investigation. By eliminating variability introduced by undefined components, such as those found in complex products like FBS, researchers can more accurately assess the true impact of substances on biological systems. The use of chemically defined media with limited batch variation of the components should enhance the reproducibility of experimental results aligning with principles of scientific accuracy and ethical considerations. It supports the pursuit of reliable data that can inform regulatory decisions regarding the safety and toxicity of chemicals, pharmaceuticals, and other substances. Ultimately, by applying chemically defined media standards, toxicologists can advance their understanding of potential hazards and contribute to improved public health and environmental safety practices.

A pivotal stride was a 2021 report leading major efforts to validate 17 in vitro mechanistic methods focused on thyroid hormone disruption and providing examples for replacing animal-derived materials in these methods (Bartnicka et al., 2021). Furthermore, an industry-sponsored initiative, launched in September 2020, aims to transition the test guidelines (TG) of the Organisation for Economic Co-operation and Development (OECD) toward animal-component-free assays (NC3Rs, 2020). This initiative seeks to enhance the reliability, reproducibility, and consistency of OECD TG by promoting the adoption of chemically defined media wherever possible. Reichstein et al. (2023) exemplified this by successfully demonstrating the replacement of animal-derived components in OECD TG 455 (estrogenic activity) and OECD TG 487 (genotoxicity) (OECD, 2021b; OECD, 2023). Similarly, Perez-Diaz et al. (2023) adapted TK6 cells to an animal product-free, chemically defined culture medium for genotoxicity studies under OECD TG 487. Other groups have also made significant strides in advancing research and testing methodologies within the field of toxicology. For instance, by refining procedures in accordance with the International Organization for Standardization (ISO) standard ISO 10993-5, which addresses in vitro cytotoxicity for the biological evaluation of medical devices. The methodology employs either L929 or CaCo-2 cells combined with a straightforward medium composed of a DMEM/F12 mixture supplemented with insulin-transferrin-selenium (Wiest, 2017; Weber et al., 2021a). A recent study is also focused on enhancing the reliability of toxicological assessments by developing a specialized culture medium for the long-term, serum-free cultivation of fish cells (Jožef et al., 2025). This medium is tailored specifically for a cell line assay using RTgill-W1 cells derived from the rainbow trout (Oncorhynchus mykiss). This assay is utilized in OECD TG 249, a standard protocol for assessing fish acute toxicity (OECD, 2021a). However, full replacement of animal-derived materials was not yet achieved in that study, but protein-free formulations supplemented with dipeptides were suggested as a viable alternative (Jožef et al., 2025). There are also studies on fully humanizing toxicological in vitro test systems. Fraser et al. (2025) used hPL for the toxicity testing strategy of a nanomaterial while Ward et al. (2025) presented the AcutoX method for predicting acute oral toxicity with human fibroblasts cultured in the presence of pooled human serum.

These advancements are paving the way for a future where toxicological research and testing become more reliable based on defined methodologies. By refining culture media and testing methodologies, researchers aim to enhance the reliability and accuracy of toxicological data. This, in turn, contributes to more robust, improved safety assessments and regulatory decision-making processes.

Not only commercial cell banks such as the American Type Culture Collection and the European Collection of Cell Cultures, but everyone who works with cell cultures, must freeze cells sooner or later, coming along with the question of how to properly thaw them to ensure successful revival. As cell lines may exhibit genetic drift after a certain number of passages, frozen cells are a useful source to refresh the cell cultures. In addition, cell lines can be accidentally lost due to infection, cross-contamination, or for various other reasons. Thus, cryopreserved master and working cell banks ensure the constant availability of cells. Proper freezing and thawing techniques are economically beneficial for each laboratory because it avoids the need to purchase new cell ampoules from vendors. Cryopreservation is used for fertility cell preservation (and future in vitro fertilization), stem cell and iPSCs preservation, as well as preservation of other cell types dedicated to research and clinical applications (Jaiswal and Vagga, 2022). Importantly, a standardized protocol is crucial for reproducible freezing, allowing different personnel to achieve consistent results.

Directly freezing cells in a medium with water and no cryoprotectant will be fatal. When a solution with a high concentration of water freezes, the water molecules will form ice crystals through hydrogen bonding resulting in increased salt concentration in the remaining solution. Ice crystals damage cell membranes and the elevated solute concentration causes cellular dehydration resulting in osmotic shock (Jang et al., 2017). Therefore, cryoprotectants that penetrate the cell membranes reduce ice formation are added to the cell freezing medium, and specific freezing protocols are applied. Often cryoprotectants like glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, and propylene glycol are used, in combination with FBS (Whaley et al., 2021). Due to its high protein content, FBS in the freezing medium provides a certain cryoprotective effect, and concentrations up to 95% are applied (Fujisawa et al., 2019).

Currently, several cryoprotective media have been developed that dispense with FBS for obvious safety reasons. Some of these media are publicly available and are based on human serum, human blood components (Hreinsson, 2003; Reuther et al., 2006; Park et al., 2018; Rafnsdóttir et al., 2023), or combinations of defined animal-component-free cryoprotectants (González Hernández and Fischer, 2007; Volbers et al., 2016; Hsieh et al., 2018; Pless-Petig and Rauen, 2018).

Several authors have demonstrated the suitability of various proprietary commercially available FBS-free cryopreservation media (Miki et al., 2016; Chary et al., 2022; Uhrig et al., 2022). However, as mentioned previously, if the composition is proprietary, the influence of the media components on the cells cannot be investigated and therefore, it must be thoroughly studied and proven by the vendors. Thus, disclosed media formulations are preferred.

The simplest cryopreservation strategy involves using high protein concentrations in any xeno-free medium. In this regard, Hreinsson (2003) used human serum albumin (HSA) at concentrations of 25 mg/mL in phosphate-buffered salt (PBS) solution to successfully cryopreserve follicles in ovarian cortical tissue. Oredsson et al. (2025) demonstrated that cryopreservation in PBS with 20 mg/mL HSA works effectively with 5% DMSO as a cryoprotectant. CaCo-2 cells and HEK293 cells adapted to a chemically defined medium were successfully frozen in this medium to which 7.5%–10% DMSO was added (J. Wiest and E. V. Wenzel, personal communication, 3 March 2025). Therefore, using the chemically defined medium in which the cells are cultured and adding DMSO to it for freezing can be easily investigated by the user.

Pakhomov et al. (2022), Pakhomov et al. (2024a), Pakhomov et al. (2024b) reported an optimal freezing medium for testis interstitial cells consisting of 100 mg/mL Dextran 40 with 0.7 M concentration of the permeating cryoprotectant Me₂SO in Ham’s F12 medium. This freezing medium was superior to FBS-supplemented medium. Interestingly, they investigated physical processes such as ice crystal formation in various freezing media by differential scanning calorimetry and thermomechanical analysis.

The final evidence for the suitability of the freezing method and medium as well as storage conditions is the viability of cells post-thawing and seeding. This has been investigated in studies by several authors regarding proprietary xeno-free freezing media (Miki et al., 2016; Uhrig et al., 2022) and disclosed media (Hreinsson, 2003).

A prominent example is albumin, the principal protein component in human blood (Ancell, 1839; Carter and Ho, 1994). In practice, bovine serum albumin (BSA) is frequently used in cell culture media (Pfeifer et al., 2024), even though bovine-derived proteins can contain substances xenogenic to humans, such as N-glycolylneuraminic acid (Neu5Gc), potentially compromising the safety of therapeutic cellular products (Rohrer et al., 1998; Heiskanen et al., 2007; Nystedt et al., 2010; Hutton et al., 2024). In many cases, it is not well-communicated that animal-derived proteins like BSA are part of a laboratory material, such as in the supplement B27 (Yao et al., 2006; Chen et al., 2008), unless explicitly stated as xeno-free B27 (Lukovic et al., 2017; Martin E.-R. et al., 2022). In the following example, BSA is hidden behind two layers of trade names: Classically, KnockOut™ Serum Replacement (KOSR or KSR) has been commonly used for adaptation, culturing, passaging, and cryopreservation (Wagner and Welch, 2010a; Wagner and Welch, 2010b) as well as improving the reprogramming efficacy of stem cells (Zhao et al., 2010). However, one of its components is AlbuMAX®, which is lipid-rich BSA (Cranmer et al., 1997; Zhang and Robinson, 2005; Garcia-Gonzalo and Izpisúa Belmonte, 2008; Ogawa et al., 2024), making KOSR problematic for culturing cells for clinical use according to Ludwig and Thomson (2007). Moreover, Martin et al. (2005) identified KOSR as a major source of the xenoantigen Neu5Gc. This illustrates that finding truly animal-component-free media always requires a peek (or sometimes a deep dive) into the specific formulations and details of supplements and media to ensure they are truly free of animal-derived materials. Rivera-Ordaz et al. (2021) state that “formulations of commercially available media remain largely undisclosed, even though reference to the original published compositions is sometimes stated by the manufacturer”, with notable exception like the fully disclosed formulation of Advanced DMEM/F12, which also contains AlbuMAX® (Thermo Fisher Scientific, 2025b). Therefore, open access databases are invaluable tools (see Table 6), but do not relieve scientists from using their critical mind. Consequently, the use of HSA and other human proteins is recommended for human-cell-based biotechnological applications over their bovine counterparts (Penhallow et al., 1986; Maier et al., 2021; Mishra and Heath, 2021). Furthermore, research into albumin-free cell culture, e.g., Chen G. et al. (2011) showed how to cultivate human ESCs and iPSCs in a medium devoid of albumin.

Further approaches to “replace” FBS with different problematic animal-derived materials include egg white extract (Lee et al., 2024), bovine ocular fluid, earthworm heat-inactivated coelomic fluid, invertebrate fluids, non-bovine sera (Subbiahanadar Chelladurai et al., 2021), and animal tissue hydrolysates (Schlaeger, 1996; Siemensma et al., 2008). Even porcine or bovine platelet lysate is considered (Johansson et al., 2003; Hahn et al., 2024). Others supplement medium containing FBS with additional animal-derived materials, such as extracts from shrimp ovaries and eye stacks (Zhao and Guo, 2023). However, all these examples will just reintroduce the mentioned disadvantages of animal-derived materials back into science, while just shifting ethical issues from bovine fetuses to other animals. Another example is sericin, a globular protein with adhesive and gelatin-like characteristics found in the cocoons of the domestic silk moth (Bombyx mori) (Zhang, 2002). While sericin has been shown to have positive effects on cell attachment, proliferation, and survival rates (Tsubouchi et al., 2005; Yanagihara et al., 2006; Liu et al., 2016), its production requires the systematic killing of large numbers of animals, making the use of sericin unethical. Thus, obtaining sericin recombinantly or identifying structurally and functionally similar proteins from plant, fungal, or microbial sources opens up animal-free possibilities.

Other options include materials derived from algae or plants, like native proteins from the mixotrophically cultured red algae Galdieria sulphuraria (Eisenberg et al., 2025), soy hydrolysates (Mattick et al., 2015), and plant-based agro-industrial wastes (Teng et al., 2023; Flaibam et al., 2024b). They contain amino acids, vitamins, and lipids essential for cell culture (Ho et al., 2021). While their limitations, such as lack of standardization and the presence of potential plant-based anti-nutrients, do not yet justify their endorsement for use in scientific research, they may still represent an option for more accessible and sustainable cultured meat production (Etemadian et al., 2021; Flaibam et al., 2024a; Eisenberg et al., 2025). Nevertheless, due to the complexity of hydrolysates, it may be necessary to conduct proteomics and peptidomics analyses to assess their quality and gather information on potential allergens (Carrasco-Castilla et al., 2012).

In general, if proteins are necessary for maintaining the functionality of cells in culture, chemically defined cell culture media with recombinant proteins are the optimal choice to reduce variability and minimize contamination risks.

Ultimately, a xeno-free approach in cell culture should be every scientist’s aim, as working with xenogenic materials creates a chimeric environment. For example, culturing simian cells with bovine serum on a murine matrix, detached with a crustacean-derived dissociation agent (Luce et al., 2022) would generate experimental results with high variability and questionable transferability toward human biology. Science must move away from using animal-derived materials, just like it is moving away from using live animals for scientific purposes.