Hi Reader, what was your favorite innovation from last week?
The ability to grow cells in petri dishes has been a major innovation enabling us to study cancer, metabolism, or the side effects of chemicals the way we do today.
But we can’t do this without FBS—and like many innovations, it has come with a significant footprint that was long overlooked.
So, where do we stand with innovations today?
Today's Lesson: Exchanging Fetal Bovine Serum
How we can make cell culture more sustainable
Number Of The Day
A widely cited study from 2010 compiled more than 450 serum-free media in an online database. This tool made it possible to search through several alternatives that were chemically defined or included animal-derived substitutes. Unfortunately, the website hosting this database is no longer available—underlining the importance of sustainable data storage. Still, more and more alternatives become available:
450
The Price of FBS-Nourishment
In 1951 tumor cells taken from Henrietta Lack would become the origin for the very first immortal human cell culture.
These cervical tumor cells opened a new chapter in biology.
How HeLa cells look like – on the left through the microscope and on the right in a beautiful multiphoton fluorescence image.
However, growing cells in vitro needs frequent media exchanges
+ maintenance of frozen backups
+ maintenance in warm, CO₂-controlled incubators
+ autoclavation of all used materials before disposal…
However, among the most impactful components of cell culture is fetal bovine serum (FBS). An often necessary addition to the medium that nourishes the cells we grow.
Why FBS?
FBS is serum harvested from the fetuses of pregnant cows that were slaughtered without knowledge of pregnancy—an estimated +2 million fetuses per year, yielding around 800,000 liters of serum.
This graphic was adapted from Munteanu et al. Since the serum is only filtrated, concerns about viruses, Mycoplasma, or prions remain – with the last being a major problem for food production e.g., when it comes to cultured meat.
FBS is used because it is rich in growth factors, vitamins, hormones, and trace elements (containing approximately 1800 proteins and more than 4000 metabolites.
It promotes cell attachment, proliferation, pH buffering, and protects against mechanical damage.
Whether we like it or not, we often still need FBS for our cell culture. These pictures come from a preprint, showing micrographs of ZEM2S cells micrographs on day 3 of culture, with the pictures in the right column being 20X enlarged.
Moreover, it has low antibody and γ-globulin levels, which otherwise inhibit growth. Finally, its near-universal applicability for any cell line made it the standard.
Unexpected Drawbacks
Nevertheless, FBS comes with some disadvantages:
An Life-Cycle-Analyses suggested that up to 90% of the environmental impact of media was attributable to FBS.
No standardized undefined composition
Since it is derived from living animals batch variability
Some argue it poses a risk by possibly containing viruses, prions, endotoxins, or mycoplasma.
Ethical issues are growing, especially where regulations prohibit fetal material use.
The supply is unstable, and price is volatile, dictated by beef consumption, disease outbreaks, and trade restrictions.
This graphic is from a paper exploring the impacts of 1 liter DMEM + 10% FBS and its alternatives. Please note that both Wali et al. and Risner et al. conclude that FBS has a rather significant impact, while the analysis by Tuomisto et al. reached a different conclusion — with FBS contributing only about 12% of the overall impact. I explore the reasons in our Slack, but in short, this has to do with the fact that Tuomisto studied a setup in a bioreactor and, more importantly, how one accounts for the impact of the cattle.
Therefore, finding an alternative to FBS would be environmentally, monetary and scientifically sustainable.
Which Alternatives Exist?
The challenge with replacing FBS isn’t a lack of options—it’s that no one option works for every cell line. That said, multiple promising paths have emerged:
This graph is taken from this paper — they conclude that between 2000-2020 approximately 233 papers were published on FBS alternatives. However, if you decide to read it, be cautious, as I found some citation errors where the wrong papers were quoted.
1. Defined Serum-Free Media
These are formulations with common medium backbones (e.g., DMEM, RPMI, or MEM) but supplemented to be serum-free alternatives. They are available for specific cell types—like HUVEC or hybridoma cell lines for antibody production.
Some projects might require the addition of compounds like TGF-β3, Insulin, transferrin, or selenium - Lee et al. provide a nice overview.
2. Human Platelet Lysate (HPL)
Platelet lysates offer a human-derived, animal-free alternative. In some studies this approach has shown great success with mesenchymal stem cells, macrophages, or Hep-2 cells. The challenges: shelf life is short, and batch consistency can vary.
PLBioScience promotes that their Human Platelet Lysate is fibrinogen-depleted and anticoagulant free – I have not yet worked with Platelet Lysate but might be a factor to consider.
3. Bovine Ocular Fluid
Collected within hours of slaughter, ocular fluid contains fibronectin, VEGF, insulin-like growth factor, and other growth-enhancing components. Studies report improved growth so some cell lines.
4. Plant- and Algae-Based Protein Hydrolysates
These are enzymatically digested protein blends from sources like soy, cottonseed, rapeseed, and microalgae. At low concentrations (0.001–0.1 mg/mL), they support proliferation and reduce apoptosis. But at higher concentrations, they become toxic or suppress cell growth. Nevertheless, most often they are feasible to reduce the amount of FBS added to a cell culture.
While some protein hydrolysates have higher GHG emissions per functional unit, their very low required concentrations mean their overall impact per liter of media is small.
5. Earthworm Coelomic Fluid
Yes, really. When heat-inactivated, coelomic fluid from P. excavatus showed no morphological changes and supported normal growth in HeLa and fibroblast cells. Still, concerns about immune cell contamination and immunogenicity remain. However, upscaling seems achievable.
In fact, many alternatives were explored due to attempts to grow cultured meat as using FBS makes upscaling challenging. There are of course, even more approaches, however, applicability to cell lines, upscaling and adoption by scientists will crown a successful alternative.
Applying The Knowledge
Switching to serum free media is not without challenges although for most cell lines, alternatives exist.
For some special applications such as Zhou et al. chondrocyte Ca-osciallation measurements where FBS can cause dampening of signals, alternatives can even improve experimental outcomes.
If you switch:
Know your cells:
There is no universal substitute. What works for fibroblasts may not for stem cells. You will have to take the time to find the right alternative for you.
Wean gradually:
Many cells won’t adapt to serum-free media immediately. Begin by reducing FBS stepwise and supplementing with alternatives as outlined by Thermo Fisher here.
Record precisely:
Record which formulations were used, their concentrations, and how the cells behaved. This data is gold—for reproducibility, future decisions, and even publication.
Upcoming Lesson:
What Is Biogenic Carbon?
How We Feel Today
References
Brunner, D., et al., 2010. Serum-free cell culture: the serum-free media interactive online database. ALTEX 27(1), 53–62. doi:10.14573/altex.2010.1.53.
Wali, M.E., et al., 2024. Life cycle assessment of culture media with alternative compositions for cultured meat production. Int J Life Cycle Assess 29, 2077–2093. https://doi.org/10.1007/s11367-024-02350-6
Risner, D., et al., 2024. Environmental impacts of cultured meat: a cradle-to-gate life cycle assessment. ACS Food Sci Technol 5(1), 61–74. doi:10.1021/acsfoodscitech.4c00281.
Tuomisto, H.L., et al., 2022. Prospective life cycle assessment of a bioprocess design for cultured meat production in hollow fiber bioreactors. Sci Total Environ 851(Pt 1), 158051. doi:10.1016/j.scitotenv.2022.158051.
Subbiahanadar Chelladurai, K., et al., 2021. Alternative to FBS in animal cell culture – an overview and future perspective. Heliyon 7(8), e07686. doi:10.1016/j.heliyon.2021.e07686.
Amirvaresi, A., et al., 2024. Evaluation of plant- and microbial-derived protein hydrolysates as substitutes for fetal bovine serum in cultivated seafood cell culture media. bioRxiv. https://doi.org/10.1101/2024.03.27.587063
Zhou, Y., et al., 2015. The effect of chemically defined medium on spontaneous calcium signaling of in situ chondrocytes during long-term culture. J Biomech 48(6), 990–996. doi:10.1016/j.jbiomech.2015.02.005.
Lee, D.Y., et al., 2022. Review of the current research on fetal bovine serum and the development of cultured meat. Food Sci Anim Resour 42(5), 775–799. doi:10.5851/kosfa.2022.e46.
If you have a wish or a question, feel free to reply to this Email. Otherwise, wish you a beatiful week! See you again the 1st : )
Edited by Patrick Penndorf Connection@ReAdvance.com Lutherstraße 159, 07743, Jena, Thuringia, Germany Data Protection & Impressum If you think we do a bad job: Unsubscribe
Personal Note From Patrick, The Editor Hello Reader, do you remember a moment when a new innovation completely stunned you? Back when I was a student, I often wondered: What are companies and institutions actually doing to support sustainable science? I never really took the time to look it up... But later, I discovered just how many exciting solutions already exist. So today, I want to share a few of them with you. Today's Lesson: Innovations that Inspire Discovering what makes our science...
Personal Note From Patrick, The Editor Hi Reader, how long is your average conversation with ChatGPT? Last week, we saw that a single query to a large language model (LLM) consumes 0.5–50 Wh, releasing 1–5 grams of CO₂. That’s about 10 times the energy required for a Google search. Whether it’s AlphaFold, ChatGPT, let’s look at how we can reduce our footprint when using these models. Today's Lesson: Lowering Impacts of AI How we can calculate more sustainably Number Of The Day Far more than 9...
Personal Note From Patrick, The Editor Hi Reader, how often do you use ChatGPT? After discussing how to reduce the energy consumption of -70°C freezers, today we’re tackling the energy demands of AI. I use AI every day, and yet, there have been at least three times when I couldn’t tell that a video I watched was entirely AI-generated. While the debate on how AI will transform science is ongoing, its energy consumption is already a pressing issue. So, let’s explore how AI impacts the...
